Antibody-drug conjugate, composition, and use thereof and method therefor

Abstract

The present application provides an antibody-drug conjugate, a composition, and use thereof and a method therefor. A drug linker has a structure represented by a formula (I) Q-L-E-D, the antibody-drug conjugate has a structure represented by a formula (II), the drug is selected from a nucleoside active compound, and the prepared antibody-drug conjugate exhibits good tumor killing effect.

Description

Antibody drug conjugates, compositions, and methods of use and methods

[0001] This application is based on Chinese Patent Application No. 202410441253.6, filed on April 12, 2024, Chinese Patent Application No. 202411241509.5, filed on September 5, 2024, and Chinese Patent Application No. 202411663505.6, filed on November 20, 2024, for which priority is claimed, and the disclosures of which are incorporated herein in their entireties. TECHNICAL FIELD

[0002] The present application belongs to the field of medicine, and relates to antibody drug conjugates, compositions thereof, and uses thereof in treating diseases associated with abnormal cell activity, including but not limited to treating neoplastic diseases, particularly Her2-expressing cancers. The present application also provides drug-linkers for conjugation to antibodies, the drug being selected from antimetabolite antineoplastic drugs. The present application also provides combinations of antibody drug conjugates and therapeutic uses thereof. BACKGROUND

[0003] Antimetabolites are commonly used antineoplastic drugs in the clinic. Antimetabolites are structurally similar to folate, purine, and pyrimidine, which are required for DNA synthesis. After activation by intracellular triphosphorylation, nucleoside antimetabolites disrupt the synthesis of intracellular triphosphate deoxynucleotides (dNTPs), interfere with the incorporation of DNA or RNA macromolecules, competitively inhibit DNA synthesis-related enzymes, and specifically interfere with nucleic acid metabolism, preventing cell division and proliferation, and ultimately leading to tumor cell death. Nucleoside antimetabolites are mostly hydrophilic molecules, and thus require specific transmembrane receptors for recognition and transport into cells. The number of receptors and their distribution in various tissues result in differences in specificity and sensitivity of this class of drugs for different cancer cells, as well as differences in drug distribution in tissues in vivo. Antimetabolites often develop drug resistance due to changes in drug transport, decreased permeability, changes in metabolic enzymes, and enhanced DNA repair capacity.

[0004] Biological macromolecular drugs (e.g., therapeutic antibodies or antibody fragments) have made important progress as anti-tumor drugs. However, biological macromolecular drugs, although strong in targeting, have limited therapeutic effect on solid tumors. In recent years, it has been found that biological macromolecular drugs (e.g., therapeutic antibodies) can be linked to cytotoxic anti-tumor drugs to form antibody drug conjugates (ADC). ADC combines the targeting effect of antibodies and the high activity of cytotoxic drugs. Antibodies guide ADC to bind to target cells, which are then internalized by cells, release drugs, kill cells, and treat diseases. Because antibodies have specificity and targeting for tumor cell-related targets, their application value not only lies in treatment, but also serves as an ideal carrier for drug targeted delivery, reducing drug side effects.

[0005] Based on the targeting and effectiveness of antibody drug conjugates, attaching antimetabolites to antibodies through appropriate linking methods is an innovative attempt. In addition, antibody conjugates using antimetabolites and other mechanism of action anti-tumor active compounds as effector molecules have the potential for synergistic effects, and are expected to improve the effectiveness and safety of tumor treatment. SUMMARY

[0006] The present application provides a novel drug-linker and its antibody drug conjugate (i.e., conjugating antimetabolites to antibodies through a linker), which has targeted killing of tumors and plays a role in reducing toxicity and increasing efficacy.

[0007] Drug-linker

[0008] In one aspect, the present application provides a drug-linker conjugated to an antibody or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, having the structure shown in Formula (I),

[0009] Q-L-E-D

[0010] Formula (I)

[0011] wherein:

[0012] Q is a precursor of a linker site connected to an antibody or an antigen-binding fragment thereof;

[0013] L is a connecting structure connecting Q and E;

[0014] E is a structure connecting L and D;

[0015] D is a biologically active molecule moiety.

[0016] In some embodiments, Q is selected from the following structures:

[0017] wherein LG represents a leaving group; Ra is selected from cyano, nitro, C 2-6 alkyl, C 1-6 haloalkyl (e.g., trifluoromethyl or trichloromethyl), -C(=O)C 1-6 alkyl, and -SO2C 1-6 alkyl; preferably cyano; p is selected from an integer between 1 and 12.

[0018] In some embodiments, Q is selected from the following structures:

[0019] wherein LG represents a leaving group; p is selected from an integer between 1 and 12.

[0020] In some embodiments, p is selected from an integer between 1 and 11, 1 and 10, 1 and 9, 1 and 8, 1 and 7, 1 and 6, 1 and 5, 1 and 4, 1 and 3, 1 and 2, for example, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0021] In some embodiments, each LG is independently selected from halogen (e.g., F, Cl, Br, I), haloC 1-6 alkyl, C 1-6 alkylsulfonyl, haloC 1-6 alkylsulfonyl, halosulfonyl, C 1-6 alkylsulfonate, haloC 1-6 alkylsulfonate, C 1-6 alkylsulfinate, C 1-6 alkylsulfoxide, haloPhO, hydroxyl, thiol, amino, nitro, azido, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments, said haloC 1-6 alkyl, C 1-6 alkylsulfonyl, haloC 1-6 alkylsulfonyl, halosulfonyl, C 1-6 alkylsulfonate, haloC 1-6 alkylsulfonate, C 1-6 alkylsulfinate, C 1-6 alkylsulfoxide, haloPhO, alkenyl, alkynyl, and alkynyl-containing structural fragments are optionally substituted with one or more suitable substituents.

[0022] In some embodiments, each LG is independently selected from halogen (e.g., F, Cl, Br, I), methylsulfonyl, fluorophenoxy, hydroxyl, thiol, or amino.

[0023] In some embodiments, LG is C 1-6 alkylsulfonate.

[0024] In some embodiments, LG is methylsulfonate.

[0025] In some embodiments, Q is selected from the following structures:

[0026] wherein LG represents a leaving group (e.g., halogen, mesyl, fluorophenoxy, hydroxyl, thiol, or amino); p is selected from an integer from 1-12, e.g., p is selected from an integer from 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, e.g., p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0027] In some embodiments, Q is selected from the following structures:

[0028] wherein LG represents a leaving group (e.g., halogen, mesyl, fluorophenoxy, hydroxyl, thiol, or amino); p is selected from an integer from 1-12, e.g., p is selected from an integer from 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, e.g., p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0029] In some embodiments, Q is selected from the following structures:

[0030] In some embodiments, Q is selected from the following structures:

[0031] In some embodiments, Q is selected from the following structures:

[0032] In some embodiments, Q is selected from the following structures:

[0033] In some embodiments, L is selected from one or more substituted or unsubstituted structural fragments consisting of: C 1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 5-12 membered heterocyclyl, -N(R')-, carbonyl, -O-, natural amino acid, or unnatural amino acid, and analogs thereof (such as Ala, Arg, Asn, Asp, Cit, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2) rOCH3)), Lys(R'), short peptides 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, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG, SEQ ID NO: 44), Gly-Gly-Val-Ala (GGVA, SEQ ID NO: 45), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 46), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO: 47), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO: 48), Ala-Ala-Glu), wherein R' represents hydrogen, C 1-6 alkyl, glucosyl, galactosyl, glucuronyl, galacturonyl, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 alkyl, -(CH2N(Me)-C(=O) r -C 1-6 alkyl, a polyethylene glycol fragment of 1-10 EO units (i.e. -(CH2CH2O 1-10 -C 1-6alkyl), -CH2N(R")-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), -CH2N(R")-DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, a-propionyl), -CH2N(R")-NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), 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, a-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue); wherein r is selected from an integer from 1-20; s is selected from an integer from 1-20; R" is selected from hydrogen or C 1-6 alkyl.

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

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

[0036] In some embodiments, "-NOTA" refers to

[0037] In some specific embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments: C 1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 5-12 membered heterocyclyl, -N(R')-, carbonyl, -O-, natural amino acid or unnatural amino acid and analogs thereof (such as Ala, Arg, Asn, Asp, Cit, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2) rOCH3)), Lys(R'), short peptides 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, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG, SEQ ID NO: 44), Gly-Gly-Val-Ala (GGVA, SEQ ID NO: 45), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 46), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO: 47), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO: 48), Ala-Ala-Glu), wherein R' represents hydrogen, C 1-6 alkyl, glucosyl, galactosyl, glucuronyl, galacturonyl, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 alkyl, -(CH2N(Me)-C(=O) r -C 1-6 alkyl, polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O 1-10 -C 1-6alkyl), -CH2N(R")-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), -CH2N(R")-DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, a-propionyl), -CH2N(R")-NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), 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, a-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue); wherein r is selected from an integer from 1-20; s is selected from an integer from 1-20; R" is selected from hydrogen or C 1-6 alkyl.

[0038] In some embodiments, r is selected from an integer from 1-15, for example 1-10, 1-8, 1-6, 1-4, 1-2, for example r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0039] In some embodiments, s is selected from an integer from 1-15, for example 1-10, 1-8, 1-6, 1-4, 1-2, 3-6, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0040] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments: C 1-6 alkylene, carbonyl, 9-12 membered nitrogen-containing heterocyclyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu, wherein s is selected from an integer from 1-20; R' represents hydrogen, -CH2N(R")-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), -CH2N(R")-DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, alpha-propionyl), or -CH2N(R")-NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue); R" is selected from hydrogen or C 1-6 alkyl.

[0041] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments: C 1-6 alkylene, carbonyl, 9-12 membered nitrogen containing heterocyclyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu, wherein s is selected from an integer from 1-20, preferably s is selected from an integer from 1-15, such as 1-10, 1-8, 1-6, 1-4, 1-2, 3-6, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0042] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments: C 1-6alkylene, carbonyl, 9-12 membered nitrogen-containing heterocyclyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu, wherein s is selected from an integer from 1 to 20, preferably s is selected from an integer from 1 to 15, such as 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 2, 3 to 6, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0043] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments: C 1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, 5-12 membered heterocyclyl, -N(R')-, carbonyl, -O-, natural amino acid or unnatural amino acid and analogs thereof (such as Ala, Arg, Asn, Asp, Cit, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2) rOCH3)), Lys(R'), a short peptide 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, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu), wherein R' represents hydrogen, C 1-6 alkyl, glucosyl, galactosyl, glucuronyl, galacturonyl, -CH2N(C 1-6 alkyl)-C(=O)-(CH2CH2O) r -C 1-6 alkyl, -(CH2N(Me)-C(=O) r -C 1-6 alkyl, a polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) 1-10 -C 1-6 alkyl), -CH2N(R")-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), -CH2N(R")-DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, alpha-propionyl), -CH2N(R")-NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue), 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, alpha-propionyl), or NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue); wherein r is selected from an integer from 1-20; s is selected from an integer from 1-20; R" is selected from hydrogen or C1-6 alkyl. In some embodiments, r is selected from an integer from 1-15, such as an integer from 1-10, 1-8, 1-6, 1-4, 1-2, for example r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, s is selected from an integer from 1-15, such as an integer from 1-10, 1-8, 1-6, 1-4, 1-2, 3-6, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0044] In some embodiments, L is selected from one or more substituted or unsubstituted structural fragments consisting of: 1-6 alkylene, carbonyl, 9-12 membered nitrogen-containing heterocyclyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu, wherein s is selected from an integer from 1-20; R' represents hydrogen, -CH2N(R")-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), -CH2N(R")-DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, alpha-propionyl), or -CH2N(R")-NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue); R" is selected from hydrogen or C 1-6 alkyl.

[0045] In some embodiments, L is selected from one or more substituted or unsubstituted structural fragments consisting of: 1-6alkylene, carbonyl, 9-12 membered nitrogen-containing heterocyclyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu, wherein s is selected from an integer from 1 to 20, preferably s is selected from an integer from 1 to 15, for example an integer from 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 2, 3 to 6, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0046] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments:

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

[0048] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments:

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

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

[0051] where s is selected from an integer from 1 to 20.

[0052] In some specific embodiments, s is selected from an integer from 1 to 15, for example, an integer from 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 2, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0053] In some specific embodiments, L is selected from In some embodiments, L is connected to Q through a nitrogen atom at one end and to E at the other end.

[0054] In some specific embodiments, L is selected from In some embodiments, L is connected to Q through a nitrogen atom at one end and to E at the other end.

[0055] In some embodiments, L is selected from

[0056] In some specific embodiments, L is selected from In some embodiments, L is connected to Q through a nitrogen atom at one end and to E at the other end.

[0057] In some embodiments, E is a single bond, -NH-CH2-, or is selected from the following structures:

[0058] In some preferred embodiments, E is connected to L at the left end and to D at the right end.

[0059] In some embodiments, E is a single bond, -NH-CH2-, or is selected from the following structures:

[0060] In some preferred embodiments, E is connected to L at the left end and to D at the right end.

[0061] In some specific embodiments, E is a single bond, -NH-CH2-, or In some preferred embodiments, E is connected to L at the left end and to D at the right end.

[0062] In some embodiments, E is a single bond.

[0063] In some embodiments, E is -NH-CH2- or In some preferred embodiments, the left end of E is attached to L and the right end of E is attached to D.

[0064] In some embodiments, E is -NH-CH2-. In some preferred embodiments, the left end of E is attached to L and the right end of E is attached to D.

[0065] In some embodiments, E is

[0066] In some embodiments, D is selected from a fragment of a pharmaceutically active molecule.

[0067] In some embodiments, D is selected from a fragment of a nucleoside or a folate antineoplastic pharmaceutically active molecule.

[0068] In some embodiments, the nucleoside antineoplastic pharmaceutically active molecule is selected from the group consisting of fluorouracil, capecitabine, gemcitabine, cytarabine, azacitidine, doxifluridine, 5-fluoro-2'-deoxyuridine, tegafur, carmofur, uridine triacetate, troxacitabine, decitabine, ancitabine, inositol triacetate, FF-10502, brivudine, fludarabine, clatridine, clofarabine, nelarabine, furogenitabine, cordycepin, pentostatin, raltitrexed, pemetrexed, methotrexate, and pharmaceutically acceptable salts, esters, and analogs thereof.

[0069] The pharmaceutically active molecules disclosed herein typically contain a variety of functional groups, such as hydroxyl (-OH), primary amino (-NH2), secondary amine (-NR1H), where R1herein represents only a non-hydrogen substituent on N, which can react with appropriate functional groups in the remainder of the conjugate to effect attachment.

[0070] In some embodiments, the pharmaceutically active molecule is attached to E in the drug-linker through an -OH, a primary amino, or a secondary amine on the pharmaceutically active molecule.

[0071] In some embodiments, D is selected from

[0072] In some embodiments, D is selected from

[0073] In some embodiments, D is selected from

[0074] In some embodiments, D is selected from

[0075] In some embodiments, D is selected from

[0076] In some embodiments, -L-E-D is selected from the structures:

[0077] In some embodiments, -L-E-D is selected from the structures:

[0078] In some embodiments, -L-E-D is selected from the structures:

[0079] In some embodiments, -L-E-D is selected from the structures:

[0080] In some embodiments, the "Drug-Linker" is selected from the structures shown below:

[0081] In some embodiments, the drug-linker described supra can optionally be substituted with one or more suitable substituents.

[0082] Synthetic intermediates

[0083] In another aspect, the present application provides an intermediate compound having the following structure:

[0084] wherein PG 1 each is independently selected from H or a hydroxyl protecting group, preferably a tert-butyldimethylsilyl group, a phosphonic acid, a benzoyl group, a trityl group, a 4-methoxyphenyldiphenylmethyl group, a dimethoxytrityl group, a 2,4-dimethoxybenzyl group, a p-methoxybenzyl group, and a benzyl group;

[0085] PG 2 each is independently H or an amino protecting group, preferably an alkoxycarbonyl amino protecting group such as benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), formyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), meth(o)ethylcarbonyl; an acyl amino protecting group such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p)-nitrophenylsulfonyl (Ns), pivaloyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-methylphenylsulfonyl, p-nitrophenylsulfonyl, tert-butyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; an alkyl amino protecting group such as trityl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn);

[0086] R 2 is selected from H, C 1-6 alkyl; and LG represents a leaving group as described in any one of the preceding embodiments.

[0087] In some embodiments, the intermediate compounds of the present application have the following structure:

[0088] wherein PG 1 , PG 2 , R 2 and LG are as defined above.

[0089] In some embodiments, the intermediate compounds of the present application have the following structure:

[0090] In another aspect, the present application provides use of an intermediate compound, a salt, a stereoisomer, a tautomer or an isotopically-labeled compound thereof in the manufacture of a drug-linker of formula (I), said intermediate compound being as described above.

[0091] Antibody drug conjugate (ADC)

[0092] In another aspect, the present application provides an antibody drug conjugate having a structure of formula (II),

[0093]

[0094] Formula (II)

[0095] wherein L, E and D are as described in any one of the above,

[0096] M is a linker moiety attached to an antibody or an antigen binding fragment thereof;

[0097] Ab is an antibody or an antigen binding fragment thereof;

[0098] x is selected from 1 to 10.

[0099] A precursor of M in formula (II) is Q as defined above.

[0100] In the antibody drug conjugate, D can be attached to the antibody or the antigen binding fragment thereof via a linker (e.g. the "M-L-E" moiety as shown in the present application).

[0101] In some embodiments, M is selected from wherein the mark 1 in the structure of M indicates the attachment of M to Ab, and the mark 2 in the structure of M indicates the attachment of M to L; p is selected from an integer from 1 to 12; and Ra is as described in any one of the above.

[0102] In some specific embodiments, M is selected from the following structures: wherein the mark 1 in the structure of M indicates the attachment of M to Ab, and the mark 2 in the structure of M indicates the attachment of M to L; p is selected from an integer from 1 to 12.

[0103] In some specific embodiments,

[0104] ​In some embodiments, p is selected from an integer from 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, for example, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0105] In some embodiments, M is selected from the following structures:

[0106] In some embodiments, M is

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

[0108] (1) a heavy chain variable region (VH) and / or a 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 3 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 3 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 3 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 3 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 according to any one of (la), (lb) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived or has one or several 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;

[0112] or,

[0113] (2) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system:

[0114] (2a) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, CDR-L3 of SEQ ID NO: 10 or a variant thereof; or,

[0115] (2b) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0116] wherein the variant according to any one of (2a), (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 to the sequence from which it is derived or has one or several 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;

[0117] or,

[0118] (3) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the Kabat numbering system:

[0119] (3a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, CDR-L3 of SEQ ID NO: 10 or a variant thereof; or,

[0120] (3b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0121] wherein the variant of any one of (3a), (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 to the sequence from which it is derived, or the variant has one or several (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;

[0122] or,

[0123] (4) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the IMGT numbering system:

[0124] (4a) a heavy chain variable region (VH) comprising 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 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 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 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 of any one of (4a), (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 to the sequence from which it is derived, or the variant has one or several (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.

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

[0128] (1) a heavy chain variable region (VH) and a 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 3 CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, CDR-H3 of SEQ ID NO: 7; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, CDR-L3 of SEQ ID NO: 10; or,

[0130] (1b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, CDR-H3 of SEQ ID NO: 22; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, CDR-L3 of SEQ ID NO: 25;

[0131] or,

[0132] (2) a heavy chain variable region (VH) and a light chain variable region (VL) wherein the CDRs are defined by the AbM numbering system:

[0133] (2a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, CDR-H3 of SEQ ID NO: 7; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, CDR-L3 of SEQ ID NO: 10; or,

[0134] (2b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, CDR-H3 of SEQ ID NO: 22; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, CDR-L3 of SEQ ID NO: 25;

[0135] or,

[0136] (3) a heavy chain variable region (VH) and a light chain variable region (VL) in which the CDRs are defined by the Kabat numbering system:

[0137] (3a) a heavy chain variable region (VH) comprising 3 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 3 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 3 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 3 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) a heavy chain variable region (VH) and a light chain variable region (VL) in which the CDRs are defined by the IMGT numbering system:

[0141] (4a) a heavy chain variable region (VH) comprising 3 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 3 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 3 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 3 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] (a) a VH as set forth in SEQ ID NO: 1 or a variant thereof, and / or, a VL as set forth in SEQ ID NO: 2 or a variant thereof; or

[0145] (b) a VH as set forth in SEQ ID NO: 3 or a variant thereof, and / or, a VL as set forth in SEQ ID NO: 4 or a variant thereof;

[0146] 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 with the sequence from which it is derived or has one or several (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.

[0147] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0148] (a) a VH as set forth in SEQ ID NO: 1 and, a VL as set forth in SEQ ID NO: 2; or

[0149] (b) a VH as set forth in SEQ ID NO: 3 and, a VL as set forth in SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof further comprises:

[0150] (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having 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

[0151] (b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof having 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.

[0152] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgGl, IgG2, IgG3, or IgG4 heavy chain constant region, e.g., a human IgGl heavy chain constant region or a human IgG4 heavy chain constant region.

[0153] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, or a variant thereof having up to 20 conservative substitutions (e.g., up to 15, up to 10, or up to 5 conservative substitutions; e.g., 1, 2, 3, 4, or 5 conservative substitutions) compared to SEQ ID NO: 35.

[0154] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 41, or a variant thereof having up to 20 conservative substitutions (e.g., up to 15, up to 10, or up to 5 conservative substitutions; e.g., 1, 2, 3, 4, or 5 conservative substitutions) compared to SEQ ID NO: 41.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as set forth in SEQ ID NO: 36, or a variant thereof having up to 20 conservative substitutions (e.g., up to 15, up to 10, or up to 5 conservative substitutions; e.g., 1, 2, 3, 4, or 5 conservative substitutions) compared to SEQ ID NO: 36.

[0156] In some embodiments, the antibody or antigen-binding fragment thereof 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.

[0157] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 41 and a light chain constant region (CL) as set forth in SEQ ID NO: 36.

[0158] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0159] (1) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, and, a light chain comprising a VL as set forth in SEQ ID NO: 2 and a light chain constant region (CL) as set forth in SEQ ID NO: 36;

[0160] (2) a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 35, and, a light chain comprising a VL of the sequence set forth in SEQ ID NO: 4 and a light chain constant region (CL) of the sequence set forth in SEQ ID NO: 36; or

[0161] (3) a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 41, and, a light chain comprising a VL of the sequence set forth in SEQ ID NO: 2 and a light chain constant region (CL) of the sequence set forth in SEQ ID NO: 36; or

[0162] (4) a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 41, and, a light chain comprising a VL of the sequence set forth in SEQ ID NO: 4 and a light chain constant region (CL) of the sequence set forth in SEQ ID NO: 36.

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

[0164] (1) a heavy chain comprising the sequence set forth in SEQ ID NO: 37, and, a light chain comprising the sequence set forth in SEQ ID NO: 38;

[0165] (2) a heavy chain comprising the sequence set forth in SEQ ID NO: 39, and, a light chain comprising the sequence set forth in SEQ ID NO: 40;

[0166] (3) a heavy chain comprising the sequence set forth in SEQ ID NO: 42, and, a light chain comprising the sequence set forth in SEQ ID NO: 38; or

[0167] (4) a heavy chain comprising the sequence set forth in SEQ ID NO: 43, and, a light chain comprising the sequence set forth in SEQ ID NO: 40.

[0168] In certain embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain can comprise a C-terminal lysine or lack a C-terminal lysine or lack a C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof can be cyclized into pyroglutamic acid.

[0169] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0170] In certain embodiments, provided herein are compositions comprising antibodies or antigen-binding fragments disclosed herein, wherein the various antibodies or antigen-binding fragments 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, N-terminal amino acid cyclization to pyroglutamic acid, or N-terminal amino acid cyclization to pyroglutamate.

[0171] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen, and can include post-translational modifications thereof (e.g., C-terminal lysine clipping in the heavy chain, N-terminal glutamine or glutamic acid conversion to pyroglutamic acid or pyroglutamate in the heavy chain or light chain), which can occur upon recombinant expression in a host cell (e.g., a CHO cell) or during purification / storage.

[0172] In certain embodiments, the N-terminal glutamine of the VH of the sequence set forth in SEQ ID NO: 1 or 3, or a variant thereof, or the heavy chain of the sequence set forth in SEQ ID NO: 37, 39, 42, or 43, or a variant thereof, is cyclized to form pyroglutamic acid or pyroglutamate.

[0173] In certain embodiments, the heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 35 or 41, or a variant thereof, or the heavy chain of the sequence set forth in SEQ ID NO: 37, 39, 42, or 43, or a variant thereof, lacks a C-terminal lysine.

[0174] In some specific embodiments, Ab is selected from an antibody or antigen-binding fragment thereof that specifically binds to a member of the ErbB family of receptor tyrosine kinases, epidermal growth factor receptor 2 (Her2).

[0175] In some specific embodiments, Ab is selected from Trastuzumab, Pertuzumab, a Trastuzumab mutant, a Pertuzumab mutant, or a bi-specific antibody constructed from Trastuzumab and Pertuzumab, or an antigen-binding fragment thereof.

[0176] In some specific embodiments, Ab is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which is assigned the query accession number (IMGT / mAb-DB ID) 97 in the IMGT database, or the amino acid sequence of which is assigned the query accession number (IMGT / mAb-DB ID) 80 in the IMGT database, respectively, or an antigen-binding fragment thereof.

[0177] Those skilled in the art will appreciate that the antibody drug conjugates described herein can be prepared modularly. For example, a "drug-linker" in free form (which can be understood as Q-L-E-D, wherein Q is the structural form of M before covalent attachment to an antibody or antigen binding fragment thereof) can be obtained first, and then covalently attached to an antibody or antigen binding fragment thereof to obtain the antibody drug conjugates described herein. Accordingly, Q in the "drug-linker" in free form is attached to one or more sulfhydryl (-SH) or amino (-NH2) groups on the antibody or antigen binding fragment thereof by substitution reaction (e.g., removal of -SO2Me or -Br, etc. structure thereon) or by addition reaction, etc.

[0178] In some embodiments, the antibody drug conjugate is selected from the group consisting of ADC A-1 to ADC A-30, ADC B-1 to ADC B-32, ADC C-1 to ADC C-64:

[0179] In some embodiments, the antibody drug conjugate is selected from the group consisting of the following structures:

[0180] wherein Ab' -(-S- represents an antibody or antigen binding fragment thereof, indicates the specific attachment of sulfhydryl in the antibody or antigen binding fragment thereof to the M fragment, and x is selected from 1 to 10.

[0181] In some embodiments, Ab' in each antibody drug conjugate represents an antibody or antigen binding fragment thereof that specifically binds to a member of the ErbB family of receptor tyrosine kinases, epidermal growth factor receptor 2 (Her2);

[0182] wherein, represents a specific linkage mode of a thiol group in the antibody or antigen binding fragment thereof to the M fragment.

[0183] In some embodiments, the antibody or antigen binding fragment thereof comprises

[0184] (1) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0185] (1a) a heavy chain variable region (VH) comprising 3 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 3 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,

[0186] (1b) a heavy chain variable region (VH) comprising 3 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 3 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;

[0187] wherein, the variant in any one of (1a), (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 to the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions;

[0188] or,

[0189] (2) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the AbM numbering system:

[0190] (2a) a heavy chain variable region (VH) comprising 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 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,

[0191] (2b) a heavy chain variable region (VH) comprising 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 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;

[0192] wherein the variant of any one of (2a), (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 to the sequence from which it is derived, or the variant has one or several (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;

[0193] or,

[0194] (3) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the Kabat numbering system:

[0195] (3a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, CDR-L3 of SEQ ID NO: 10 or a variant thereof; or,

[0196] (3b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0197] wherein the variant of any one of (3a), (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 several (e.g., 1, 2, or 3) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution;

[0198] or,

[0199] (4) a heavy chain variable region (VH) and / or a light chain variable region (VL) wherein the CDRs are defined according to the IMGT numbering system:

[0200] (4a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, CDR-L3 of SEQ ID NO: 10 or a variant thereof; or,

[0201] (4b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0202] wherein the variant of any one of (4a), (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or several (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.

[0203] In some embodiments, the Ab’ comprises

[0204] (1) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the Chothia numbering system:

[0205] (1a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, CDR-H3 of SEQ ID NO: 7; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, CDR-L3 of SEQ ID NO: 10; or,

[0206] (1b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 20, CDR-H2 of SEQ ID NO: 21, CDR-H3 of SEQ ID NO: 22; and, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, CDR-L3 of SEQ ID NO: 25;

[0207] (2) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system:

[0208] (2a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, CDR-H3 of SEQ ID NO: 7; and, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, CDR-L3 of SEQ ID NO: 10; or,

[0209] (2b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, CDR-H3 of SEQ ID NO: 22; and, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, CDR-L3 of SEQ ID NO: 25;

[0210] (3) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the Kabat numbering system:

[0211] (3a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, CDR-H3 of SEQ ID NO: 7; and, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, CDR-L3 of SEQ ID NO: 10; or,

[0212] (3b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, CDR-H3 of SEQ ID NO: 22; and, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, CDR-L3 of SEQ ID NO: 25.

[0213] (3a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, CDR-H3 of SEQ ID NO: 7; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, CDR-L3 of SEQ ID NO: 10; or,

[0214] (3b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, CDR-H3 of SEQ ID NO: 22; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, CDR-L3 of SEQ ID NO: 25;

[0215] or,

[0216] (4) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system:

[0217] (4a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, CDR-H3 of SEQ ID NO: 15; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, CDR-L3 of SEQ ID NO: 10; or,

[0218] (4b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 28, CDR-H2 of SEQ ID NO: 29, CDR-H3 of SEQ ID NO: 30; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 31, CDR-L2 of SEQ ID NO: 32, CDR-L3 of SEQ ID NO: 25.

[0219] In some embodiments, the Ab' comprises:

[0220] (a) a VH as depicted in SEQ ID NO: 1 or a variant thereof, and / or, a VL as depicted in SEQ ID NO: 2 or a variant thereof; or

[0221] (b) a VH as depicted in SEQ ID NO: 3 or a variant thereof, and / or, a VL as depicted in SEQ ID NO: 4 or a variant thereof;

[0222] wherein said 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 with the sequence from which it is derived, or said variant has one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably said substitutions are conservative substitutions.

[0223] In some embodiments, said Ab' comprises:

[0224] (a) a VH as depicted in SEQ ID NO: 1, and, a VL as depicted in SEQ ID NO: 2; or

[0225] (b) a VH as depicted in SEQ ID NO: 3, and, a VL as depicted in SEQ ID NO: 4.

[0226] In some embodiments, said Ab' further comprises:

[0227] (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having 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

[0228] (b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof having 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.

[0229] In some embodiments, said heavy chain constant region is an IgG heavy chain constant region, e.g. an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, e.g. a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

[0230] In some embodiments, the Ab' comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, or a variant thereof having up to 20 conservative substitutions (e.g., up to 15, up to 10, or up to 5 amino acid conservative substitutions; e.g., 1, 2, 3, 4, or 5 amino acid conservative substitutions) compared to SEQ ID NO: 35.

[0231] In some embodiments, the Ab' comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 41, or a variant thereof having up to 20 conservative substitutions (e.g., up to 15, up to 10, or up to 5 amino acid conservative substitutions; e.g., 1, 2, 3, 4, or 5 amino acid conservative substitutions) compared to SEQ ID NO: 41.

[0232] In some embodiments, the Ab' comprises a light chain constant region (CL) as set forth in SEQ ID NO: 36, or a variant thereof having up to 20 conservative substitutions (e.g., up to 15, up to 10, or up to 5 amino acid conservative substitutions; e.g., 1, 2, 3, 4, or 5 amino acid conservative substitutions) compared to SEQ ID NO: 36.

[0233] In some embodiments, the Ab' 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.

[0234] In some embodiments, the Ab' comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 41 and a light chain constant region (CL) as set forth in SEQ ID NO: 36.

[0235] In some embodiments, the Ab' comprises:

[0236] (1) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, and a light chain comprising a VL as set forth in SEQ ID NO: 2 and a light chain constant region (CL) as set forth in SEQ ID NO: 36;

[0237] (2) a heavy chain comprising a VH as set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 35, and a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 36; or

[0238] (3) a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 41, and, a light chain comprising a VL of the sequence set forth in SEQ ID NO: 2 and a light chain constant region (CL) of the sequence set forth in SEQ ID NO: 36; or

[0239] (4) a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 41, and, a light chain comprising a VL of the sequence set forth in SEQ ID NO: 4 and a light chain constant region (CL) of the sequence set forth in SEQ ID NO: 36.

[0240] In some embodiments, the Ab' comprises:

[0241] (1) a heavy chain comprising the sequence set forth in SEQ ID NO: 37, and, a light chain comprising the sequence set forth in SEQ ID NO: 38;

[0242] (2) a heavy chain comprising the sequence set forth in SEQ ID NO: 39, and, a light chain comprising the sequence set forth in SEQ ID NO: 40;

[0243] (3) a heavy chain comprising the sequence set forth in SEQ ID NO: 42, and, a light chain comprising the sequence set forth in SEQ ID NO: 38; or

[0244] (4) a heavy chain comprising the sequence set forth in SEQ ID NO: 43, and, a light chain comprising the sequence set forth in SEQ ID NO: 40.

[0245] In certain embodiments, the N-terminal glutamine of the VH of the sequence set forth in SEQ ID NO: 1 or 3 or a variant thereof or the heavy chain of the sequence set forth in SEQ ID NO: 37, 39, 42 or 43 or a variant thereof is subjected to cyclization to form pyroglutamic acid or pyroglutamate.

[0246] In certain embodiments, the heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 35 or 41 or a variant thereof or the heavy chain of the sequence set forth in SEQ ID NO: 37, 39, 42 or 43 or a variant thereof lacks a C-terminal lysine.

[0247] In some specific embodiments, Ab' is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which is query accession number (IMGT / mAb-DB ID) 97 in IMGT database, or Pertuzumab, the amino acid sequence of which is query accession number (IMGT / mAb-DB ID) 80 in IMGT database.

[0248] In some embodiments, Ab' in each antibody drug conjugate represents Trastuzumab, Pertuzumab, or an antigen binding fragment thereof.

[0249] In some embodiments, Ab' in each antibody drug conjugate represents the following antibodies or antigen binding fragments:

[0250] (1) a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 35, and a light chain comprising a VL of the sequence set forth in SEQ ID NO: 2 and a light chain constant region (CL) of the sequence set forth in SEQ ID NO: 36;

[0251] (2) a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 35, and a light chain comprising a VL of the sequence set forth in SEQ ID NO: 4 and a light chain constant region (CL) of the sequence set forth in SEQ ID NO: 36; or

[0252] (3) a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 41, and a light chain comprising a VL of the sequence set forth in SEQ ID NO: 2 and a light chain constant region (CL) of the sequence set forth in SEQ ID NO: 36; or

[0253] (4) a heavy chain comprising a VH of the sequence set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 41, and a light chain comprising a VL of the sequence set forth in SEQ ID NO: 4 and a light chain constant region (CL) of the sequence set forth in SEQ ID NO: 36.

[0254] In certain embodiments, the N-terminal glutamine of the VH of the sequence set forth in SEQ ID NO: 1 or 3 or a variant thereof is cyclized to form pyroglutamic acid or a pyroglutamate salt.

[0255] In certain embodiments, the heavy chain constant region (CH) of the sequence set forth in SEQ ID NO: 35 or 41 lacks a C-terminal lysine.

[0256] In some embodiments, x in the conjugate represented by Ab-[M-L-E-D]x is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 6-8.

[0257] In some embodiments, x in the conjugate represented by Ab-[M-L-E-D]x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0258] In some embodiments, x in the conjugate represented by Ab-[M-L-E-D]x is 1.0-10.0, for example: 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5-6.0, 4.0-4.5, 4.0-5.0, 4.0-5.5, 4.0-6.0, 4.5-5.0, 4.5-5.5, 4.5-6.0, 5.0-5.5, 5.0-6.0, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.0-9.0, 7.5-8.5, 7.0-8.0, or 7.5-8.0.

[0259] In some embodiments, x in the conjugate represented by Ab-[M-L-E-D]x is 4.0-8.0.

[0260] In some embodiments, x in the conjugate represented by Ab-[M-L-E-D]x is 6.0-8.0.

[0261] In some embodiments, x in the conjugate represented by Ab-[M-L-E-D]x is 7.0-7.5.

[0262] In some embodiments, x in the conjugate represented by Ab-[M-L-E-D]x is 7.0-8.0.

[0263] In some embodiments, x in the conjugate represented by Ab-[M-L-E-D]x is 7.0-9.0.

[0264] In some embodiments, x in the conjugate represented by Ab-[M-L-E-D]x is 7.5-8.5.

[0265] In some embodiments, x in the conjugate of Ab-[M-L-E-D]x is 7.5-8.0.

[0266] In some embodiments, the conjugate described herein is an antibody drug conjugate (ADC).

[0267] In some embodiments, the conjugate described herein is optionally substituted with one or more suitable substituents.

[0268] Linker

[0269] In another aspect, the present application provides a linker having a structure of -E-L-M-; wherein E, L and M are as described in any one of the above.

[0270] In some embodiments, E is used to connect to D as described in any one of the above, and M is used to connect to an antibody or antigen binding fragment thereof as described in any one of the above.

[0271] In some embodiments, the linker has a structure as shown below, wherein position 1 is connected to D as described in any one of the above, and position 2 is connected to an antibody or antigen binding fragment thereof as described in any one of the above:

[0272] In some embodiments, the present application provides a linker having a structure of -E-L-Q; wherein E, L and Q are as described in any one of the above.

[0273] In some embodiments, E is used to connect to D as described in any one of the above.

[0274] In some embodiments, the linker has a structure as shown below, wherein position 1 is connected to D as described in any one of the above:

[0275] In some embodiments, a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof is provided, comprising a linker structure of -M-L-E-.

[0276] In some embodiments, there is provided a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, comprising a linker structure of the structure shown by -M-L-E- and Ab and / or D, said linker structure being attached to Ab and / or D; wherein Ab or D is as defined in any of the preceding embodiments.

[0277] In some embodiments, there is provided a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, comprising a linker structure of the structure shown by Q-L-E.

[0278] In some embodiments, there is provided a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, comprising a linker structure of the structure shown by -M-L-E- and Ab and / or D, said linker structure being attached to Ab and / or D; wherein Ab or D is as defined in any of the preceding embodiments.

[0279] In some embodiments, the present application provides a linking unit of the structure shown by -L-E-D, wherein L, E and D are as defined in any of the preceding embodiments. In some embodiments, the linking unit is used to link an antibody or an antigen binding fragment thereof via a linker to obtain an antibody drug conjugate.

[0280] In some embodiments, the present application provides a linking unit of the structure shown by -M-L-E-D, wherein M, L, E and D are as defined in any of the preceding embodiments. In some embodiments, the linking unit is used to link an antibody or an antigen binding fragment thereof to obtain an antibody drug conjugate.

[0281] In some embodiments, there is provided a compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, comprising a linking unit of the structure shown by -L-E-D or -M-L-E-D.

[0282] In some embodiments, the linking unit described in the preceding embodiments can be optionally substituted with one or more suitable substituents.

[0283] Linker

[0284] In some embodiments, the present application provides a linker structure of the structure shown by:

[0285] The linker is attached to Ab at label 1 and to L at label 2; p is selected from an integer from 1-12; s is selected from an integer from 1-20, Ra is as described in any one of the preceding embodiments.

[0286] In some embodiments, For

[0287] In some embodiments, p is selected from an integer from 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, for example, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0288] In some embodiments, the linker is selected from the following structures:

[0289] In some embodiments, the linker is selected from the following structures:

[0290] In some embodiments, the linker is selected from the following structures:

[0291] ADC compositions

[0292] In another aspect, the present application provides a composition of an antibody drug conjugate (ADC) as described herein. Such a composition can comprise a plurality of ADCs described herein, wherein each ADC comprises a drug-linker described herein, wherein x is independently 1-10. In other words, each antibody molecule in the composition can be, on average, conjugated to 1-10 drug-linkers. Thus, the composition is characterized by a drug-antibody ratio (DAR) in the range of about 1 to about 10. Methods of determining DAR are well known to the skilled person, including methods using reverse phase chromatography or HPLC-MS.

[0293] For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 10 or any sub-range therebetween, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, 9-10.

[0294] For example, in any embodiment, the ADC compositions described herein have a DAR of about 1 to about 10, or any sub-range therebetween, for example: about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10, or about 9 to 10.

[0295] In certain embodiments, the DAR of the ADC compositions described herein is about 1 to 8, for example about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 3.5 to 7.0, about 3.5 to 7.5, about 3.5 to 8.0, 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 7.5, 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 4.5 to 8.0, about 5.0 to 5.5, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.0, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 7.0 to 7.5, about 7.0 to 8.0, about 7.5 to 8.0.

[0296] In certain embodiments, the DAR of the ADC compositions described herein is about 6.0 to 10.0, e.g., about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6.1, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19, about 6.2, about 6.21, about 6.22, about 6.23, about 6.24, about 6.25, about 6.26, about 6.27, about 6.28, about 6.29, about 6.3, about 6.31, about 6.32, about 6.33, about 6.34, about 6.35, about 6.36, about 6.37, about 6.38, about 6.39, about 6.4, about 6.41, about 6.42, about 6.43, about 6.44, about 6.45, about 6.46, about 6.47, about 6.48, about 6.49, about 6.5, about 6.51, about 6.52, about 6.53, about 6.54, about 6.55, about 6.56, about 6.57, about 6.58, about 6.59, about 6.6, about 6.61, about 6.62, about 6.63, about 6.64, about 6.65, about 6.66, about 6.67, about 6.68, about 6.69, about 6.7, about 6.71, about 6.72, about 6.73, about 6.74, about 6.75, about 6.76, about 6.77, about 6.78, about 6.79, about 6.8, about 6.81, about 6.82, about 6.83, about 6.84, about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, about 6.9, about 6.91, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.0, about 7.01, about 7.02, about 7.03, about 7.04, about 7.05, about 7.06, about 7.07, about 7.08, about 7.09, about 7.1, about 7.11, about 7.12, about 7.13, about 7.14, about 7.15, about 7.16, about 7.17, about 7.18, about 7.19, about 7.2, about 7.21, about 7.22, about 7.23, about 7.24, about 7.25, about 7.26, about 7.27, about 7.28, about 7.29, about 7.3, about 7.31, about 7.32, about 7.33, about 7.34, about 7.35, about 7.36, about 7.37, about 7.38, about 7.39, about 7.4, about 7.41, about 7.42, about 7.43, about 7.44, about 7.45, about 7.46, about 7.47, about 7.48, about 7.49, about 7.5, about 7.51, about 7.52, about 7.53, about 7.54, about 7.55, about 7.56, about 7.57, about 7.58, about 7.59, about 7.6, about 7.61, about 7.62, about 7.63, about 7.64, about 7.65, about 7.66, about 7.67, about 7.68, about 7.69, about 7.7, about 7.71, about 7.72, about 7.73, about 7.74, about 7.75, about 7.76, about 7.77, about 7.78, about 7.79, about 7.8, about 7.81, about 7.82, about 7.83, about 7.84, about 7.85, about 7.86, about 7.87, about 7.88, about 7.89, about 7.9, about 7.91, about 7.92, about 7.93, about 7.94, about 7.95, about 7.96, about 7.97, about 7.98, about 7.99, about 8.0, about 8.01, about 8.02, about 8.03, about 8.04, about 8.05, about 8.06, about 8.07, about 8.08, about 8.09, about 8.1, about 8.11, about 8.12, about 8.13, about 8.14, about 8.15, about 8.16, about 8.17, about 8.18, about 8.19, about 8.2, about 8.21, about 8.22, about 8.23, about 8.24, about 8.25, about 8.26, about 8.27, about 8.28, about 8.29, about 8.3, about 8.31, about 8.32, about 8.33, about 8.34, about 8.35, about 8.36, about 8.37, about 8.38, about 8.39, about 8.4, about 8.41, about 8.42, about 8.43, about 8.44, about 8.45, about 8.46, about 8.47, about 8.48, about 8.49, about 8.5, about 8.51, about 8.52, about 8.53, about 8.54, about 8.55, about 8.56, about 8.57, about 8.58, about 8.59, about 8.6, about 8.61, about 8.62, about 8.63, about 8.64, about 8.65, about 8.66, about 8.67, about 8.68, about 8.69, about 8.7, about 8.71, about 8.72, about 8.73, about 8.74, about 8.75, about 8.76, about 8.77, about 8.78, about 8.79, about 8.8, about 8.81, about 8.82, about 8.83, about 8.84, about 8.85, about 8.86, about 8.87, about 8.88, about 8.89, about 8.9, about 8.91, about 8.92, about 8.93, about 8.94, about 8.95, about 8.96, about 8.97, about 8.98, about 8.99, about 9.0, about 6.0-8.0, about 6.0-8.5, about 6.0-9.0, about 6.0-9.5, about 6.0-10.0, about 6.5-8.0, about 6.5-8.5, about 6.5-9.0, about 6.5-9.5, about 6.5-10.0, about 7.0-8.0, about 7.0-8.5, about 7.0-9.0, about 7.0-9.5, about 7.0-10.0, about 7.5-8.0, about 7.5-8.5, about 7.5-9.0, about 7.5-9.5, about 7.5-10.0, about 8.0 to 8.5, about 8.0 to 9.0, about 8.0 to 9.5, about 8.0 to 10.0.

[0297] In certain embodiments, the DAR of the ADC compositions described herein is about 4.0 to 8.0.

[0298] In certain embodiments, the DAR of the ADC compositions described herein is about 6.0 to 8.0.

[0299] In certain embodiments, the DAR of the ADC compositions described herein is about 7.0 to 8.0.

[0300] In certain embodiments, the DAR of the ADC compositions described herein is about 7.0 to 7.5.

[0301] In certain embodiments, the DAR of the ADC compositions described herein is about 7.2 to 8.0.

[0302] In certain embodiments, the DAR of the ADC compositions described herein is about 7.5 to 8.0.

[0303] In certain embodiments, the DAR of the ADC compositions described herein is about 7.24, about 7.46, about 7.56, about 7.7, about 7.8, about 7.83, about 7.9, or about 7.99.

[0304] In certain embodiments, the DAR of the ADC compositions described herein is about 6.79, about 7.24, about 7.46, about 7.56, about 7.9, or about 7.99.

[0305] In certain embodiments, the DAR of the ADC compositions described herein is 7.24, 7.46, 7.56, 7.7, 7.8, 7.83, 7.9, or 7.99.

[0306] In certain embodiments, the DAR of the ADC compositions described herein is 6.79, 7.24, 7.46, 7.56, 7.9, or 7.99.

[0307] Pharmaceutical composition or pharmaceutical combination

[0308] In another aspect, the application provides a pharmaceutical composition comprising the antibody drug conjugate of any one of the preceding, the ADC composition of any one, or the drug-linker of any one, and one or more pharmaceutical excipients.

[0309] The antibody drug conjugates, ADC compositions, or drug-linkers described herein are typically 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.

[0310] In some embodiments, the pharmaceutical composition of the present disclosure further comprises one or more optional other therapeutic agents. In some embodiments, the other therapeutic agent is an antibody drug conjugate targeting Her2. In some embodiments, the other therapeutic agent is an antibody drug conjugate (ADC) of the present invention.

[0311] In some embodiments, the additional therapeutic agent is DS8201 or Trastuzumab-Z-1.

[0312] In another aspect, the present disclosure provides a pharmaceutical combination comprising:

[0313] Component a: any of the above-mentioned antibody-drug conjugates, any of the above-mentioned ADC compositions, or any of the above-mentioned drug-linkers, and one or more pharmaceutical excipients; and

[0314] Component b: one or more other therapeutic agents, and one or more pharmaceutical excipients; preferably, the other therapeutic agent is an antibody-drug conjugate, such as an antibody-drug conjugate targeting Her2, such as any one of the antibody-drug conjugates, ADC compositions, or drug-linkers described herein other than component a, and another example is DS8201 or Trastuzumab-Z-1.

[0315] In some embodiments, the pharmaceutical combination is a kit.

[0316] In some embodiments, component a and component b are in separate containers.

[0317] In some embodiments, the dose of component a or component b is selected from the group consisting of 0.1-2000 mg, such as 0.1-1900 mg, 0.1-1800 mg, 0.1-1700 mg, 0.1-1600 mg, 0.1-1500 mg, 0.1-1400 mg, 0.1-1300 mg, 0.1-1200 mg, 0.1-1100 mg, 0.1-900 mg, 0.1-900 mg, 0.1-800 mg, 0.1-700 mg, 0.1-600 mg, 0.1-500 mg, 0.1-400 mg, 0.1-300 mg, 0.1-200 mg, 0.1-100 mg, 0.1-50 mg, 0.1-30 mg, 0.1-20 mg, 0.1-10 mg; and for example 0.1 mg, 1.0 mg, 2.0 mg, 3.0 mg, 4.0 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg.

[0318] In some embodiments, the antibody drug conjugate, drug-linker, ADC composition, drug combination, or pharmaceutical composition is formulated for local or systemic administration. Systemic administration includes enteral administration (i.e., absorption through the gastrointestinal tract) or parenteral administration. In the present context, "parenteral administration" means administration by any route other than through the gastrointestinal tract, such as by intravenous injection or infusion. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, systemic administration is by intravenous injection.

[0319] Applications

[0320] The antibody drug conjugate, drug-linker, ADC composition, drug combination, or pharmaceutical composition described herein can be used for the treatment of a variety of diseases or disorders, such as Her2 expressing (Her2 positive) cancer, including solid tumors or hematological malignancies, such as ovarian cancer, pancreatic cancer, nasopharyngeal cancer, head and neck cancer, urothelial cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, in particular lung adenocarcinoma), or lymphoma.

[0321] Accordingly, the present application provides use of the antibody drug conjugate, drug-linker, ADC composition, drug combination or pharmaceutical composition of any one of the preceding in the manufacture of a medicament for treating a Her2-expressing cancer.

[0322] Meanwhile, the present application provides the antibody drug conjugate, drug-linker, ADC composition, drug combination or pharmaceutical composition of any one of the preceding for use in treating a Her2-expressing cancer.

[0323] Meanwhile, the present application also provides a method of treating a Her2-expressing cancer, comprising the step of administering to a subject in need thereof an effective amount of the antibody drug conjugate, drug-linker, ADC composition, drug combination or pharmaceutical composition of any one of the preceding.

[0324] The antibody drug conjugate, drug-linker, ADC composition or pharmaceutical composition described herein can be used in combination with one or more other therapeutic agents for treating a variety of diseases or conditions, such as a Her2-expressing cancer, including solid tumors or hematological malignancies, such as ovarian cancer, pancreatic cancer, nasopharyngeal cancer, head and neck cancer, urothelial cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, in particular lung adenocarcinoma), or lymphoma.

[0325] In some embodiments, the "in combination with one or more other therapeutic agents" includes simultaneous (concurrent) or sequential administration or application of the pharmaceutical composition or drug combination of the preceding in any order. In some embodiments, the other therapeutic agent is an antibody drug conjugate.

[0326] Meanwhile, the present application also provides use of the antibody drug conjugate, drug-linker, ADC composition or pharmaceutical composition described herein in combination with one or more other therapeutic agents in the manufacture of a medicament for treating a variety of diseases or conditions, such as a Her2-expressing cancer, including solid tumors or hematological malignancies, such as ovarian cancer, pancreatic cancer, nasopharyngeal cancer, head and neck cancer, urothelial cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, in particular lung adenocarcinoma), or lymphoma.

[0327] Meanwhile, the present application also provides a method of treating a Her2-expressing cancer, comprising administering to a subject in need thereof:

[0328] a) an effective amount of the antibody drug conjugate, drug-linker, ADC composition, drug combination or pharmaceutical composition of any one of the preceding;

[0329] b) an effective amount of the other therapeutic agent.

[0330] In some embodiments, the other therapeutic agent is an antibody drug conjugate targeting Her2.

[0331] In some embodiments, the other therapeutic agent is an antibody drug conjugate described herein.

[0332] In some embodiments, the other therapeutic agent is, for example, DS8201 or Trastuzumab-Z-1.

[0333] In some embodiments, the present application also provides a method of treating a Her2 expressing cancer, comprising administering to a subject in need thereof:

[0334] a) an effective amount of an antibody drug conjugate in the present application;

[0335] b) an effective amount of an other therapeutic agent.

[0336] In some embodiments, the other therapeutic agent is an antibody drug conjugate targeting Her2.

[0337] In some embodiments, the other therapeutic agent is an anti-human HER2 antibody drug conjugate, the anti-human HER2 antibody is Trastuzumab.

[0338] In some embodiments, the other therapeutic agent is an antibody drug conjugate, the drug is a topoisomerase inhibitor.

[0339] In some embodiments, the topoisomerase inhibitor is a topoisomerase I inhibitor.

[0340] In some embodiments, the topoisomerase I inhibitor is a camptothecin compound, for example, Topotecan, Irinotecan, Belotecan, Dxd, SN38 or

[0341] In some embodiments, the antibody drug conjugate is selected from Trastuzumab-A-1, Trastuzumab-A-2, Trastuzumab-C-2, Trastuzumab-C-2, Trastuzumab-C-3, Trastuzumab-C-5, Trastuzumab-C-10, Trastuzumab-C-13, Trastuzumab-C-28; the other therapeutic agent is selected from DS8201 or Trastuzumab-Z-1.

[0342] In some embodiments, the antibody drug conjugate is selected from Trastuzumab-A-2, the other therapeutic agent is selected from DS8201.

[0343] In some embodiments, the antibody drug conjugate is selected from Trastuzumab-C-3 and the other therapeutic agent is selected from Trastuzumab-Z-1.

[0344] In some embodiments, the antibody drug conjugate is administered at a dose per administration selected from 0.1-100 mg / kg, preferably from 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg, based on the body weight of the subject to which the antibody drug conjugate is administered.

[0345] In some embodiments, the other therapeutic agent is administered at a dose per administration selected from 0.1-100 mg / kg, preferably from 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg, based on the body weight of the subject to which the other therapeutic agent is administered.

[0346] In some embodiments, the antibody drug conjugate, the drug-linker, the ADC composition, the drug combination, or the pharmaceutical composition is administered at a frequency of 1-20 times within a period of 1-4 weeks; for example, once a week, twice a week, three times a week, once every two weeks, three times every two weeks, once every three weeks, twice every three weeks, once every four weeks, or three times every four weeks.

[0347] In some embodiments, the other therapeutic agent is administered at a frequency of 1-20 times within a period of 1-4 weeks; for example, once a week, twice a week, three times a week, once every two weeks, three times every two weeks, once every three weeks, twice every three weeks, once every four weeks, or three times every four weeks.

[0348] In some embodiments, the antibody drug conjugate, the drug-linker, the ADC composition, or the pharmaceutical composition is sufficient (e.g., in a subject) to:

[0349] (1) inhibit proliferation of a cell (e.g., a tumor cell);

[0350] (2) inhibit tumor growth;

[0351] (3) inducing and / or increasing antibody-dependent cellular cytotoxicity activity;

[0352] (4) inhibiting Her2-mediated signal transduction;

[0353] (5) preventing and / or treating Her2-mediated diseases / disorders; or

[0354] (6) any combination of (1)-(5) above.

[0355] In some embodiments, the cancer is selected from a solid tumor or a hematological malignancy; for example from ovarian cancer, pancreatic cancer, nasopharyngeal cancer, head and neck cancer, urothelial cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, in particular lung adenocarcinoma) or lymphoma.

[0356] In some embodiments, the cancer is selected from a digestive tract tumor, for example gastric cancer or colorectal cancer.

[0357] Definitions

[0358] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. References herein to technical terms used herein are intended to refer to technical terms as commonly understood in the art, including variations or replacements of those technical terms that are obvious to one of ordinary skill in the art. Also, the laboratory operations steps of genomics, nucleic acid chemistry, molecular biology, etc. used herein are all conventional steps widely used in the corresponding fields. Although it is believed that the following terms are well understood by one of ordinary skill in the art, the following definitions are set forth to better explain the present application.

[0359] The term "antibody" refers to an immunoglobulin molecule that is generally comprised of two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a different class of antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antigen binding site. Assignment 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 variable region of the antibody has been cyclized to a pyroglutamate. Thus, in compositions comprising antibodies disclosed herein, the various antibodies therein 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 or an N-terminal amino acid cyclized to a pyroglutamate.

[0360] The term "complementarity determining region" or "CDR" refers to amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy and light chains, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, e.g., as defined by the Rabat numbering system (Rabat 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, one of skill in the art will readily be able to identify the CDRs defined by each numbering system. Moreover, 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).

[0361] In the present application, the CDRs contained by an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art, e.g., by the Rabat, Chothia, IMGT, or AbM numbering system. In certain embodiments, the CDRs contained by an antibody or antigen-binding fragment thereof are defined by the Chothia numbering system.

[0362] The following general rules (published at www.bioinf.org.uk: the research group of Professor Andrew C. R. Martin) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with the amino acids making up the epitope of the antigen to which the antibody binds. In rare cases, these general constant features do not appear; but Cys residues are the most conserved feature.

[0363] V H The entire amino acid sequence of V H The amino acid positions in V H The amino acid positions in V L The amino acid positions in V

[0364] The amino acid positions in the heavy chain constant region can be numbered sequentially starting from amino acid position 1 through the end of the sequence, or according to Eu. The amino acid sequence of an IgGl heavy chain constant region is 330 amino acids in length, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu starts at position 118 and ends at position 447. Unless otherwise specified, the amino acid positions of the heavy and light chains described herein are defined according to the sequential numbering.

[0365] The term "framework region" or "FR" residues refer to those amino acid residues in a variable region of an antibody that are not CDR residues as defined above.

[0366] The term "antibody" is not limited by the method in which the antibody is produced. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotype, for example, an IgG (e.g., IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibody.

[0367] The term "antigen binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, e.g., a polypeptide of a fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen binding fragments of an antibody 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 (sdAb, nanobodies), and polypeptides comprising at least a portion of an antibody that is sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0368] The term "Fd" means an antibody fragment consisting of a VH and CHI domain; the term "dAb fragment" means an antibody fragment that consists of a VH domain (Ward et al., Nature 341 :544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of a VL, VH, CL and CHI domain; the term "F(ab')2 fragment" means an antibody fragment that comprises two Fab fragments linked by disulfide bridges on the hinge region; the term "Fab' fragment" means the fragment obtained by reducing the disulfide bonds of a F(ab')2 fragment, consisting of an intact light chain and a Fd fragment of a heavy chain (consisting of a VH and CHI domain).

[0369] The term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is commonly considered the smallest fragment of an antibody that is capable of forming a complete antigen binding site. It is generally considered that the six CDRs confer antigen binding specificity to an antibody. However, even a single variable domain (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although it may do so at a lower affinity than the entire binding site.

[0370] The term "Fc" means an antibody fragment formed by disulfide bonds between the second, third constant regions of the first heavy chain and the second, third constant regions of the second heavy chain of an antibody. The Fc fragment of an antibody has various diverse functions, but is not involved in antigen binding.

[0371] The term "scFv" refers to a single polypeptide chain comprising a VL and a 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, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS (SEQ ID NO: 49) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4(SEQ ID NO: 50) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present application 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 can also be present between the VHand VLof the scFv. In certain embodiments, the VHand VLdomains can be positioned relative to each other in any suitable arrangement. For example, scFv comprising NH2-VH-VH-COOH, NH2-VH-VL-COOH, NH2-VL-VH-COOH, or NH2-VL-VL-COOH are contemplated. 2- VL-VL-COOH.

[0372] The term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment consisting of a single monomeric variable antibody domain, such as a single heavy chain variable region, that retains the ability to specifically bind the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0373] Each of the above-described antibody fragments is capable of specifically binding to the same antigen bound by the full-length antibody from which it is derived, and / or competes with the full-length antibody for specific binding to the antigen.

[0374] In the present context, the term "antibody" when used in reference to a term "antibody" includes not only intact antibodies, but also antigen binding fragments of antibodies, unless otherwise clearly indicated by context.

[0375] Antigen binding fragments of antibodies (e.g., the above-described antibody fragments) can be obtained using conventional techniques known to those of skill in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage) from a given antibody (e.g., an antibody provided herein) and screened for specificity in the same manner as is done for intact antibodies.

[0376] The term "murine antibody" refers to an antibody obtained by fusing B cells of an immunized mouse with myeloma cells, screening for murine hybridoma cells that both proliferate indefinitely and secrete antibody, followed by screening, antibody production, and antibody purification, or refers to an antibody secreted by a plasma cell that developed from a B cell that proliferated in response to an antigen invading a mouse.

[0377] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered to have an amino acid sequence that is modified to increase homology to the sequence of a human antibody. Typically, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from a human immunoglobulin (recipient antibody). A humanized antibody typically retains the desired properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance an immune response, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody that has the desired properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity, and / or ability to enhance an immune response).

[0378] The term "identity" is used in reference to the matching of sequences between two polypeptides or between two nucleic acids. When a position in each sequence that is being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules that is occupied by adenine, or a position in each of two polypeptides that is occupied by lysine), then the molecules are identical at that position. The "percentage of identity" between two sequences is the 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 of 10 positions in two sequences are matched then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Typically, the comparison is made over the full length of the two sequences. Such a comparison can be conveniently accomplished by use of the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, as implemented in the computer program Align (DNAstar, Inc.), or by use of the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as integrated 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 percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) as implemented in the GAP program, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6, as incorporated into the GCG software package (available at www.gcg.com).

[0379] The term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a 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 of an amino acid residue with an amino acid residue having a similar side chain, e.g., substitutions made between residues within the same family that are physicochemically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having 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, a preference is given to substituting the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative 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).

[0380] The nomenclature used herein for the twenty conventional amino acids follows the conventional usage. See, e.g., 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 application, the amino acids are generally represented by the one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0381] The term "linker" refers to a structural moiety that links a cytotoxic drug to an antibody or antigen binding fragment. For example, to the -M-L-E- structural moiety in the formula Ab-[M-L-E-] x .

[0382] The term "drug-linker" refers to the structure of the cytotoxic drug and linker of the present application before it is attached to the antibody or antigen-binding fragment thereof. For example, "drug-linker" refers to Q-L-E-D, where Q is the structure of M before it is covalently attached to the antibody or antigen-binding fragment thereof. Covalent attachment of the "drug-linker" to the antibody or antigen-binding fragment thereof results in the antibody drug conjugate of the present application.

[0383] The "drug-linker" also includes all pharmaceutically acceptable isotopically-labeled compounds thereof, which are identical to the "drug-linker" compounds of the present application, but for the fact 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 usually found in nature. Examples of isotopes suitable for inclusion in the present application include, but are not limited to, isotopes of hydrogen, such as 2 H, 3 H, deuterium D, tritium T); isotopes of carbon, such as 11 C, 13 C and 14 C); isotopes of chlorine, such as 37 Cl); isotopes of fluorine, such as 18 F); isotopes of iodine, such as 123 I and 125 I); isotopes of nitrogen, such as 13 N and 15 N); isotopes of oxygen, such as 15 O, 17 O and 18 O); and isotopes of sulfur, such as 35 S).

[0384] The terms "comprise", "comprising", "have", "having", "include", "including", "contain", "containing", or "involve" and other variants thereof are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0385] The term "alkyl" denotes a straight or branched hydrocarbon group derived from a straight or branched chain alkane by removal of one hydrogen atom, for example "C 1-20 alkyl", "C 1-10 alkyl", "C 1-6 alkyl", "C 1-4 alkyl", "C 1-3"alkyl" and the like, specific examples of which include, but are not limited to: methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, i-hexyl, 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, and the like.

[0386] The term "haloalkyl" denotes a group in which one or more of the hydrogen atoms in the above-described alkyl groups are replaced with a halogen (e.g., F, Cl, Br, I), including mono-substituted, poly-substituted, or per-substituted forms, such as "haloC 1-6 "alkyl," "C 1-6 The term "haloalkyl" denotes a group in which one or more of the hydrogen atoms in the above-described alkyl groups are replaced with a halogen (e.g., F, Cl, Br, I), including mono-substituted, poly-substituted, or per-substituted forms, such as "haloC

[0387] The term "alkylsulfonyl" denotes a group in which the hydrogen atoms of a sulfonyl group are replaced with an alkyl group, of the formula "alkyl-S(O)2-", such as "C 1- 6alkylsulfonyl," "C 1-4 The term "alkylsulfonyl" denotes a group in which the hydrogen atoms of a sulfonyl group are replaced with an alkyl group, of the formula "alkyl-S(O)2-", such as "C

[0388] The term "haloalkylsulfonyl" denotes a group in which the hydrogen atoms of a sulfonyl group are replaced with a haloalkyl group, of the formula "haloalkyl-S(O)2-", such as "C 1-6 haloalkylsulfonyl," specific examples of which include, but are not limited to: trifluoromethylsulfonyl (CF3-S(O)2-), 2-chloroethylsulfonyl, 1,2-difluoropropylsulfonyl, perfluorobutylsulfonyl, and the like.

[0389] The term "halosulfonyl" denotes a group in which the hydrogen atoms of a sulfonyl group are replaced with a halogen atom (e.g., F, Cl, Br, I), of the formula "-SO2X", specific examples of which include, but are not limited to: sulfonyl chloride (-SO2Cl), sulfonyl fluoride (-SO2F), and the like.

[0390] The term "alkylsulfonate" denotes a group in which the hydrogen atoms of a sulfonate group are replaced with an alkyl group, of the formula "alkyl-S(O)2-O-", such as "C 1-6 alkylsulfonate," specific examples of which include, but are not limited to: methylsulfonate (CH3-S(O)2-), ethylsulfonate, isopropylsulfonate, t-butylsulfonate, and the like.

[0391] The term "haloalkylsulfonate" denotes a group in which the hydrogen atom of a sulfonate group is replaced by a haloalkyl group, of the formula "haloalkyl-S(O)2-O-", for example "trifluoromethylsulfonate (CF3-S(O)2-O-)", "2-chloroethylsulfonate", "1,1-difluoropropylsulfonate", and the like. 1-6 The term "alkylsulfonate" denotes a group in which the hydrogen atom of a sulfonate group is replaced by an alkyl group, of the formula "alkyl-S(O)2-O-", for example "methylsulfonate (CH3-S(O)2-O-)", "ethylsulfonate", "isopropylsulfonate", and the like.

[0392] The term "alkylsulfinyl" denotes a group in which the hydrogen atom of a sulfinyl group is replaced by an alkyl group, of the formula "alkyl-S(O)-O-", for example "C 1-6 The term "alkylsulfinyl" denotes a group in which the hydrogen atom of a sulfinyl group is replaced by an alkyl group, of the formula "alkyl-S(O)-O-", for example "C

[0393] The term "alkylsulfoxyl" denotes a group in which the hydrogen atom of a sulfoxyl group is replaced by an alkyl group, of the formula "alkyl-S(O)-", for example "C 1- The term "alkylsulfoxyl" denotes a group in which the hydrogen atom of a sulfoxyl group is replaced by an alkyl group, of the formula "alkyl-S(O)-", for example "C

[0394] The term "halophenoxy" denotes a group in which one or more hydrogens on the phenyl ring of a phenoxy group are replaced by halogen, for example in mono-, poly-, or perhalo form, specific examples including but not limited to "4-fluorophenoxy", "2,6-dichlorophenoxy", "3-bromo-5-iodophenoxy", "pentafluorophenoxy", and the like.

[0395] The term "alkenyl" denotes a straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond, for example "C 2-20 The term "alkenyl" denotes a straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond, for example "C 2-6 The term "alkenyl" denotes a straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond, for example "C

[0396] The term "alkynyl" denotes a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond, for example "C 2-20 The term "alkynyl" denotes a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond, for example "C 2-6 The term "alkynyl" denotes a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond, for example "C

[0397] The term "aryl" denotes a monocyclic or bicyclic aromatic hydrocarbon group, for example "6-10 membered aryl" and the like, specific examples including but not limited to "phenyl", "1-naphthyl", "2-naphthyl", "3-naphthyl", and the like.

[0398] The term "heteroaryl" denotes a monovalent aromatic radical of a 5-, 6-, or 7-membered ring, and includes fused ring systems of 5-20 atoms (where at least one is a heteroatom) containing from 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, e.g., "5-6 membered heteroaryl" and the like, specific examples include, but are not limited to: thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, thiadiazolyl, and the like.

[0399] The term "nitrogen-containing heterocyclyl" denotes a heterocyclyl group in which the heteroatom is nitrogen, e.g., a heterocyclyl group consisting of 2 to 12 carbon atoms and 1 to 4 nitrogen atoms, e.g., "9-12 membered nitrogen-containing heterocyclyl" and the like, specific examples include, but are not limited to: piperidinyl, pyrrolidinyl, azetidinyl, dihydropyrazolyl, azabicyclo[3.1.0]hexanyl, azaspiro[2.4]heptanyl, and the like.

[0400] The term "isotopically-labeled compound" denotes a compound of the present application which contains an atom that has the same atomic number but a different atomic mass or mass number than the atom that predominates in nature. Examples of isotopes suitable for inclusion in the compounds of the present application include, but are not limited to, isotopes of hydrogen, such as 2 H, 3 deuterium D, tritium T); isotopes of carbon, such as 11 C, 13 C and 14 C); isotopes of chlorine, such as 37 Cl); isotopes of fluorine, such as 18 F); isotopes of iodine, such as 123 I and 125 I); isotopes of nitrogen, such as 13 N and 15 N); isotopes of oxygen, such as 15 O, 17 O and 18 O); and isotopes of sulfur, such as 35 S).

[0401] A solid line (-), a solid wedge ( ), or a dashed wedge ( ) depict the chemical bonds of the compounds of the invention. The use of a solid line to depict a bond to an asymmetric carbon atom is intended to indicate that all possible stereoisomers (e.g., particular enantiomers, racemic mixtures, etc.) are included at that carbon atom. The use of a solid or dashed wedge to depict a bond to an asymmetric carbon atom is intended to indicate that the depicted stereoisomer is present. When present in a racemic mixture, the solid and dashed wedges are used to define the relative stereochemistry, not the absolute stereochemistry. Unless otherwise indicated, the compounds of the invention are intended to exist in the form of stereoisomers, which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the invention can exhibit more than one type of isomerism, and consist of mixtures (e.g., racemic mixtures and diastereomeric pairs) thereof.

[0402] The term "substituted" means that one or more (e.g., 1, 2, 3, 4, or 5) hydrogens on the designated compound or structural fragment are replaced with a substituent, provided that the designated atom's normal valence is not exceeded and the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds. By way of example, and without limitation, the substituents can each independently be one or more of the following: NR 8 R 9 , -O-, -S-, -NR'-, halogen, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -CN, =O, C1-C6(alkyl)ene, C1-C6halo(alkyl)ene, C1-C6alkoxy, C2-C6(alkyl)enylene, C2-C6(alkyn)enylene, C3-C8(cycloalkyl)ene, 3-10 membered (hetero)cycloalkyl)ene, C6-C 10 (aryl)ene, and 5-10 membered (hetero)aryl)ene, and the like, wherein R 8 , R 9 , and R' are as previously defined. For example, the substituents can be suitable substituents as described above.

[0403] If a functional group or structural fragment is described as "substituted or unsubstituted," that functional group or structural fragment can be (1) unsubstituted or (2) substituted.

[0404] As used herein, the term "suitable substituents" means modifications that one of skill in the art could make to a compound according to the needs of the compound's substituents. "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), C1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkyl-O-C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 3-6 cycloalkyl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl, C 6-10 aryl, benzyl, hydroxyl substituted benzyl, indolylmethyl 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 heterocyclyl, 5-10 membered heteroaryl, C 6-10 aryl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, halogen, hydroxyl, carboxyl and ester group (e.g., -C 1-6 alkyl-C(=O)-OC 1-6 alkyl).

[0405] Whether explicitly stated or not, the numerical values of the present application are modified by the term "about". The term "about" means within ±10% of the stated value, preferably within ±5%, more preferably within ±2%.

[0406] As used herein, the term "DAR" or "drug-to-antibody ratio" means: (a) the number of linkers / drug moieties attached to an antibody in a single antibody-drug conjugate molecule, which is an integer from 0 to 10, e.g., an integer from 1 to 10; or (b) the average number of linkers / drug moieties attached to an antibody in a composition comprising more than one antibody-drug conjugate molecule, which is an integer or decimal number from 0 to 10, e.g., an integer or decimal number from 1 to 10. BRIEF DESCRIPTION OF DRAWINGS

[0407] Figure 1 shows the results of the efficacy test of the anti-human Her2 antibody drug conjugate of the present application on a JIMT-1 cell subcutaneous tumor-bearing mouse model.

[0408] Figure 2 shows the changes in the body weight of mice in each treatment group in the human breast cancer cell JIMT-1 CDX model.

[0409] Figures 3 and 4 show the results of the efficacy test of the anti-human Her2 antibody drug conjugate of the present application on a JIMT-1 cell subcutaneous tumor-bearing mouse model.

[0410] Figure 5 shows the changes in the body weight of mice in each treatment group in the human breast cancer cell JIMT-1 CDX model. DETAILED DESCRIPTION

[0411] The present application is further described through the description of specific embodiments, but this is not a limitation of the present application. Those skilled in the art can make various modifications or improvements according to the teachings of the present application without departing from the basic idea and scope of the present application.

[0412] The information of the sequences involved in the present application is described in the following table:

[0413] The abbreviations used herein have the following meanings:

[0414] The structure of the compounds described in the following examples is determined by nuclear magnetic resonance (1H NMR) or mass spectrometry (MS). 1 H NMR) or mass spectrometry (MS).

[0415] The determination of nuclear magnetic resonance (1H NMR) uses a Bruker 400MHz nuclear magnetic resonance instrument; the deuterated reagent is hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard substance is tetramethylsilane (TMS).

[0416] The abbreviations in the nuclear magnetic resonance (NMR) spectrum used in the examples are shown below.

[0417] s: singlet; d: doublet; t: triplet; q: quartet; m: multiplet; br: broad; J: coupling constant; Hz: hertz; DMSO-d6: deuterated dimethyl sulfoxide. The δ value is expressed in ppm value.

[0418] The determination of mass spectrometry (MS) uses an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0419] Intermediate Preparation Example One: Preparation of 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosan-24-yne acid (INT-1)

[0420] Step One: Preparation of 1-amino-3,6,9,12,15-pentaoctadecan-18-oic acid (INT-1-2)

[0421] Dissolve 2,2-dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azatricosane-23-oic acid (0.50 g, 1.22 mmol) in dichloromethane (3 mL), add trifluoroacetic acid (3 mL), and stir the reaction at room temperature for 3 hours. After the reaction is complete, concentrate the reaction under reduced pressure to give the crude trifluoroacetate salt of the title compound (650 mg, 1.21 mmol).

[0422] The structural characterization data thereof are as follows:

[0423] MS m / z (ESI): 310.2 [M+H]+

[0424] Step two: Preparation of 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16- pentaaoxa-19-azapentacos-24-ynoic acid (INT-1)

[0425] Dissolve 1-amino-3,6,9,12,15-pentaoctadecan-18-oic acid trifluoroacetate salt (650 mg, 1.21 mmol) in DMF (5.0 mL), add DIPEA (781.61 mg, 6.05 mmol), then add 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (441.91 mg, 1.21 mmol) portionwise, and stir the reaction at room temperature for 1 hour. After the reaction is complete, purify the reaction directly by reverse-phase column chromatography (acetonitrile / 0.05% aqueous sodium bicarbonate = 0-60%) and then lyophilize to give the title compound (646 mg, 1.15 mmol).

[0426] The structural characterization data thereof are as follows:

[0427] MS m / z (ESI): 560.3 [M+H]+

[0428] Intermediate Preparation Example Two: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-alanyl-L-alanine (INT-2)

[0429] Step one: Preparation of L-alanyl-L-alanine (INT-2-1)

[0430] Dissolve (tert-butoxycarbonyl)-L-alanyl-L-alanine (100 mg, 384.19 μmol) in dichloromethane (2 mL), add trifluoroacetic acid (0.5 mL), and react at 20 °C for 1 hour. After the reaction is complete, concentrate the reaction under reduced pressure to remove the solvent to give the pale yellow title compound (160 mg, 370.93 μmol).

[0431] The structural characterization data thereof are as follows:

[0432] MS m / z (ESI): 161.1 [M+H] +

[0433] Step two: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-alanyl-L-alanine (INT-2)

[0434] L-alanyl-L-alanine (160 mg, 412.14 μmol) and 2,5-dioxopyrrolidin-1-yl 6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (150.58 mg, 412.14 μmol) were dissolved in N,N-dimethylacetamide (2 mL), N,N-diisopropylethylamine (266.33 mg, 2.06 mmol) was added, and the reaction was carried out at 20 °C for 1 hour. After the reaction was completed, the title compound (125 mg, 304.55 μmol) was obtained by freeze-drying after column chromatography (C18, water / acetonitrile = 2 / 1).

[0435] The structural characterization data thereof are as follows:

[0436] MS m / z (ESI): 428.1 [M+H2O] +

[0437] Intermediate Preparation Example Three: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)glycylglycine (INT-3)

[0438] Glycylglycine (1 g, 7.57 mmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (2.77 g, 7.57 mmol) were dissolved in dimethyl sulfoxide (10 mL), and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the title compound (2.52 g, 6.59 mmol) was obtained by freeze-drying after column chromatography (C18, water / acetonitrile = 2 / 1).

[0439] The structural characterization data thereof are as follows:

[0440] MS m / z (ESI): 383.2 [M+H] +

[0441] Intermediate Preparation Example Four: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)glycylglycyl-L-phenylalanine (INT-4)

[0442] Step one: Preparation of Glycylglycyl-L-phenylalanine (INT-4-1)

[0443] ((Benzyloxy)carbonyl)glycylglycyl-L-phenylalanine (200 mg, 483.77 μmol) was dissolved in methanol (8 mL), after nitrogen replacement, palladium on carbon (20 mg) was added. Hydrogen gas was replaced three times, stirred at room temperature for 3 hours. After the reaction was completed, the palladium on carbon was removed by filtration, and the filtrate was concentrated to remove the solvent to obtain the title compound (135 mg, 483.37 μmol).

[0444] The structural characterization data thereof are as follows:

[0445] MS m / z (ESI): 280.2 [M+H] +

[0446] Step two: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)glycylglycyl-L-phenylalanine (INT-4)

[0447] Glycylglycyl-L-phenylalanine (130 mg, 465.46 μmol) and 2,5-dioxopyrrolidin-1-yl 6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (170.06 mg, 465.46 μmol) were dissolved in N,N- dimethylformamide (8 mL), N,N-diisopropylethylamine (120.32 mg, 930.93 μmol) was added, and stirring was performed at room temperature for 1 hour. After the reaction was completed, the title compound (60 mg, 0.11 mmol) was obtained by freeze-drying after flash column chromatography (C18, water / acetonitrile = 2 / 1).

[0448] The structural characterization data thereof are as follows:

[0449] MS m / z (ESI): 530.3 [M+H] +

[0450] Intermediate Preparation Example Five: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)alaninate (INT-5)

[0451] Step one: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-2- (hydroxymethyl)benzyl)(methyl)alaninate (INT-5-2)

[0452] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)(methyl)carbamate (6.45 g, 8.25 mmol) was dissolved in DMF (60 mL), and pyridinium hydrofluoride (15.40 g, 155.39 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. After completion of the reaction, the reaction solution was added with water and extracted with ethyl acetate. The organic phase was washed with brine and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate = 10-100%) and concentrated again under reduced pressure to obtain the title compound (4.4 g, 8.09 mmol).

[0453] Its structural characterization data are as follows:

[0454] MS m / z(ESI):561.4[M+H2O] + ;526.4[M-H2O+H] +

[0455] Step 2: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5)

[0456] Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (1.0 g, 1.84 mmol) was dissolved in DMF (5 mL), DIPEA (475.49 mg, 3.68 mmol, 640.82 μL) was added, followed by bis(4-nitrophenyl) carbonate (671.54 mg, 2.21 mmol, FR), and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the reaction solution was added to water and extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product, which was then purified by silica gel column chromatography (petroleum ether / ethyl acetate = 0-80%) and concentrated under reduced pressure to obtain the title compound (1.3 g, 1.83 mmol).

[0457] Its structural characterization data are as follows:

[0458] MS m / z(ESI):726.1[M+H2O] + ;731.3[M+Na] +

[0459] Example 1: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoylamido)butanamido)propanamido)benzyl (l-((2R,4R,5R)-3,3-difluoro-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyrimidin-4-yl)carbamate (A-1)

[0460] Step 1: Preparation of (9H-fluoren-9-yl)methyl (S)-(l-((4- (hydroxymethyl)phenyl)amino)-l-oxopropan-2-yl)carbamate (A-1-2)

[0461] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine (5 g, 16.06 mmol), p-aminobenzyl alcohol (2.77 g, 22.49 mmol) and EEDQ (7.49 g, 30.29 mmol) were dissolved with dichloromethane (90 mL) and methanol (30 mL), and reacted at room temperature for 3 hours. After vacuum concentration, the title compound (6.69 g, 16.06 mmol) was obtained by filtering the solid after slurry with methyl tert-butyl ether.

[0462] The structural characterization data thereof are as follows:

[0463] MS m / z (ESI): 417.4 [M+H] +

[0464] Step 2: Preparation of (S)-(l-((4-(hydroxymethyl)phenyl)amino)-l-oxopropan-2- yl)carbamate allyl ester (A-1-3)

[0465] (9H-fluoren-9-yl)methyl (S)-(l-((4-(hydroxymethyl)phenyl)amino)-l-oxopropan-2- yl)carbamate (1 g, 2.40 mmol) was dissolved with DMF (9.77 mL), and diethylamine (1.77 g, 24.17 mmol, 2.50 mL) was added to react at room temperature for one hour. After the system was lowered to zero degree, water (976.82 μL), potassium carbonate (663.70 mg, 4.80 mmol) was added, and Alloc-Cl (578.84 mg, 4.80 mmol, 510.44 μL) was added to stir at room temperature for 2 hours. After filtration, the filtrate was collected, and vacuum concentration was performed to obtain the crude product. The crude product was purified by silica gel column chromatography (EA / PE = 0%~60%), and vacuum concentration was performed to obtain the title compound (246 mg, 883.93 μmol).

[0466] The structural characterization data thereof are as follows:

[0467] MS m / z (ESI): 279.1 [M+H] +

[0468] Step three: Preparation of allyl (S)-(1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1- oxopropan-2-yl)carbamate (A-1-4)

[0469] After (S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)carbamate (406 mg, 1.46 mmol) and bis(4-nitrophenyl) carbonate (887.59 mg, 2.92 mmol) were dissolved with DMF (7 mL), DIPEA (565.62 mg, 4.38 mmol) was added, and after nitrogen replacement, it was reacted at room temperature for 2 hours. The solvent was removed to obtain a crude product, which was purified by silica gel column chromatography (EA / PE = 0% ~ 60%) and concentrated under vacuum and reduced pressure to obtain the title compound (571 mg, 1.29 mmol).

[0470] The structural characterization data thereof are as follows:

[0471] MS m / z (ESI): 444.4 [M+H] +

[0472] Step four: Preparation of 4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one (A-1-6)

[0473] After gemcitabine hydrochloride (4 g, 13.35 mmol) and TBSCl (5.08 g, 61.69 mmol) and imidazole (3.64 g, 53.39 mmol) were dissolved with dry DMF (16.29 mL), nitrogen replacement was performed, triethylamine (2.70 g, 26.70 mmol, 3.71 mL) was added, and the reaction was performed at room temperature for 16 hours. Water and ethyl acetate were added for extraction, the organic phase was collected and dried, and then concentrated under vacuum and reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (EA / PE = 0% ~ 60%) and concentrated under vacuum and reduced pressure to obtain the title compound (5.00 g, 10.16 mmol).

[0474] The structural characterization data thereof are as follows:

[0475] MS m / z (ESI): 492.6 [M+H] +

[0476] Step five: Preparation of 4-((S)-2-(((allyloxy)carbonyl)amino)propanamido)benzyl (1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)carbamate (A-1-7)

[0477] After 4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)- one (187.97 mg, 382.27 μmol) was dissolved in dry THF (5.79 mL), the temperature was lowered to -78 °C, at this temperature, LiHMDS (1 M, 382.27 μL) was added, after the temperature was naturally raised to room temperature, the reaction was carried out for 30 min, (S)-(1-((4-((((4- nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)carbamate allyl ester dissolved in THF (5.79 mL) was added, and the reaction was carried out at room temperature for 18 h. The solvent was removed under vacuum and reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (EA / PE = 0% ~ 60%) and concentrated under vacuum and reduced pressure to obtain the title compound (85 mg, 106.78 μmol).

[0478] The structural characterization data thereof are as follows:

[0479] MS m / z (ESI): 752.7 [M-44] +

[0480] Step six: Preparation of 4-((S)-2-aminopropanamido)benzyl (1-((2R,4R,5R)-4- ((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3- difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (A-1-8)

[0481] Step 1: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 3-methylbutanamido)propanamido)benzyl (1-((2R,4R,5R)-4-(((tert-butyldimethylsilyl)oxy)- 5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)carbamate (A-1-9)

[0482] The structural characterization data thereof are as follows:

[0483] MS m / z (ESI): 710.2 [M-H] -

[0484] Step 1: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 3-methylbutanamido)propanamido)benzyl (1-((2R,4R,5R)-4-(((tert-butyldimethylsilyl)oxy)- 5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)carbamate (A-1-9)

[0485] Step 1: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 3-methylbutanamido)propanamido)benzyl (1-((2R,4R,5R)-4-(((tert-butyldimethylsilyl)oxy)- 5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)carbamate (A-1-9)

[0486] The structural characterization data thereof are as follows:

[0487] MS m / z (ESI): 1034.3 [M-44] +

[0488] Step Eight: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutylamino)propylamino)benzyl (1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (A-1-10)

[0489] To the reaction solution of the previous step, diethylamine (19.11 mg, 261.30 μmol) was added directly and reacted at room temperature for one hour. After removing the solvent by vacuum concentration, the crude product was purified by high performance preparative chromatography column and freeze-dried to obtain the title compound 8 (10 mg, 12.33 μmol).

[0490] The structural characterization data thereof are as follows:

[0491] MS m / z (ESI): 812.07 [M+H] +

[0492] The preparation method thereof is as follows:

[0493] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid aqueous solution)

[0494] Step Nine: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ylamino)butylamino)propylamino)benzyl (1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (A-1-11)

[0495] 4-((S)-2-((S)-2-amino-3-methylbutylamino)propylamino)benzyl (1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (8 mg, 9.86 μmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-ynanoic acid hexyl ester (5.41 mg, 14.80 μmol) were dissolved in DMF (1 mL) and DIPEA (3.82 mg, 29.59 μmol) was added, and the mixture was reacted at room temperature for one hour. Water and ethyl acetate were added for extraction, and the collected organic phases were dried and concentrated under reduced pressure to give a crude product of the title compound (10.47 mg, 9.86 μmol), which was used directly in the next step without purification.

[0496] Its structural characterization data are as follows:

[0497] MS m / z(ESI):1062.3[M+H] +

[0498] Step 10: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-amido)butyramido)propionamido)benzyl(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (A-1)

[0499] 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amino)butylamino)propylamino)benzyl(1-((2R,4R,(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2 The crude product of 4-dihydropyrimidin-4-yl)carbamate (10.47 mg, 9.86 μmol) was dissolved in DMF (1.04 mL) and pyridine hydrofluoride (46.69 mg, 471.10 μmol, 42.44 μL) was added. The mixture was allowed to react at room temperature for 16 hours. The solvent was removed to obtain the crude product, which was purified by high-performance preparative chromatography and freeze-dried to obtain the title compound (10 mg, 12.33 μmol).

[0500] Its structural characterization data are as follows:

[0501] MS m / z(ESI):833.8[M+H] +

[0502] The preparation method thereof is as follows:

[0503] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid aqueous solution)

[0504] Example 2: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoylamido)butyramido)propanamido)benzyl (l-((2R,4R,5R)-3,3-difluoro-4- hydroxy-5-((phosphoryloxy)methyl)tetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyrimidin-4- yl)carbamate (A-2)

[0505] Step one: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 3-methylbutyramido)propanamido)benzyl (l-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyrimidin-4-yl)carbamate (A-2- 1)

[0506] After 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido) propanamido)benzyl (l-((2R,4R,5R)-4-(((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-l,2- dihydropyrimidin-4-yl)carbamate (137 mg, 132.58 μmol) was dissolved in DMF (4 mL), HFPyr (656.99 mg, 6.63 mmol) was added and the reaction was allowed to proceed at room temperature for 16 hours. The solvent was removed under vacuum and reduced pressure to obtain a crude product, which was purified by high performance preparative chromatography column and freeze-dried to obtain the title compound (28 mg, 34.79 μmol).

[0507] The structural characterization data thereof are as follows:

[0508] MS m / z (ESI): 762.3 [M-42] +

[0509] The preparation method thereof is as follows:

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

[0511] Step two: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (1- ((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-((phosphonooxy)methyl)tetrahydrofuran-2- yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (A-2-2)

[0512] Phosphorous acid trimethyl ester (236.23 mg, 1.69 mmol, 197.35 μL) and 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2- oxo-1,2-dihydropyrimidin-4-yl)carbamate (20 mg, 24.85 μmol) were dissolved together at 0 °C, phosphorous oxychloride (36.07 mg, 235.25 μmol, 21.93 μL) was added at -10 °C and stirred for one hour, then at room temperature for one hour. The reaction was quenched with water, the crude was purified with high performance preparative chromatography column and lyophilized to give the title compound (8 mg, 9.04 μmol).

[0513] The structural characterization data thereof are as follows:

[0514] MS m / z (ESI): 883.5 [M-H] -

[0515] The preparation method thereof is as follows:

[0516] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid in water)

[0517] Step three: Preparation of 4-((S)-2-((S)-2-amino-3-methylbutylamino)propanamido)benzyl (1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-((phosphonooxy)methyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (A-2-3)

[0518] Step 1: Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 3-methylbutanamido)propanamido)benzyl (1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5- ((phosphonooxy)methyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (A-1)

[0519] The structural characterization data thereof are as follows:

[0520] MS m / z (ESI): 661.5 [M-H] -

[0521] Step 4: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoylamido)butynoylamido)propiolamido)benzyl (1-((2R,4R,5R)-3,3-difluoro- 4-hydroxy-5-((phosphonooxy)methyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)carbamate (A-2)

[0522] The crude 4-((S)-2-((S)-2-amino-3-methylbutylamino)propanamido)benzyl (1-((2R,4R,5R)- 3,3-difluoro-4-hydroxy-5-((phosphonooxy)methyl)tetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)carbamate (6.00 mg, 9.06 μmol) and 2,5-dioxopyrrolidin-1-yl 6-(2- (methylsulfonyl)pyrimidin-5-yl)-5-ynoate (4.96 mg, 13.58 μmol) were dissolved in DMF (1 mL), and stirred at room temperature for one hour. The solvent was removed under vacuum and reduced pressure to obtain a crude product, which was purified by high-performance preparative chromatography column and freeze-dried to obtain the title compound (8 mg, 9.04 μmol).

[0523] The structural characterization data thereof are as follows:

[0524] MS m / z (ESI): 911.5 [M-H] -

[0525] The preparation method thereof is as follows:

[0526] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid aqueous solution)

[0527] Example 3: Preparation of 2,2',2"-(10-(2-((2-((((1-((2R,4R,5R)-3,3-difluoro-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4- yl)aminoformyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamino)butyramido)propanamido)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-1)

[0528] Step 1: Preparation of allyl (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino) propanamido)-2-((((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (B-1-1)

[0529] After 4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one (A-1-6) (45.79 mg, 93.13 µmol) was dissolved in THF (2 mL), the temperature was reduced to -78 °C with dry ice and ethanol, LiHMDS (1 M, 93.13 µL) was added slowly, and after the temperature was allowed to rise to room temperature, it was reacted for another half hour. Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-2-((((4- nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5) (44 mg, 62.08 µmol) dissolved in THF (2 mL) was added and the reaction was continued at room temperature for 18 hours. The solvent was removed to obtain the crude product. The crude product was directly used in the next step without purification.

[0530] The structural characterization data thereof are as follows:

[0531] MS m / z (ESI): 1062.3 [M+H]+

[0532] Step two: Preparation of allyl (5-((S)-2-((((1-((2R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3- difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)oxy) methyl)benzyl)(methyl)carbamate (B-1-2)

[0533] Allyl (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)- 2-((((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (B-1-1) (65 mg, 61.24 μmol, FR) was dissolved in DMF (2 mL) and diethylamine (61.24 μmol, 1 mL) was added. It was stirred at room temperature for one hour. After that, the solvent was removed to give a crude product. The crude product was purified by high performance preparative chromatography column and freeze-dried to give the title compound 3 (30 mg, 35.75 μmol).

[0534] The structural characterization data thereof are as follows:

[0535] MS m / z (ESI): 812.07 [M+H] +

[0536] The preparation method thereof is as follows:

[0537] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid aqueous solution)

[0538] Step three: Preparation of allyl (5-((S)-2-((S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-((((1-((2R,4R,5R)- 4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3- difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)oxy) methyl)benzyl)(methyl)carbamate (B-1-3)

[0539] Allyl (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3- methylbutanamido)propanamido)-2-((((1-((2R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3- difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)oxy) methyl)benzyl (methyl)carbamate (B-1-3) (41 mg, 35.33 μmol) was dissolved in DMF (2 mL) and diethylamine (35.33 μmol, 1 mL) was added. The reaction was stirred at room temperature for one hour. The solvent was then removed to give a crude product. The crude product was purified by high performance preparative chromatography and lyophilized to give the title compound 5 (33 mg, 35.17 μmol).

[0540] Structural characterization data thereof are as follows:

[0541] MS m / z (ESI): 1161.4 [M+H] +

[0542] Step Four: Preparation of Allyl (5-((S)-2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)-2-((((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)oxy)methyl))(methyl)carbamate (B-1-4)

[0543] Allyl (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3- methylbutanamido)propanamido)-2-((((1-((2R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3- difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)oxy) methyl)benzyl (methyl)carbamate (B-1-3) (41 mg, 35.33 μmol) was dissolved in DMF (2 mL) and diethylamine (35.33 μmol, 1 mL) was added. The reaction was stirred at room temperature for one hour. The solvent was then removed to give a crude product. The crude product was purified by high performance preparative chromatography and lyophilized to give the title compound 5 (33 mg, 35.17 μmol).

[0544] Structural characterization data thereof are as follows:

[0545] MS m / z (ESI): 939.5 [M+H] +

[0546] It is prepared as follows:

[0547] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid in water)

[0548] Step five: Preparation of allyl (2-((((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamino)butyramido)propanamido)benzyl)(methyl)carbamate (B-1-5)

[0549] Benzyl allyl (5-((S)-2-((S)-2-amino-3-methylbutyramido)propanamido)-2-((((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)oxy)methyl))(methyl)carbamate (B-1-4) (33 mg, 35.17 μmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (23.37 mg, 63.95 μmol) were dissolved in DMF (2 mL) and DIPEA (16.53 mg, 127.90 μmol) was added. The mixture was stirred at room temperature for three hours. The solvent was then removed to give a crude product. The crude product was purified by reverse phase column (acetonitrile / 0.05% formic acid in water = 0% - 60%) and lyophilized to give the title compound (28 mg, 23.56 μmol).

[0550] The structural characterization data thereof are as follows:

[0551] MS m / z (ESI): 1189.5 [M+H] +

[0552] Step six: Preparation of allyl (2-((((l-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyrimidin-4-yl)carbamoyl)oxy) methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl amino)butyramido)propanamido)benzyl)(methyl)carbamate (B-l-6)

[0553] Allyl (2-((((l-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-l,2- dihydropyrimidin-4-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamino)butyramido)propanamido)benzyl)(methyl)carbamate (B-l-5) (28 mg, 23.56 μmol) was dissolved in DMF (2 mL) and pyridine hydrofluoride (118.14 mg, 1.19 mmol, 107.40 μL) was added. The reaction was stirred at room temperature overnight. The solvent was removed to give a crude product. The crude product was purified by reverse phase column (acetonitrile / 0.05% formic acid in water = 0% - 60%) and lyophilized to give the title compound 7 (17 mg, 17.71 μmol).

[0554] The structural characterization data thereof are as follows:

[0555] MS m / z (ESI): 960.4 [M+H] +

[0556] Step seven: Preparation of 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoylamino)butyramido)propanamido)-2-((methylamino)methyl)benzyl(l- ((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo- 1,2-dihydropyrimidin-4-yl)carbamate (B-l-7)

[0557] Allyl (2-((((1 -((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran- 2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl- 2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamino)butyramido)propanamido) benzyl)(methyl)carbamate (B-1-6) (17 mg, 17.71 μmol) and N-methylmorpholine (53.74 mg, 531.27 μmol, 58.41 μL), formic acid (30.97 mg, 672.94 μmol) were dissolved in DMF (2 mL) and then palladium tetraphenylphosphine (20.46 mg, 17.71 μmol) was added. The reaction was stirred under nitrogen at room temperature for 30 minutes. The solvent was then removed to give a crude product. The crude product was purified by high performance preparative chromatography and then lyophilized to give the title compound (5 mg, 5.71 μmol).

[0558] The structural characterization data thereof are as follows:

[0559] MS m / z (ESI): 876.4 [M+H] +

[0560] The preparation method thereof is as follows:

[0561] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid solution)

[0562] Step eight: preparation of 2,2',2"-(10-(2-((2-((((1 -((2R,4R,5R)-3,3-difluoro-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl) carbamoyl)oxy)methyl)-5-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoylamino)butyramido)propanamido)benzyl)(methyl)amino)-2-oxoethyl)- 1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (B-1 )

[0563] 4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamido)butanamido)propanamido)-2-((methylamino)methyl)benzyl (1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (B-1-7) (5 mg, 5.71 μmol) 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 (5.73 mg, 11.42 μmol) were dissolved in DMF and DIPEA (2.21 mg, 17.13 μmol) was added. The reaction was allowed to proceed at room temperature overnight. The solvent was then removed to give a crude product. The crude product was purified by high performance preparative chromatography and lyophilized to give the title compound (1.1 mg, 0.85 μmol).

[0564] The structural characterization data thereof are as follows:

[0565] MS m / z (ESI): 1263.2 [M+H] +

[0566] The preparation method thereof is as follows:

[0567] Mobile phase A: acetonitrile; mobile phase B: water (0.05% aqueous formic acid)

[0568] Example Four: Preparation of N-((S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-9-benzyl-5,8,11,14-tetraoxo-2-oxa-4,7,10,13-tetraazapentadec-15-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamine (C-1)

[0569] Step One: Preparation of (9H-fluoren-9-yl)methyl (2-(((((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-2-oxoethyl)carbamate (C-1-1)

[0570] Dissolve gemcitabine hydrochloride (1 g, 3.80 mmol) and methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (1.40 g, 3.80 mmol) in DMF (38 mL), then add HC1 / 1,4-dioxane (2 M, 3.80 mL) and react at room temperature for 8 hours. Remove the solvent under reduced pressure in vacuo to obtain a crude product, purify the crude product by reverse phase column chromatography (acetonitrile / 0.05% trifluoroacetic acid aqueous solution = 0~50%) and freeze-dry to obtain the title compound (873 mg, 1.53 mmol).

[0571] The structural characterization data thereof are as follows:

[0572] MS m / z (ESI): 572.3 [M+H] +

[0573] Step two: Preparation of 2-amino-N-((((2R,3R,5R)-5-(4-amino-2-oxo- pyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)acetamide (C-1-2)

[0574] Dissolve (9H-fluoren-9-yl)methyl (2-(((((2R,3R,5R)-5-(4-amino-2-oxo- pyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-2- oxoethyl)carbamate (162 mg, 283.45 μmol) in DMF (3 mL), then add diethylamine (283.45 μmol, 1 mL) and react at room temperature for one hour. Remove the solvent under reduced pressure in vacuo to obtain a crude product, purify the crude product by reverse phase column chromatography (acetonitrile / 0.05% trifluoroacetic acid aqueous solution = 0~70%) and freeze-dry to obtain the title compound (99 mg, 283.43 μmol).

[0575] The structural characterization data thereof are as follows:

[0576] MS m / z (ESI): 350.3 [M+H] +

[0577] Step three: Preparation of N-((S)-1-((2R,3R,5R)-5-(4-amino-2-oxo- pyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-9-benzyl-5,8,11,14- tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl- 5-ynoic amide (C-1)

[0578] (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)glycylglycyl-L-phenylalanine (180.11 mg, 340.12 μmol) were dissolved in DMF (3 mL), then HATU (129.32 mg, 340.12 μmol) and DIPEA (73.26 mg, 566.86 μmol) were added, and the reaction was allowed to proceed at room temperature for two hours. The solvent was removed under reduced pressure in vacuo to obtain a crude product, which was purified by high-performance preparative chromatography and freeze-dried to obtain the title compound (43.05 mg, 48.01 μmol).

[0579] The structural characterization data thereof are as follows:

[0580] MS m / z (ESI): 861.5 [M+H] +

[0581] The preparation method thereof is as follows:

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

[0583] Example Five: Preparation of N-((S)-1-((S)-1-(((((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide (C-5)

[0584] Step One: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-(((((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropan-2-yl)carbamate (C-5-1)

[0585] Dissolve gemcitabine hydrochloride (1 g, 3.80 mmol) and (S)-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanamido)methyl acetate (1.45 g, 3.80 mmol) in DMF (38 mL), then add HC1 / 1,4-dioxane (2 M, 3.80 mL) and react at room temperature overnight. Concentrate under reduced pressure in vacuo to remove the solvent to obtain a crude product, which is purified by a reverse-phase chromatographic column (acetonitrile / 0.05% trifluoroacetic acid aqueous solution = 0-70%) and then freeze-dried to obtain the title compound (1.34 g, 2.29 mmol).

[0586] The structural characterization data thereof are as follows:

[0587] MS m / z (ESI): 586.3 [M+H] +

[0588] Step two: Preparation of (S)-2-amino-N-((((2R,3R,5R)-5-(4-amino-2-oxo- pyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)propanamide (C-5-2)

[0589] Dissolve (9H-fluoren-9-yl)methyl ((S)-1-(((((2R,3R,5R)-5-(4-amino-2-oxo- pyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)- 1-oxopropan-2-yl)carbamate (289 mg, 493.55 μmol) in DMF (5 mL), then add diethylamine (493.55 μmol, 2 mL) and react at room temperature for 2 hours. Concentrate under reduced pressure in vacuo to remove the solvent to obtain a crude product, which is not purified but directly subjected to the next step.

[0590] The structural characterization data thereof are as follows:

[0591] MS m / z (ESI): 364.3 [M+H] +

[0592] Step three: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-((S)-1-(((((2R,3R,5R)-5-(4- amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (C-5-3)

[0593] To a solution of (S)-2-amino-N-((((2R,3R,5R)-5-(4-amino-2-oxo- pyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl) propanamide (crude) and ((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (167.21 mg, 492.68 μmol) in DMF (5 mL) was added DIPEA (191.02 mg, 1.48 mmol) and the reaction mixture was stirred at room temperature for 4 hours. The solvent was removed to give a crude product, which was purified by reverse phase column (acetonitrile / 0.05% trifluoroacetic acid in water = 0~70%) and lyophilized to give the title compound (194 mg, 283.34 μmol).

[0594] The structural characterization data thereof are as follows:

[0595] MS m / z (ESI): 685.3 [M+H] +

[0596] Step four: Preparation of (S)-2-amino-N-((S)-1-(((((2R,3R,5R)-5-(4-amino-2-oxo- pyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)- 1-oxopropan-2-yl)-3-methylbutanamide (C-5-4)

[0597] To a solution of (9H-fluoren-9-yl)methyl ((S)-1-((S)-1-(((((2R,3R,5R)-5-(4-amino-2- oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)- 1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (194 mg, 283.34 μmol) in DMF (3 mL) was added diethylamine (207.23 mg, 2.83 mmol, 293.11 μL) and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed under vacuum to give a crude product, which was used directly in the next step without purification.

[0598] The structural characterization data thereof are as follows:

[0599] MS m / z (ESI): 463.2 [M+H] +

[0600] Step five: Preparation of N-((S)-1-((S)-1-(((((2R,3R,5R)-5-(4-amino-2-oxo- pyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)- 1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoic amide (C-5)

[0601] The crude (S)-2-amino-N-((S)-1-(((((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)- 4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropan-2-yl)- 3-methylbutanamide (65.5 mg, 141.64 μmol) and 2,5-dioxopyrrolidin-1-yl 6-(2- (methylsulfonyl)pyrimidin-5-yl)-5-ynehexanoate (65.5 mg, 141.64 μmol) were dissolved in DMF (1 mL), followed by the addition of DIPEA (27.46 mg, 212.46 μmol) and reaction at room temperature for 2 hours. The solvent was removed under vacuum and reduced pressure to obtain a crude product, which was purified by high performance preparative chromatography column and freeze-dried to obtain the title compound (25 mg, 34.38 μmol).

[0602] The structural characterization data thereof are as follows:

[0603] MS m / z (ESI): 713.3 [M+H] +

[0604] The preparation method thereof is as follows:

[0605] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid aqueous solution)

[0606] Example six: Preparation of N-((6S,9S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)- yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-9-isopropyl-6-methyl-5,8,11,14- tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)-6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoic amide (C-2)

[0607] (S)-2-amino-N-((S)-1-(((((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)- 4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropan- 2-yl)-3-methylbutanamide (50.00 mg, 108.12 μmol) and (6-(2-(methylsulfonyl) pyrimidin-5-yl)hex-5-ynoyl)glycylglycine (62.02 mg, 162.18 μmol) were dissolved with DMF (2 mL), then DIPEA (41.92 mg, 324.36 μmol) and HATU (61.68 mg, 162.18 μmol) were added, and the reaction was allowed to proceed at room temperature for 1 hour. The solvent was removed under reduced pressure in vacuo to obtain a crude product, which was purified by high-performance preparative chromatography and freeze-dried to obtain the title compound (20 mg, 22.98 μmol).

[0608] The structural characterization data thereof are as follows:

[0609] MS m / z (ESI): 827.4 [M+H] +

[0610] The preparation method thereof is as follows:

[0611] Mobile phase A: acetonitrile; mobile phase B: water (0.05% aqueous formic acid)

[0612] Example Seven: Preparation of N-((6S,9S,12S)-1-((2R,3R,5R)-5-(4-amino-2-oxopyrimidin- 1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2- oxo-4,7,10-triazatridecan-12-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)amide (C-3)

[0613] (S)-2-amino-N-((((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4- difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)propanamide (119 mg, 327.54 pmol) and (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-alanyl-L-alanine (201.65 mg, 491.31 pmol) were dissolved in DMF (3 mL) and HATU (186.81 mg, 491.31 pmol) and DIPEA (126.99 mg, 982.61 pmol) were added. The reaction was allowed to proceed at room temperature for one hour. The solvent was removed under reduced pressure in vacuo to give a crude product, which was purified by high performance preparative chromatography and lyophilized to give the title compound (86.11 mg, 111.66 pmol).

[0614] The structural characterization data thereof are as follows:

[0615] MS m / z (ESI): 756.3 [M+H] +

[0616] The preparation method thereof is as follows:

[0617] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid in water)

[0618] Example Eight: Preparation of N-((6S,9S,12S)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11- trioxo-2-oxa-4,7,10-triazatridecan-12-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5- ynamide (C-10)

[0619] Step One: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-(((((2R,3S,5R)-5-(5-fluoro-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)- 1-oxopropan-2-yl)carbamate (C-10-2)

[0620] (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl 5- fluoro-l-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine- 2,4(lH,3H)-dione (C-10-1) (500.00 mg, 2.03 mmol) and (S)-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanamido)methyl 5-fluoro-l-((2R,4S,5R)-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (776.65 mg, 2.03 mmol) were dissolved in DMF (20 mL) and HCl / 1,4-dioxane (2M, 2.03 mL) was added. The reaction was stirred at room temperature overnight. After the reaction was completed, the system was added with saturated aqueous sodium chloride solution and extracted with ethyl acetate. The organic phase was collected and dried and concentrated to obtain the crude product. After the reaction was completed, the title compound (140 mg, 246.24 μmol) was obtained by purification on a silica gel column (methanol / dichloromethane = 0% to 10%, 40 min).

[0621] Step two: Preparation of (S)-2-amino-N-((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)propanamide (C-10-3)

[0622] (9H-fluoren-9-yl)methyl [(S)-l-(((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)- 1-oxopropan-2-yl]carbamate (120 mg, 211.06 μmol) was dissolved in DMF (2 mL) and diethylamine (1 mL) was added. The reaction was stirred at room temperature for two hours. After the reaction was completed, the solvent was removed to obtain the crude product, which was quickly used in the next step without purification.

[0623] Step three: Preparation of N-((6S,9S,12S)-l-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,l l- trioxo-2-oxa-4,7,10-triazatridecan-l2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5- yneyl)amide (C-10)

[0624] (S)-2-amino-N-((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)- 3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)propanamide (100.74 mg, 245.44 μmol) and (5-(2-(methylsulfonyl)pyrimidin-5-yl)pent-4-ynoyl)-L-alanyl-L-alanine (85 mg, 245.44 μmol) were dissolved in DMF (2 mL), followed by the addition of HATU (111.99 mg, 294.53 μmol), and finally DIPEA (95.16 mg, 736.33 μmol), and stirred at room temperature for two hours. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (108.21 mg, 142.08 μmol).

[0625] The structural characterization data thereof are as follows:

[0626] MS m / z (ESI): 761.3 [M+Na] +

[0627] The preparation method thereof is as follows:

[0628] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0629] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0630] Example Nine: N-((6S,9S,12S)-1-((2R,3S,5R)-5-(2,4-dioxo-5-fluoro-3,4-dihydropyrimidin-1(2H)-yl)- 3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecan-12-yl)-6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)amide (C-10')

[0631] Step One: Preparation of (9H-fluoren-9-yl)methyl [(S)-1-(((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo- 3,4-dihydropyrimidin-1(2H)-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methyl)amino)-1- oxopropan-2-yl]carbamate (C-10'-2)

[0632] (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl 5- fluoro-l-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine- 2,4(lH,3H)-dione (C-10-1) (500.00 mg, 2.03 mmol) and HC1 / 1,4-dioxane (2M, 2.03 mL) were added after dissolving in DMF (20 mL) and reacted at room temperature overnight. After the reaction was completed, the system was added with saturated aqueous sodium chloride solution and extracted with ethyl acetate. The organic phase was collected and dried and concentrated to obtain a crude product. After the reaction was completed, the title compound (100 mg, 175.89 μmol) was obtained by purification through a silica gel chromatographic column (methanol / methylene chloride = 0% to 10%, 40 min).

[0633] Step two: Preparation of (S)-2-amino-N-((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methyl)propanamide (C-10'-3)

[0634] (9H-fluoren-9-yl)methyl [(S)-l-(((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methyl)amino)- 1-oxopropan-2-yl]carbamate (100.00 mg, 175.89 μmol) was dissolved in DMF (2 mL) and diethylamine (1 mL) was added and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed to obtain a crude product which was quickly subjected to the next step without purification.

[0635] Step three: Preparation of N-((6S,9S,12S)-l-((2R,3S,5R)-5-(2,4-dioxo-5-fluoro-3,4- dihydropyrimidin-l(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,l l- trioxo-2-oxa-4,7,10-triazatridecan-l2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5- ynamide (C-10')

[0636] (S)-2-amino-N-((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin- 1(2H)-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methyl)propanamide (60.90 mg, 175.85 μmol) and (5-(2-(methylsulfonyl)pyrimidin-5-yl)pent-4-ynoyl)-L- alaninyl-L-alanine (86.61 mg, 211.02 μmol) were dissolved in DMF (2 mL), followed by the addition of HATU (80.24 mg, 211.02 μmol), and finally DIPEA (68.18 mg, 527.56 μmol), and stirred at room temperature for two hours. After the reaction was completed, the reaction solution was directly purified by preparative high-performance liquid chromatography and freeze-dried to obtain the title compound (55.65 mg, 85.35 μmol).

[0637] The structural characterization data thereof are as follows:

[0638] MS m / z (ESI): 761.3 [M+Na] +

[0639] The preparation method thereof is as follows:

[0640] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0641] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0642] Example Ten: Preparation of N-((6S,9S,12S)-1-((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)- 4-hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10- triazatridecan-12-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamine (C-13)

[0643] Step One: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-(((((2S,4R,5R)-5-(6-amino- 9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1- oxopropan-2-yl)carbamate (C-13-2)

[0644] Cordycepin (500 mg, 1.31 mmol) and (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl acetate (328.50 mg, 1.31 mmol) were dissolved in DMF (13 mL), then HCl / 1,4-dioxane (2M, 653.75 μL) was added and the reaction was allowed to proceed at room temperature overnight. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (195 mg, 339.96 μmol).

[0645] The structural characterization data thereof are as follows:

[0646] MS m / z (ESI): 574.2 [M+H] +

[0647] The preparation method thereof is as follows:

[0648] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

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

[0650] Step two: preparation of (S)-2-amino-N-((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)methyl)propanamide (C-13-3)

[0651] ((S)-1-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropan-2-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (25 mg, 43.58 μmol) was dissolved in DMF (1 mL), then diethylamine (0.5 mL) was added and the reaction was allowed to proceed at room temperature for two hours. After the reaction was completed, the solvent was removed to obtain a crude product, which was quickly subjected to the next step without purification.

[0652] Step three: preparation of N-((6S,9S,12S)-1-((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecan-12-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide (C-13)

[0653] (S)-2-amino-N-((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)methyl)propanamide (15 mg, 42.69 μmol) and (5-(2-(methylsulfonyl)pyrimidin-5-yl)pent-4-ynoyl)-L-alanyl-L-alanine (21.03 mg, 51.23 μmol) were dissolved in DMF (2 mL), followed by the addition of DIPEA (16.55 mg, 128.07 μmol) and stirring for ten minutes, followed by the addition of HATU (19.48 mg, 51.23 μmol) and continued reaction at room temperature for one hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (6.54 mg, 8.62 μmol).

[0654] The structural characterization data thereof are as follows:

[0655] MS m / z (ESI): 744.2 [M+H] +

[0656] The preparation method thereof is as follows:

[0657] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0658] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

[0659] Example Eleven: Preparation of N-((6S,9S,12S)-1-((2R,3S,5R)-5-(2,4-dioxo-5- (trifluoromethyl)-3,4-dihydropyrimidin-1 (2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9- dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecan-12-yl)-6-(2-(methylsulfonyl)pyrimidin- 5-yl)hex-5-ynoic amide (C-28)

[0660] Step One: Preparation of (9H-fluoren-9-yl)methyl [(S)-1-(((((2R,3S,5R)-5-(2,4-dioxo-5- (trifluoromethyl)-3,4-dihydropyrimidin-1 (2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropan-2-yl]carbamate (C-28-1)

[0661] Trifluorothymidine (500 mg, 1.31 mmol) and (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl fluoride (387.28 mg, 1.31 mmol) were dissolved in DMF (6.5 mL) and HCl / 1,4-dioxane (2M, 326.88 μL) was added and reacted at room temperature overnight. After the reaction was completed, the system was added with saturated aqueous sodium chloride solution and extracted with ethyl acetate. The organic phase was collected and dried and concentrated to obtain the crude product. After the reaction was completed, the title compound (161 mg, 260.28 μmol) was obtained by purification through a silica gel chromatographic column (methanol / dichloromethane = 0% - 10%, 40 min).

[0662] Step two: Preparation of (S)-2-amino-N-((((2R,3S,5R)-5-(2,4-dioxo-5- (trifluoromethyl)-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)propanamide (C-28-2)

[0663] (9H-fluoren-9-yl)methyl [(S)-1-(((((2R,3S,5R)-5-(2,4-dioxo-5-(trifluoromethyl)- 3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)- 1-oxopropan-2-yl]carbamate (161 mg, 260.28 μmol) was dissolved in DMF (2 mL) and diethylamine (1 mL) was added and reacted at room temperature for two hours. After the reaction was completed, the solvent was removed to obtain the crude product which was quickly subjected to the next step without purification.

[0664] Step three: Preparation of N-((6S,9S,12S)-1-((2R,3S,5R)-5-(2,4-dioxo-5- (trifluoromethyl)-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-6,9- dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecan-12-yl)-6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoic amide (C-28)

[0665] (S)-2-amino-N-((((2R,3S,5R)-5-(2,4-dioxo-5-(trifluoromethyl)-3,4- dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl) propanamide (103 mg, 259.89 μmol) and (5-(2-(methylsulfonyl)pyrimidin-5-yl)pent-4- ynyl)-L-alanyl-L-alanine (106.67 mg, 259.89 μmol) were dissolved in DMF (2 mL), followed by the addition of DIPEA (100.77 mg, 779.68 μmol) and stirred for ten minutes, then HATU (98.82 mg, 259.89 μmol) was added and the reaction was continued at room temperature for one hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (57.37 mg, 73.79 μmol).

[0666] The structural characterization data thereof are as follows:

[0667] MS m / z (ESI): 789.8 [M+H] +

[0668] The preparation method thereof is as follows:

[0669] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

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

[0671] Example Twelve: Preparation of N-((S)-9-benzyl-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo- 3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-5,8,11,14- tetraoxo-2-oxa-4,7,10,13-tetraazapentadecan-15-yl)-6-(2-(methylsulfonyl)pyrimidin- 5-yl)hex-5-ynoic amide (C-8)

[0672] Step One: Preparation of (9H-fluoren-9-yl)methyl (2-(((((2R,3S,5R)-5-(5-fluoro-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl) amino)-2-oxoethyl)carbamate (C-8-2)

[0673] (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methyl acetic acid ester (120.29 mg, 488.62 μmol) was dissolved in tetrahydrofuran (2 mL), and trifluoroacetic acid (185.71 mg, 1.63 mmol, 124.72 μL) was added. The reaction was allowed to proceed at room temperature for four hours. The reaction was concentrated directly to give a crude product, which was purified by silica gel column chromatography (methanol / dichloromethane = 0% to 10%) and concentrated under vacuum to give the title compound (105 mg, 189.35 μmol).

[0674] The structure was characterized as follows:

[0675] ESI-MS (m / z): 572.3 [M + H2O] + .

[0676] Step two: Preparation of 2-amino-N-((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-1 (2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)acetamide (C-8-3)

[0677] (9H-fluoren-9-yl)methyl (2-(((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-1 (2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-2- oxoethyl)carbamate (C-8-2) (110.53 mg, 189.35 μmol) was dissolved in DMF (2 mL), and diethylamine (0.5 mL) was added. The reaction was allowed to proceed at room temperature for two hours. The reaction was concentrated directly to give a crude product, which was used in the next step without purification.

[0678] Step three: Preparation of N-((S)-9-benzyl-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-1 (2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-5,8,11,14-tetraoxo-2-oxa-4,7,10,13- tetraazapentadecan-15-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (C-8)

[0679] To a solution of 2-amino-N-((((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methoxy)methyl)acetamide crude and (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)glycylglycyl-L- phenylalanine (98.81 mg, 186.59 μmol) in DMF (2 mL) was added HATU (70.95 mg, 186.59 μmol) followed by DIPEA (72.34 mg, 559.76 μmol) and stirred at room temperature for 2 h. The reaction mixture was purified by preparative HPLC and lyophilized to give the title compound (54.22 mg, 61.04 μmol).

[0680] The structural characterization data thereof are as follows:

[0681] MS m / z (ESI): 842.3 [M-H] +

[0682] The preparation method thereof is as follows:

[0683] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0684] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0685] Example Thirteen: Preparation of N-((6S,9S,12S)-1-((2R,3S,4R,5R)-5-(4-amino-2- oxopyrimidin-1(2H)-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)-6,9- dimethyl-5,8,11-trioxo-2-oxa-4,7,10-triazatridecan-12-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide (C-58)

[0686] Step One: Preparation of (9H-fluoren-9-yl)methyl [(S)-1-(((((2R,3S,4R,5R)-5-(4- amino-2-oxopyrimidin-1(2H)-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-1-oxopropan-2-yl]carbamate (C-58-2)

[0687] Dissolve 4-amino-l-((2R,3R,4S,5R)-4-ethynyl-3,4-dihydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(lH)-one (50 mg, 187.10 μmol) and (S)-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl benzoate (78.70 mg, 205.81 μmol) in DMSO (2 mL), then add HC1 / 1,4-dioxane (4 M, 93.55 μL), and react at room temperature for four hours. Concentrate the reaction mixture directly to obtain a crude product, which is purified by a reverse-phase chromatographic column (acetonitrile / 0.05% formic acid aqueous solution = 0% to 50%) and lyophilized to obtain the title compound (23 mg, 39.01 μmol).

[0688] The structure thereof is characterized as follows:

[0689] ESI-MS (m / z): 590.3 [M-H]+.

[0690] Step two: Preparation of (S)-2-amino-N-((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-l(2H)-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)propanamide (C-58-3)

[0691] Dissolve (9H-fluoren-9-yl)methyl [(S)-l-(((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-l(2H)-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)amino)-l-oxopropan-2-yl]carbamate (C-58-2) (23 mg, 39.01 μmol) in DMF (2 mL), then add diethylamine (14.46 mg, 195.05 μmol), and react at room temperature for two hours. Concentrate the reaction mixture directly to obtain a crude product, which is used directly in the next step without purification.

[0692] Step three: Preparation of N-((6S,9S,12S)-l-((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-l(2H)-yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)-6,9-dimethyl-5,8,l l-trioxo-2-oxa-4,7,10-triazatridecan-l2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide (C-58)

[0693] To a solution of (S)-2-amino-N-((((2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)- yl)-3-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl)propanamide crude and (5-(2-(methylsulfonyl)pyrimidin-5-yl)pent-4-ynoyl)-L-alanyl-L-alanine (18.77 mg, 45.73 μmol) in DMF (2 mL) was added HATU (17.39 mg, 45.73 μmol) followed by DIPEA (9.85 mg, 76.22 μmol) and stirred at room temperature for 2 h. After completion of the reaction, the reaction mixture was purified by preparative high performance liquid chromatography followed by lyophilization to afford the title compound (7.0 mg, 8.75 μmol).

[0694] The structural characterization data thereof are as follows:

[0695] MS m / z (ESI): 760.3 [M+Na] +

[0696] The preparation method thereof is as follows:

[0697] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0698] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0699] Example Fourteen: Preparation of N-((S)-1-(((S)-1-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1- oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin- 5-yl)hex-5-ynoic amide (B-13)

[0700] Step One: Preparation of tert-butyl ((S)-1-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5- (((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin- 4-yl)amino)-1-oxopropan-2-yl)carbamate (B-13-2)

[0701] To tert-butyl ((S)-1-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)amino)-1-oxopropan-2-yl)carbamate (210 mg, 0.32 mmol) was added dichloromethane (5 mL), then trifluoroacetic acid (1 mL), stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to remove the solvent, and the crude product was purified by reverse phase column chromatography (acetonitrile-0.05% aqueous formic acid solution = 0-90%) and freeze-dried to obtain the title compound (45 mg, 0.08 mmol).

[0702] The structural characterization data thereof are as follows:

[0703] MS m / z (ESI): 562.2 [M+H] +

[0704] Step two: preparation of (S)-2-amino-N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)propanamide (B-13-3):

[0705] To tert-butyl ((S)-1-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)amino)-1-oxopropan-2-yl)carbamate (210 mg, 0.32 mmol) was added dichloromethane (5 mL), then trifluoroacetic acid (1 mL), stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to remove the solvent, and the crude product was purified by reverse phase column chromatography (acetonitrile-0.05% aqueous formic acid solution = 0-90%) and freeze-dried to obtain the title compound (45 mg, 0.08 mmol).

[0706] The structural characterization data thereof are as follows:

[0707] MS m / z (ESI): 562.2 [M+H] +

[0708] Step three: Preparation of N-((S)-1-(((S)-1-(((S)-1-((1-((2R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (B-13-4):

[0709] (S)-2-amino-N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2- dihydropyrimidin-4-yl)propanamide (45 mg, 0.08 mmol) and (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-alanyl-L-alanine (36.1 mg, 0.09 mmol) were dissolved in DMF (2 mL), DIPEA (31 mg, 0.24 mmol) was added dropwise, followed by the addition of HATU (45.60 mg, 0.12 mmol) in portions, and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was purified by reverse phase column chromatography (acetonitrile-0.05% aqueous formic acid = 0-90%) and freeze-dried to obtain the title compound (50 mg, 0.05 mmol).

[0710] The structural characterization data thereof are as follows:

[0711] MS m / z (ESI): 955.3 [M+H] +

[0712] Step four: Preparation of N-((S)-1-(((S)-1-(((S)-1-((1-((2R,4R,5R)-3,3-difluoro-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-1- oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (B-13):

[0713] N-((S)-1-(((S)-1-(((S)-1-((1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin- 4-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide (50 mg, 0.05 mmol) was dissolved in DMF (2 mL), hydrofluoric acid pyridine salt (0.5 mL) was added, stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (28.91 mg, 24.66 μmol, 98.5% purity).

[0714] The structural characterization data thereof are as follows:

[0715] MS m / z (ESI): 727.2 [M+H] +

[0716] The preparation method thereof is as follows:

[0717] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0718] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0719] Example Fifteen: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxa- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-1-yl)amino)- 7,10-dimethyl-1,6,9,12-tetraoxa-3-oxa-5,8,11-triazatetradecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin- 5-yl)hex-5-ynoic amide (Z-1)

[0720] Step One:

[0721] Compound Z-1-1 (657 mg, 1.22 mmol) and compound Z-1-2 (500 mg, 1.11 mmol) were dissolved in N,N-dimethylformamide (10 mL), followed by the addition of HATU (630.67 mg, 1.66 mmol) and N,N-diisopropyl ethylamine (428 mg, 3.32 mmol), and stirring at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain 700 mg of compound Z-1-3.

[0722] The preparation method is as follows:

[0723] Column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

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

[0725] Step two:

[0726] Compound Z-1-3 (500 mg, 0.513 mmol) was dissolved in N,N-dimethylformamide (2 mL), diethylamine (75.05 mg, 1.03 mmol) was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain 307 mg of compound Z-1-4.

[0727] The preparation method is as follows:

[0728] Column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

[0729] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0730] Step three:

[0731] Z-1-4 (170 mg, 0.226 mmol) and compound Z-1-5 (90.83 mg, 0.249 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N,N-diisopropyl ethylamine (29.21 mg, 0.226 mmol) was added. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain 50.56 mg of compound Z-1.

[0732] The structural characterization data are as follows:

[0733] MS m / z (ESI): 1002.4 [M+H] + .

[0734] The separation and purification method is as follows:

[0735] Column: Waters SunFire Prep C18 OBD (5 pm*19 mm*150 mm)

[0736] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0737] 1 H NMR (400 MHz, DMSO) δ 9.11 (s, 2H), 8.68 (t, J = 6.4 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.16 (s, 1H), 8.10 (d, J = 7.2 Hz, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.91 (d, J = 6.8 Hz, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.65-5.55 (m, 1H), 5.43 (s, 2H), 5.21 (s, 2H), 4.67-4.55 (m, 2H), 4.29-4.15 (m, 3H), 3.98 (s, 2H), 3.41 (s, 3H), 3.25-3.15 (m, 2H), 2.57-2.56 (m, 2H), 2.35-2.27 (m, 2H), 2.22-2.12 (m, 2H), 1.91-1.75 (m, 4H), 1.23-1.09 (m, 9H), 0.87 (t, J = 7.2 Hz, 3H).

[0738] Preparation of antibody drug conjugate

[0739] 1. Preparation of Trastuzumab-A-1

[0740] 2. 41 mL of Trastuzumab (14.7 mg / mL) was diluted with 102 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then the pH was adjusted to 7.60 with 1 M Na2HPO4solution, 10 mM TCEP (tris(2-carboxyethyl)phosphine, 113.7 μL, pH 7.60) solution was added and mixed, and it was placed at room temperature for 1.5 h. Then a drug-linker A-1 (271 μL, 10 mM, 13-fold equivalent of the antibody) solution dissolved in dimethyl sulfoxide was added and mixed, and it was placed at room temperature for 2 h. After completion, the buffer was replaced with a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva), to obtain the antibody drug conjugate (i.e. Trastuzumab-A-1). The DAR value was determined by mass spectrometry to be 7.46.

[0741] 2. Preparation of Trastuzumab-A-2

[0742] Take 1.422 mL trastuzumab (14.7 mg / mL), dilute with 71 μL 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust pH to 7.60 with 1 M Na2HP04solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 79.2 μL, pH 7.60) solution and mix, stand at room temperature for 1.5 h. Then add the drug-linker A-2 (163 μL, 10 mM, 11-fold equivalent of antibody) solution dissolved in dimethyl sulfoxide, mix, stand at room temperature for 2 h, and after completion, replace the buffer with 20 mM histidine buffer solution (pH 6.0) using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e. Trastuzumab-A-2). The DAR value is determined by mass spectrometry to be 7.56.

[0743] 3. Preparation of Trastuzumab-C-1

[0744] Take 0.34 mL trastuzumab (14.7 mg / mL), dilute with 17 μL 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust pH to 7.60 with 1 M Na2HP04solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 18.95 μL, pH 7.60) solution and mix, stand at room temperature for 1.5 h. Then add the drug-linker C-1 (35.88 μL, 10 mM, 10-fold equivalent of antibody) solution dissolved in dimethyl sulfoxide, mix, stand at room temperature for 2 h, and after completion, replace the buffer with 20 mM histidine buffer solution (pH 6.0) using a NAP gel column (Cytiva) to obtain the antibody drug conjugate (i.e. Trastuzumab-C-1). The DAR value is determined by mass spectrometry to be 7.24.

[0745] 4. Preparation of Trastuzumab-C-2

[0746] Take 0.204 mL trastuzumab (14.7 mg / mL), dilute with 10.2 μL 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust pH to 7.60 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 11.36 μL, pH 7.60) solution and mix, stand at room temperature for 1.5 h. Then add a drug-linker C-2 (21.76 μL, 10 mM, 10-fold equivalent of antibody) solution dissolved in dimethyl sulfoxide and mix, stand at room temperature for 2 h. After completion, replace the buffer with 20 mM histidine buffer solution (pH 6.0) using a NAP gel column (Cytiva) to obtain an antibody drug conjugate (i.e. Trastuzumab-C-2). Mass spectrometry determines the DAR value to be 7.9.

[0747] 5. Preparation of Trastuzumab-C-3

[0748] Take 2.027 mL trastuzumab (14.8 mg / mL), dilute with 101 μL 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust pH to 7.60 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 113.7 μL, pH 7.60) solution and mix, stand at room temperature for 1.5 h. Then add a drug-linker C-3 (253 μL, 10 mM, 12-fold equivalent of antibody) solution dissolved in dimethyl sulfoxide and mix, stand at room temperature for 2 h. After completion, replace the buffer with 20 mM histidine buffer solution (pH 6.0) using a NAP gel column (Cytiva) to obtain an antibody drug conjugate (i.e. Trastuzumab-C-3). Mass spectrometry determines the DAR value to be 7.8.

[0749] 6. Preparation of Trastuzumab-C-5

[0750] Take 2.041 mL trastuzumab (14.7 mg / mL), dilute with 102 μL 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust pH to 7.60 with 1 M Na2HPO4solution, mix with 10 mM TCEP (tris(2-carboxyethyl)phosphine, 113.7 μL, pH 7.60) solution, and let stand at room temperature for 1.5 h. Then mix with a drug-linker C-5 (253 μL, 10 mM, 12-fold equivalent of antibody) solution dissolved in dimethyl sulfoxide, let stand at room temperature for 2 h, and after completion, replace the buffer with 20 mM histidine buffer solution (pH 6.0) using a NAP gel column (Cytiva) to obtain an antibody drug conjugate (i.e., Trastuzumab-C-5). Mass spectrometry was used to determine the DAR value to be 7.99.

[0751] 7. Preparation of Trastuzumab-C-10

[0752] Take 1.024 mL trastuzumab (24.4 mg / mL), dilute with 51.2 μL 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust pH to 7.60 with 1 M Na2HPO4solution, mix with 10 mM TCEP (tris(2-carboxyethyl)phosphine, 94.7 μL, pH 7.60) solution, and let stand at room temperature for 1.5 h. Then mix with a drug-linker C-10 (177.6 μL, 10 mM, 10-fold equivalent of antibody) solution dissolved in dimethyl sulfoxide, let stand at room temperature for 2 h, and after completion, replace the buffer with 20 mM histidine buffer solution (pH 6.0) using a NAP gel column (Cytiva) to obtain an antibody drug conjugate (i.e., Trastuzumab-C-10). Mass spectrometry was used to determine the DAR value to be 7.83.

[0753] 8. Preparation of Trastuzumab-C-13

[0754] Take 1.024 mL trastuzumab (24.4 mg / mL), dilute with 51.2 uL 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust pH to 7.60 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 94.7 uL, pH 7.60) solution, mix well, and stand at room temperature for 1.5 h. Then add a drug-linker C-28 (176 uL, 10 mM, 10-fold equivalent of the antibody) solution dissolved in dimethyl sulfoxide, mix well, stand at room temperature for 2 h, and after completion, replace the buffer with a 20 mM histidine buffer solution (pH 6.0) using a NAP gel column (Cytiva) to obtain an antibody drug conjugate (i.e., Trastuzumab-C-28). The DAR value is 7.8 as determined by mass spectrometry.

[0755] 9. Preparation of Trastuzumab-C-28

[0756] Take 1.024 mL trastuzumab (24.4 mg / mL), dilute with 51.2 uL 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust pH to 7.60 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 94.7 uL, pH 7.60) solution, mix well, and stand at room temperature for 1.5 h. Then add a drug-linker C-28 (176 uL, 10 mM, 10-fold equivalent of the antibody) solution dissolved in dimethyl sulfoxide, mix well, stand at room temperature for 2 h, and after completion, replace the buffer with a 20 mM histidine buffer solution (pH 6.0) using a NAP gel column (Cytiva) to obtain an antibody drug conjugate (i.e., Trastuzumab-C-28). The DAR value is 7.8 as determined by mass spectrometry.

[0757] 10. Preparation of Trastuzumab-Z-1

[0758] Take 0.957 mL of Trastuzumab antibody (20.9 mg / mL), dilute with 52.0 uL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.60 with a 1 M Na2HPO4 solution, add 10 mM TCEP (tris (2-carboxyethyl) phosphine, 75.8 uL, pH 7.60) solution and mix well, and stand at room temperature for 1.5 h. Then add 10 times the amount of Z-1 dissolved in dimethyl sulfoxide (143.5 uL, 10 mM) solution and mix well, stand at room temperature for 2 h, and after completion, replace the buffer 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-Z-1). Mass spectrometry determines the DAR value to be 8.0.

[0759] Biological evaluation

[0760] I. Evaluation of the tumor growth inhibition effect of the antibody drug conjugate on a mouse subcutaneous xenograft model

[0761] The ADC of the application was administered to a mouse CDX model of subcutaneous transplantation of human breast cancer cells JIMT-1 by tail vein injection, respectively, and the tumor volume and animal body weight changes were determined once a week to calculate the tumor inhibition effect of the ADC of the application on tumor-bearing mice.

[0762] Test drug

[0763] Take an appropriate amount of ADC and administer at 3 mg / kg and 10 mg / kg, and the specific dosages are shown below. Dilute the mother liquor (20 mM histidine buffer solution at pH 6.0 of each antibody drug conjugate obtained in the preparation example of the antibody drug conjugate) with 0.9% NaCl injection solution to the administration solution. Use 0.9% NaCl injection solution as the vehicle control (Vehicle).

[0764] Experimental animals and cell lines

[0765] NOD SCID mice (Jiangsu Jicui Yekang Biotechnology Co., Ltd.)

[0766] Human breast cancer cells JIMT-1 (Nanjing Kebai)

[0767] Experimental grouping and evaluation method

[0768] The average tumor volume was selected to be about 150 mm 3The tumor-bearing mouse individuals were randomly grouped (the number of groups was determined according to the sample quantity). According to the groups, 0.9% NaCl injection solution (hereinafter referred to as a vehicle control, Vehicle), ADC were respectively given, the administration frequency was seen in the specific examples, the administration mode was tail vein injection, and the administration volume was 10 ml / kg. After administration, the tumor diameter was measured once a week with a vernier caliper, and the tumor volume was calculated according to the following calculation formula: V = 0.5a x b 2 , wherein a and b respectively represent the long diameter and the short diameter of the tumor. The animal death was observed and recorded every day.

[0769] The tumor growth inhibition rate TGI (%) was calculated according to the following formula, which was used to evaluate the tumor inhibition effect of the ADC:

[0770] V T末 >V T0 , TGI (%) = [1- (V T末 -V T0 ) / V C末 -V C0 ] * 100%, or

[0771] V T末 ≤V T0 , TGI (%) = [1- (V T末 -V T0 ) / V T0 ] * 100%.

[0772] , wherein V T末 : the mean tumor volume of the treatment group at the end of the experiment;

[0773] V T0 : the mean tumor volume of the treatment group at the beginning of administration;

[0774] V C末 : the mean tumor volume of the vehicle control group at the end of the experiment;

[0775] V C0 : the mean tumor volume of the vehicle control group at the beginning of administration;

[0776] The tumor relative proliferation rate T / C (%) was calculated according to the following formula, which was used to evaluate the tumor inhibition effect of the ADC:

[0777] T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 ) * 100%.

[0778] (1) Pharmacodynamic detection of anti-human Her2 antibody drug conjugate in JIMT-1 model

[0779] JIMT-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in 5% CO2. Exponentially growing JIMT-1 cells were collected and resuspended to an appropriate concentration with PBS containing 50% Matrigel, and then subcutaneously inoculated into female NOD SCID mice to establish a breast cancer model. When the average tumor volume was about 150mm 3 When the average tumor volume was about 150mm

[0780] The ADC of the present application significantly inhibited the growth of JIMT-1 breast cancer xenograft tumor model. Compared with the Vehicle group, the tumor growth inhibition rates (TGI) of the Trastuzumab-A-1, Trastuzumab-A-2, DS8201+Trastuzumab-A-2 and DS8201 groups were 112.81%, 95.27%, 147.73% and 58.90%, respectively. On Day 25, no animal death and no significant animal weight loss were observed in each treatment group, and no obvious drug toxicity reaction was observed. During the treatment period, the mice tolerated the ADC of the present application well. The specific results are shown in Table 1, Figure 1 and Figure 2.

[0781] Table 1 Human breast cancer cell JIMT-1 CDX model

[0782] II. Evaluation of the tumor growth inhibition effect of antibody-drug conjugates on a mouse subcutaneous xenograft tumor model

[0783] The ADC of the present application was administered to the mouse CDX model of subcutaneously transplanted human breast cancer cells JIMT-1 by tail vein injection, respectively. The tumor volume and animal weight change were determined once a week, and the tumor inhibition effect of the ADC of the present application on tumor-bearing mice was calculated.

[0784] Test drug

[0785] Take the appropriate amount of ADC of the application, according to the administration of 10 mg / kg, the specific dose is given below. Using 0.9% NaCl injection dilution mother liquor to the administration solution. 0.9% NaCl injection as a solvent control (Vehicle).

[0786] Experimental animals and cell lines

[0787] NOD SCID mice (Jiangsu Jicui Yekang Biotechnology Co., Ltd.)

[0788] Human breast cancer cells JIMT-1 (Nanjing Kebai)

[0789] Experimental grouping and evaluation method

[0790] Select tumor average volume about 150mm 3 The tumor-bearing mice were randomly divided into groups (the number of groups was determined according to the number of samples). According to the group, 0.9% NaCl injection (hereinafter referred to as vehicle control, Vehicle), ADC was given, the administration frequency was seen in the specific examples, the administration mode was tail vein injection, and the administration volume was 10ml / kg. After administration, the tumor diameter was measured once a week with a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a x b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. The animal death was observed and recorded every day.

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

[0792] V T末 >V T0 , TGI (%) = [1-(V T末 -V T0 ) / V C末 -V C0 ]*100%, or

[0793] V T末 ≤V T0 , TGI (%) = [1-(V T末 -V T0 ) / V T0 ]*100%.

[0794] Where V T末 : the mean tumor volume at the end of the experiment in the treatment group

[0795] V T0 : the mean tumor volume at the beginning of administration in the treatment group

[0796] V C末 : the mean tumor volume at the end of the experiment in the vehicle control group

[0797] V C0 : Mean tumor volume at the beginning of vehicle control group administration

[0798] The tumor relative proliferation rate T / C (%) was calculated using the following formula to evaluate the tumor inhibition effect of the ADC:

[0799] T / C = (V T末 / V T0 ) / (V C末 / V C0 ).

[0800] (1) Pharmacodynamic detection of anti-human Her2 antibody conjugate drugs in JIMT-1 model

[0801] JIMT-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2. Exponentially growing JIMT-1 cells were collected and resuspended to an appropriate concentration with PBS containing 50% Matrigel, and subcutaneously inoculated into female NOD SCID mice to establish a breast cancer model. When the average tumor volume was about 150 mm 3 around, the mice were randomly divided into groups according to the tumor size, and the groups were as follows: vehicle control group (i.e. negative control, Vehicle group), Trastuzumab-C-3 10 mg / kg group, Trastuzumab-C-10 10 mg / kg group, Trastuzumab-C-13 10 mg / kg group, and Trastuzumab-C-28 10 mg / kg group. Each group was administered by tail vein injection (i.v.) at Day 0 and Day 14, for a total of 2 administrations.

[0802] The ADC of the present application had a significant tumor growth inhibition effect on the JIMT-1 breast cancer xenograft model. Compared with the Vehicle group, the tumor growth inhibition rates (TGI) of the Trastuzumab-C-3 10 mg / kg group, Trastuzumab-C-10 10 mg / kg group, Trastuzumab-C-13 10 mg / kg group, and Trastuzumab-C-28 10 mg / kg group were 88.78%, 70.96%, 89.64%, and 79.45%, respectively. No animal deaths or significant weight loss were observed in each treatment group on Day 27, and no obvious drug toxicity reactions were observed. The mice tolerated the ADC of the present application well during the treatment period. The specific results are shown in Table 2 and Figures 3-5.

[0803] Table 2 Human breast cancer cell JIMT-1 CDX model

[0804] III. Evaluation of the tumor growth inhibition effect of the antibody drug conjugate on a mouse subcutaneous xenograft model

[0805] The ADCs of the present application were administered to a mouse CDX model of subcutaneously transplanted human gastric cancer cells NCI-N87 via tail vein injection, respectively, once a week, and the tumor volume and the change in animal body weight were determined to calculate the tumor inhibition effect of the ADCs of the present application on tumor-bearing mice.

[0806] Test drug

[0807] An appropriate amount of ADC was administered at 3 mg / kg, and the specific administration dose is shown below. The mother solution was diluted to the administration solution using 0.9% NaCl injection solution. 0.9% NaCl injection solution was used as a vehicle control (Vehicle).

[0808] Experimental animals and cell lines

[0809] Balb / c Nude mice (Sichuan VITROLIFE Experimental Animal Technology Co., Ltd.)

[0810] Human gastric cancer cells NCI-N87 (ATCC)

[0811] Experimental grouping and evaluation method

[0812] The tumor-bearing mice with an average tumor volume of about 150 mm 3 were randomly grouped (the number of groups was determined according to the number of samples). 0.9% NaCl injection solution (hereinafter referred to as vehicle control, Vehicle), ADC were administered according to the groups, and the administration frequency is shown in the specific examples. The administration mode was tail vein injection, and the administration volume was 10 ml / kg. The tumor diameter was measured once a week after administration using a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a x b 2 , where a and b represent the long diameter and the short diameter of the tumor, respectively. The animal death was observed and recorded every day.

[0813] The tumor growth inhibition rate TGI (%) was calculated using the following formula to evaluate the tumor inhibition effect of the ADC:

[0814] V T末 >V T0 , TGI (%) = [1-(V T末 -V T0 ) / (V C末 -V C0 )]*100%, or

[0815] V T末 ≤V T0 , TGI (%) = [1-(V T末 -V T0 ) / V T0* 100%.

[0816] wherein V T末 : mean tumor volume at the end of the experiment for the treatment group;

[0817] V T0 : mean tumor volume at the start of dosing for the treatment group;

[0818] V C末 : mean tumor volume at the end of the experiment for the vehicle control group;

[0819] V C0 : mean tumor volume at the start of dosing for the vehicle control group;

[0820] The relative tumor proliferation rate T / C (%) was calculated using the following formula to evaluate the anti-tumor efficacy of ADCs:

[0821] T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 )* 100%.

[0822] (1) Anti-human Her2 ADC efficacy detection in NCI-N87 model

[0823] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in 5% CO2. Exponentially growing NCI-N87 cells were collected, 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 was about 150 mm 3 around, the mice were randomly divided into groups according to tumor size, including a vehicle control group (i.e., negative control, Vehicle group), Trastuzumab-C-3 3 mg / kg, Trastuzumab-C-10 3 mg / kg, Trastuzumab-C-13 3 mg / kg, and Trastuzumab-C-28 3 mg / kg, each group was injected intravenously (i.v.), and dosing was performed on Day 0 and Day 7, for a total of 2 doses.

[0824] The ADC of the present application has a significant tumor growth inhibition effect on NCI-N87 gastric cancer xenograft model. Compared with the Vehicle group, the tumor growth inhibition rates (TGI) of Trastuzumab-C-3 3mg / kg, Trastuzumab-C-10 3mg / kg, Trastuzumab-C-13 3mg / kg and Trastuzumab-C-28 3mg / kg of the present application were 64.16%, 58.15%, 56.07% and 27.22% respectively, no animal death and no significant animal weight loss was observed in each treatment group on Day 27, no obvious drug toxicity reaction was observed, and the mice tolerated the ADC of the present application well during the treatment period. The specific results are shown in Table 3.

[0825] Table 3 Human gastric cancer cell NCI-N87 CDX model

[0826] Four, evaluation of the tumor growth inhibition effect of antibody drug conjugate on mouse subcutaneous xenograft model

[0827] The ADC of the present application was administered to the mouse CDX model of subcutaneously transplanted human breast cancer cells JIMT-1 by tail vein injection, the tumor volume and animal weight change were determined once a week, and the tumor inhibition effect of the ADC of the present application on tumor-bearing mice was calculated.

[0828] Test drug

[0829] An appropriate amount of ADC was administered at a dose of 3mg / kg and 10mg / kg, and the specific administration dose is shown below. The mother solution was diluted to the administration solution using 0.9% NaCl injection solution. 0.9% NaCl injection solution was used as a solvent control (Vehicle).

[0830] Experimental animals and cell lines

[0831] NOD SCID mice (Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd.)

[0832] Human breast cancer cells JIMT-1 (Nanjing Kebai)

[0833] Experimental grouping and evaluation method

[0834] The tumor-bearing mice with an average tumor volume of about 150mm 3 were randomly divided into groups (the number of groups was determined according to the number of samples). 0.9% NaCl injection solution (hereinafter referred to as solvent control, Vehicle), ADC were administered according to the groups, the administration frequency is shown in the specific examples, the administration mode was tail vein injection, and the administration volume was 10ml / kg. The tumor diameter was measured once a week after administration using a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a x b 2 ​​​​​​​​​​, where a and b represent the long diameter and short diameter of the tumor respectively. The death of animals was observed and recorded every day.

[0835] The tumor growth inhibition rate TGI (%) was calculated using the following formula for evaluating the anti-tumor effect of ADC:

[0836] V T末 >V T0 , TGI (%) = [1- (V T末 -V T0 ) / (V C末 -V C0 )] * 100%, or

[0837] V T末 ≤V T0 , TGI (%) = [1- (V T末 -V T0 ) / V T0 ] * 100%.

[0838] where V T末 : the mean tumor volume of the treatment group at the end of the experiment;

[0839] V T0 : the mean tumor volume of the treatment group at the beginning of the administration;

[0840] V C末 : the mean tumor volume of the solvent control group at the end of the experiment;

[0841] V C0 : the mean tumor volume of the solvent control group at the beginning of the administration;

[0842] The tumor relative proliferation rate T / C (%) was calculated using the following formula for evaluating the anti-tumor effect of ADC:

[0843] T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 ) * 100%.

[0844] (1) Anti-human Her2 ADC in JIMT-1 model for efficacy detection

[0845] The JIMT-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2. The JIMT-1 cells in the exponential growth phase were collected, resuspended to an appropriate concentration with PBS containing 50% Matrigel, and inoculated subcutaneously into female NOD SCID mice to establish a breast cancer model. When the average tumor volume was about 150mm 3The mice were randomly divided into groups according to the tumor size, and the groups were in turn: a vehicle control group (i.e. a negative control, Vehicle group), Trastuzumab-A-1 3 mg / kg, Trastuzumab-A-1 10 mg / kg, Trastuzumab-C-5 10 mg / kg, Trastuzumab-C-2 10 mg / kg, Trastuzumab-C-3 10 mg / kg, and Trastuzumab-C-1 10 mg / kg, which were administered at Day 0 and Day 14, for a total of 2 administrations.

[0846] The ADCs of the present application had a significant inhibitory effect on the growth of tumors in the JIMT-1 breast cancer xenograft model. Compared with the Vehicle group, the tumor growth inhibition rates (TGI) of the Trastuzumab-A-1 3 mg / kg, Trastuzumab-A-1 10 mg / kg, Trastuzumab-C-5 10 mg / kg, Trastuzumab-C-2 10 mg / kg, Trastuzumab-C-3 10 mg / kg, and Trastuzumab-C-1 10 mg / kg groups were 44.16%, 96.75%, 92.99%, 86.47%, 124.80%, and 90.15%, respectively. No animal deaths or significant weight loss was observed in the treatment groups on Day 33, and no obvious drug toxicity was observed. The mice tolerated the ADCs of the present application well during the treatment period. The specific results are shown in Table 4.

[0847] Table 4 Human breast cancer cell JIMT-1 CDX model

[0848] V. Evaluation of the inhibitory effect of the antibody drug conjugate on tumor growth in a mouse subcutaneous xenograft model

[0849] The ADCs of the present application were administered to the JIMT-1 human breast cancer cell subcutaneously transplanted mouse CDX model via tail vein injection, and the tumor volume and animal weight changes were measured once a week to calculate the tumor inhibition effect of the ADCs of the present application on tumor-bearing mice.

[0850] Test drug

[0851] An appropriate amount of ADC was administered at a dose of 3 mg / kg, and the specific administration dose is shown below. The mother solution was diluted to the administration solution using 0.9% NaCl injection solution. 0.9% NaCl injection solution was used as the vehicle control (Vehicle).

[0852] Experimental animals and cell lines

[0853] NOD SCID mice (Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd.)

[0854] Human breast cancer cell JIMT-1 (Nanjing Kebai)

[0855] Experimental grouping and evaluation method

[0856] Select tumor-bearing mice with an average tumor volume of about 150 mm 3 The tumor-bearing mice were randomly divided into groups (the number of groups was determined according to the number of samples). According to the groups, 0.9% NaCl injection solution (hereinafter referred to as vehicle control, Vehicle), ADC were administered, the administration frequency was as shown in the specific examples, the administration mode was tail vein injection, and the administration volume was 10 ml / kg. After administration, the tumor diameter was measured once a week using a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a x b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. The animal death was observed and recorded every day.

[0857] The tumor growth inhibition rate TGI (%) was calculated using the following formula to evaluate the tumor inhibition effect of ADC:

[0858] V T末 >V T0 , TGI (%) = [1-(V T末 -V T0 ) / (V C末 -V C0 )]*100%, or

[0859] V T末 ≤V T0 , TGI (%) = [1-(V T末 -V T0 ) / V T0 ]*100%.

[0860] Wherein V T末 : the average tumor volume at the end of the experiment in the treatment group;

[0861] V T0 : the average tumor volume at the beginning of administration in the treatment group;

[0862] V C末 : the average tumor volume at the end of the experiment in the vehicle control group;

[0863] V C0 : the average tumor volume at the beginning of administration in the vehicle control group;

[0864] The tumor relative proliferation rate T / C (%) was calculated using the following formula to evaluate the tumor inhibition effect of ADC:

[0865] T / C (%) = (V T末 / V T0 ) / (VC末 V C0 )* 100%.

[0866] (1) Anti-human Her2 antibody drug conjugate in JIMT-1 model of efficacy testing

[0867] JIMT-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2. The JIMT-1 cells in the exponential growth phase were collected and resuspended in PBS containing 50% Matrigel to an appropriate concentration, and inoculated subcutaneously in female NOD SCID mice to establish a breast cancer model. When the average tumor volume was about 150 mm 3 around, the mice were randomly divided into groups according to the tumor size, and the groups were as follows: vehicle control (i.e. negative control, Vehicle group), Trastuzumab-Z-1 3 mg / kg, Trastuzumab-C-3 3 mg / kg, Trastuzumab-Z-1 3 mg / kg + Trastuzumab-C-3 3 mg / kg, administered on Day 0 and Day 14, for a total of 2 administrations.

[0868] The ADC of the present application had a significant inhibitory effect on the growth of tumor in the JIMT-1 breast cancer xenograft model. Compared with the Vehicle group, the tumor growth inhibition rates (TGI) of the Trastuzumab-Z-1 3 mg / kg, Trastuzumab-C-3 3 mg / kg, and Trastuzumab-Z-1 3 mg / kg + Trastuzumab-C-3 3 mg / kg groups were 43.99%, 30.60%, and 97.01%, respectively. On Day 28, there was no animal death or significant weight loss in each treatment group, and no obvious drug toxicity was observed. The mice tolerated the ADC of the present application well during the treatment period. The specific results are shown in Table 5.

[0869] Table 5 Human breast cancer cell JIMT-1 CDX model

[0870] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details without departing from the spirit of the disclosed teachings, and such changes are intended to be within the scope of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A drug-linker or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof having a structure represented by formula (I): in: Q is a precursor of a linker site that is attached to an antibody or antigen-binding fragment thereof; L is the connecting structure connecting Q and E; E is the structure connecting L and D; D is the biologically active molecule portion.

2. The drug-linker of claim 1, wherein Q is composed of one or more of the following structures: Amino acid residues or polypeptide fragments consisting of 2-10 amino acids, Wherein LG represents a leaving group, preferably, LG is independently selected from halogen (such as F, Cl, Br, I), methanesulfonyl, fluorophenoxy, hydroxyl, thiol or amino; Ra is selected from cyano, nitro, C 2-6 Alkynyl, C 1-6 Haloalkyl (e.g., trifluoromethyl or trichloromethyl), -C(=O)C 1-6 Alkyl, and -SO2C 1-6 Alkyl; preferably cyano; p is independently selected from an integer of 1-12 at each occurrence; Preferably, Q is selected from the following structures: wherein LG represents a leaving group; p is an integer selected from 1 to 12; Preferably, Q is selected from the following structures: Preferably, Q is selected from the following structures: Preferably, Q is selected from the following structures: Preferably, Q is selected from the following structures:

3. The drug-linker of claim 1 or 2, wherein 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, 5-12 membered heterocyclic group, -N(R')-, carbonyl, -O-, natural amino acid or non-natural amino acid and its analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), short peptide 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-Ly s, 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, Gly-Val-Ala, Glu- Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu), 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, polyethylene glycol fragment containing 1-10 EO units (i.e. -(CH2CH2O) 1-10 -C 1-6 alkyl), -CH2N(R")-DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), -CH2N(R")-DOTAGA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), -CH2N(R")-NOTA(1,4,7-triazacyclononane-N,N',N"-triacetic acid residue) , 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; R" is selected from hydrogen or C 1-6 alkyl; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following: C 1-6 Alkylene, carbonyl, 9-12 membered nitrogen-containing heterocyclic group, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Se r-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gl y-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu, wherein s is selected from an integer of 1 to 20; R' represents hydrogen, -CH2N(R")-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), -CH2N(R")-DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or -CH2N(R")-NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid residue); R" is selected from hydrogen or C 1-6 alkyl; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following: C 1-6 Alkylene, carbonyl, 9-12 membered nitrogen-containing heterocyclic group, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Se r-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gl y-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu, wherein s is selected from an integer of 1 to 20; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following: wherein s is selected from an integer of 1 to 20; Preferably, L is selected from substituted or unsubstituted structural fragments consisting of one or more of the following: Preferably, L is selected from Preferably, L is selected from 4. The drug-linker according to any one of claims 1 to 3, wherein E is selected from a single bond, -NH-CH2-, or the following structures: Preferably, E is a single bond, -NH-CH2- or Preferably, E is -NH-CH2- or 5. The drug-linker according to any one of claims 1 to 4, wherein D is selected from the active molecular portion of a nucleoside or folic acid anti-tumor drug; Preferably, D is selected from the active molecular part of nucleoside anti-tumor drugs; Preferably, the active nucleoside anti-tumor drug molecule is selected from fluorouracil, capecitabine, gemcitabine, cytarabine, azacitidine, doxifluridine, 5-fluoro-2'-deoxyuridine, tegafur, carmofur, uridine triacetate, troxacitabine, decitabine, ancitabine, enocitabine, FF-10502, brivudine, fludarabine, cladribine, clofarabine, nelarabine, forodesine, cordycepin, pentostatin, raltitrexed, pemetrexed, methotrexate, and pharmaceutically acceptable salts, esters, and analogs thereof; Preferably, the above-mentioned active pharmaceutical molecule is connected to E in the drug-linker through the -OH, primary amino, or secondary amine group on it; Preferably, D is selected from 6. The drug-linker of any one of claims 1 to 5, wherein -LED is selected from the following structures: Preferably, the LED is selected from the following structures:

7. The drug-linker according to any one of claims 1 to 6, having the following structure:

8. An antibody-drug conjugate having a structure represented by formula (II): in: Ab is an antibody or its antigen-binding fragment; M is a linker site with an antibody or antigen-binding fragment thereof; x is selected from 1 to 10; L, E and D are as described in any one of claims 1-7.

9. The antibody-drug conjugate according to claim 8, wherein Ab is selected from 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; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 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; or, (2) The following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18, CDR-H2 of SEQ ID NO: 19, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33, CDR-H2 of SEQ ID NO: 34, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25; or, (3) the following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26, CDR-H2 of SEQ ID NO: 27, and CDR-H3 of SEQ ID NO: 22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25; or, (4) The following heavy chain variable region (VH) and light chain variable region (VL), where the CDRs are defined according to the IMGT numbering system: (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13, CDR-H2 of SEQ ID NO: 14, and CDR-H3 of SEQ ID NO: 15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 10; or (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 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; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4; Preferably, the antibody or antigen-binding fragment thereof comprises: (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; (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; or (3) a heavy chain comprising the VH sequence of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 41, 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 (4) a heavy chain comprising the VH sequence of SEQ ID NO: 3 and the heavy chain constant region (CH) of SEQ ID NO: 41, 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; Preferably, the antibody or antigen-binding fragment thereof comprises: (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; (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; (3) a heavy chain comprising the sequence shown in SEQ ID NO: 42, and a light chain comprising the sequence shown in SEQ ID NO: 38; or (4) a heavy chain comprising the sequence shown in SEQ ID NO: 43, and a light chain comprising the sequence shown in SEQ ID NO: 40; Optionally, the N-terminal glutamine of the VH or variant thereof of the sequence shown in SEQ ID NO: 1 or 3 or the heavy chain or variant thereof of the sequence shown in SEQ ID NO: 37, 39, 42 or 43 undergoes cyclization to form pyroglutamate or pyroglutamate salt; Optionally, the heavy chain constant region (CH) of SEQ ID NO: 35 or 41 or a variant thereof, or the heavy chain of SEQ ID NO: 37, 39, 42 or 43 or a variant thereof lacks a C-terminal lysine; Preferably, Ab is selected from trastuzumab, pertuzumab, a trastuzumab mutant, a pertuzumab mutant, or a bi-epitope antibody or antigen-binding fragment thereof constructed from trastuzumab and pertuzumab.

10. The antibody-drug conjugate according to claim 8 or 9, wherein M is selected from the following structures: Wherein, the position marked 1 in the M structure is where M and Ab are connected, and the position marked 2 in the M structure is where M and L are connected; p is an integer selected from 1-12; s is an integer selected from 1-20, and Ra is as described in any one of claims 1-7; Preferably, M is selected from the following structures: Preferably, M is selected from the following structures:

11. The antibody-drug conjugate according to any one of claims 8 to 10, wherein: The "drug-linker" represented by formula (I) (eg, the following structures: A-1 to A-30, B-1 to B-32, C-1 to C-64) is obtained by connecting to the antibody via a substitution reaction (eg, removal of the -SO2Me structure).

12. The antibody-drug conjugate according to any one of claims 8 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 8 to 12, which is selected from the group consisting of ADC A-1 to ADC A-30, ADC B-1 to ADC B-32, and ADC C-1 to ADC C-64; in, Ab'-(S- represents an antibody or an antigen-binding fragment thereof; represents the specific connection mode between the thiol group in the antibody or antigen-binding fragment thereof and the M fragment, and x is selected from 1-10.

14. An antibody drug conjugate composition comprising the antibody drug conjugate according to any one of claims 8 to 13, wherein the DAR value (drug-antibody conjugate ratio) of the antibody drug conjugate composition is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, 9-10; for example, DAR is about 7.24, about 7.46, about 7.56, about 7.7, about 7.8, about 7.83, about 7.9, about 7.

99.

15. A pharmaceutical composition comprising the drug-linker according to any one of claims 1 to 7, the antibody drug conjugate according to any one of claims 8 to 13, or the combination of the antibody drug conjugate according to claim 14, and one or more pharmaceutical excipients.

16. A pharmaceutical combination comprising: Component a: a combination of the drug-linker according to any one of claims 1 to 7, the antibody-drug conjugate according to any one of claims 8 to 13, or the antibody-drug conjugate according to claim 14, and one or more pharmaceutical excipients; and Component b: one or more other therapeutic agents, and one or more pharmaceutical excipients; preferably, the other therapeutic agent is an antibody-drug conjugate, such as an antibody-drug conjugate targeting Her2, such as DS8201 or Trastuzumab-Z-1; Preferably, the drug combination is a kit; Preferably, component a and component b are in separate containers.

17. Use of the drug-linker according to any one of claims 1 to 7, the antibody drug conjugate according to any one of claims 8 to 13, the antibody drug conjugate composition according to claim 14, the pharmaceutical composition according to claim 15, or the pharmaceutical combination according to claim 16 in the preparation of a medicament for treating Her2-expressing cancer.

18. The method of claim 17, wherein the cancer disease is selected from solid tumors or hematological malignancies; for example, selected from ovarian cancer, pancreatic cancer, nasopharyngeal cancer, head and neck cancer, urothelial carcinoma, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma) and lymphoma.

19. The drug-linker of any one of claims 1 to 7, the antibody drug conjugate of any one of claims 8 to 13, the antibody drug conjugate composition of claim 14, the pharmaceutical composition of claim 15, or the pharmaceutical combination of claim 16, for use in treating Her2-expressing cancer.

20. The drug-linker, antibody drug conjugate, antibody drug conjugate composition, pharmaceutical composition or pharmaceutical combination of claim 19, wherein: The cancer is selected from solid tumors or hematological malignancies; for example, selected from ovarian cancer, pancreatic cancer, nasopharyngeal cancer, head and neck cancer, urothelial carcinoma, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma) and lymphoma.

21. A method for treating Her2-expressing cancer, comprising administering to a subject in need thereof an effective amount of the drug-linker of any one of claims 1-7, the antibody drug conjugate of any one of claims 8-13, the antibody drug conjugate composition of claim 14, the pharmaceutical composition of claim 15, or the pharmaceutical combination of claim 16.

22. The method of claim 21, wherein The cancer is selected from solid tumors or hematological malignancies; for example, selected from ovarian cancer, pancreatic cancer, nasopharyngeal cancer, head and neck cancer, urothelial carcinoma, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma) and lymphoma.

23. A method of treating a Her2-expressing cancer comprising administering to a subject in need thereof: a) the drug-linker according to any one of claims 1 to 7, the antibody-drug conjugate according to any one of claims 8 to 13, the antibody-drug conjugate composition according to claim 14, the pharmaceutical composition according to claim 15, or the pharmaceutical combination according to claim 16; b) an effective amount of another therapeutic agent; Preferably, the antibody-drug conjugate is selected from Trastuzumab-A-1, Trastuzumab-A-2, Trastuzumab-C-2, Trastuzumab-C-2, Trastuzumab-C-3, Trastuzumab-C-5, Trastuzumab-C-10, Trastuzumab-C-13, and Trastuzumab-C-28; and the other therapeutic agent is selected from DS8201 or Trastuzumab-Z-1.

24. An intermediate compound having the following structure, or a salt, stereoisomer, tautomer, or isotope-labeled compound thereof: in, PG 1 Each is independently selected from H or a hydroxyl protecting group, wherein the hydroxyl protecting group is preferably tert-butyldimethylsilyl, phosphoric acid, benzoyl, trityl, 4-methoxyphenyldiphenylmethyl, dimethoxytrityl, 2,4-dimethoxybenzyl, p-methoxybenzyl and benzyl; PG 2 Each is independently H or an amino protecting group, and the amino protecting group is preferably an alkoxycarbonyl amino protecting group, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methyl (or ethyl)oxycarbonyl; an acyl amino protecting group, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p-toluenesulfonyl) ) nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, tert-butyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; alkyl amino protecting groups, such as trityl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn); R 2 Selected from H, C 1-6 Alkyl; LG represents a leaving group, preferably, LG are each independently selected from halogen (such as F, Cl, Br, I), methylsulfonyl, fluorophenoxy, hydroxyl, mercapto or amino; Preferably, the intermediate compound has the following structure:

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