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

Abstract

Provided in the present application are an antibody-drug conjugate, a composition and the use thereof and a method therefor. Specifically provided are an antibody-drug conjugate as shown in (I), and a drug-linker molecule for coupling to an antibody. The drug-linker molecule has a structure as shown in Q-L-E-D, wherein the drug is selected from a poly(ADP-ribose)polymerase inhibitor active compound. The antibody-drug conjugate has a better tumor-killing effect.

Description

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

[0001] This application is based on and claims priority to CN application No. 202410440848.X, filed on April 12, 2024, the disclosure of which is incorporated herein in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of medicine, and relates to antibody drug conjugates, compositions thereof and use thereof in treating diseases associated with abnormal cell activity, including but not limited to treating tumor diseases, in particular Her2-expressing cancers. The present application also provides drug-linker molecules for conjugation to the antibodies, the drugs being selected from the group consisting of poly (ADP-ribose) polymerase (PARP) inhibitor anti-tumor drugs. The present application also provides combinations of antibody drug conjugates and therapeutic uses thereof. BACKGROUND

[0003] Biological macromolecular drugs such as therapeutic antibodies or antibody fragments have made important progress as anti-tumor drugs. However, biological macromolecular drugs, although highly targeted, have limited therapeutic effect on solid tumors. In recent years, it has been found that biological macromolecular drugs such as therapeutic antibodies can be linked to cytotoxic anti-tumor drugs to form antibody drug conjugates (ADCs). ADCs combine the targeting effect of antibodies and the high activity of cytotoxic drugs. Antibodies guide the binding of ADCs 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 makes them ideal carriers for drug targeted delivery, reducing the side effects of drugs.

[0004] Poly (ADP-ribose) polymerase (PARP) inhibitors inhibit tumor cell DNA damage repair and promote tumor cell apoptosis. SUMMARY

[0005] Based on the targeting and effectiveness of antibody drug conjugates, mounting PARP inhibitor anti-tumor drugs on antibodies through suitable linking methods is an innovative attempt. In addition, antibody conjugates using PARP inhibitors and other anti-tumor active compounds with different mechanisms of action as effector molecules have the potential for synergistic effects, which are expected to improve the effectiveness and safety of tumor treatment.

[0006] The present application provides a novel drug-linker and its antibody drug conjugate (i.e. PARP inhibitor anti-tumor drugs conjugated to antibodies through a linker), which has a targeted killing effect on tumors and an anti-tumor effect.

[0007] Drug-linker

[0008] In one aspect, the present application provides a compound represented by Formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, Q-L-E-D Formula (I)

[0009] wherein:

[0010] Q is a structure before covalent attachment to an antibody or antigen binding fragment thereof;

[0011] L is a linking structure connecting Q and E;

[0012] E is a structure connecting L and D;

[0013] D is an active molecular moiety.

[0014] In some embodiments, D is a pharmaceutically active molecular moiety.

[0015] In some embodiments, Q is selected from the following structures:

[0016] wherein p is selected from an integer from 1-12, and LG represents a leaving group.

[0017] In some embodiments, Q is selected from the following structures:

[0018] wherein p is selected from an integer from 1-12, and LG represents a leaving group.

[0019] 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.

[0020] In some embodiments, each LG is independently selected from the group consisting of halogen (e.g., F, Cl, Br, I), haloC 1-6 alkyl, C 1-6 alkylsulfonyl, haloC 1-6 alkylsulfonyl, haloC 1-6 alkylsulfonate, haloC 1- 6alkylsulfonate, C 1-6 alkylsulfinate, C 1-6 alkylsulfoxide, haloC 1-6 alkyl, C 1- 6alkylsulfonate, C 1-6 alkylsulfonyl, haloC1-6 alkylsulfonate, haloC 1-6 alkylsulfonate, C 1-6 alkylsulfinate, C 1-6 alkylsulfinate, halo phenoxy, alkenyl, alkynyl, and structures containing alkynyl groups are optionally substituted with one or more suitable substituents.

[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, halo sulfonyl, C 1-6 alkylsulfonate, haloC 1- 6alkylsulfonate, C 1-6 alkylsulfinate, C 1-6 alkylsulfinate, halo phenoxy, hydroxyl, thiol, amino, nitro, azido, cyano, alkenyl, alkynyl, and structures containing alkynyl groups.

[0022] In some embodiments, Q is selected from the following structures:

[0023] wherein LG represents a leaving group (e.g., halogen (e.g., F, Cl, Br, I), haloC 1-6 alkyl, C 1-6 alkylsulfonyl, haloC 1-6 alkylsulfonyl, halo sulfonyl, C 1-6 alkylsulfonate, haloC 1-6 alkylsulfonate, C 1-6 alkylsulfinate, C 1-6 alkylsulfinate, halo phenoxy, hydroxyl, thiol, amino, nitro, azido, cyano, alkenyl, alkynyl, and structures containing alkynyl groups, said haloC 1-6 alkyl, C 1-6 alkylsulfonyl, haloC 1-6 alkylsulfonyl, halo sulfonyl, C 1-6 alkylsulfonate, haloC 1-6 alkylsulfonate, C 1-6 alkylsulfinate, C 1-6 alkylsulfinate, halo phenoxy, alkenyl, alkynyl, and structures containing alkynyl groups are optionally substituted with one or more suitable substituents.

[0024] p is selected from an integer from 1-12, for example, 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.

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

[0026] In some embodiments, Q is selected from the following structures:

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

[0028] In some embodiments, Q is

[0029] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structures: C 1-6 alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, -N(R')-, 9-12 membered nitrogen-containing heterocyclyl, carbonyl, -0-, a natural amino acid or a non-natural 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) rLys(R'), and 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 containing 1-10 ethoxy (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.

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

[0031] In some embodiments, "-DOTAGA" refers to

[0032] In some embodiments, "-NOTA" refers to

[0033] 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.

[0034] 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.

[0035] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structures: 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; 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.

[0036] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structures: 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; R’ represents hydrogen, glucosyl, galactosyl, glucuronyl, galacturonyl, -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 1-4 alkyl.

[0037] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structures: 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.

[0038] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structures: 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. In some preferred embodiments, the imino end of L is connected to Q.

[0039] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structures: 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. In some preferred embodiments, the imino end of L is connected to Q.

[0040] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structures: wherein s is selected from an integer from 1-20, preferably 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. In some preferred embodiments, the imino end of L is connected to Q.

[0041] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structures: wherein s is selected from an integer from 1-20, preferably 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. In some preferred embodiments, the imino end of L is connected to Q.

[0042] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structures:

[0043] wherein s is selected from an integer from 1-20, preferably s is selected from an integer from 1-10, for example 1-8, 1-6, 1-4, 1-2, 3-6, for example s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In some preferred embodiments, the imino end of L is connected to Q.

[0044] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structures:

[0045] wherein s is selected from an integer from 1-20, preferably 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. In some preferred embodiments, the imino end of L is connected to Q.

[0046] In some embodiments, L is -L1-L2-L3-, wherein L1is absent or selected from wherein s is selected from an integer from 1-20, preferably 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;

[0047] L2is selected from the group consisting of amino acids and short peptides of amino acids, such as 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;

[0048] L3is absent or selected from the group consisting of wherein 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. In some preferred embodiments, the imino end of L and Q are connected.

[0049] In some embodiments, L1is absent or selected from the group consisting of 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.

[0050] In some embodiments, L2is selected from the group consisting of Val-Ala, Val-Cit, Ala-Ala-Ala, Gly-Gly-Phe-Gly, and Gly-Gly-Val-Ala.

[0051] In some embodiments, L3is absent or selected from the group consisting of

[0052] In some embodiments, L is selected from the group consisting of:

[0053] In some embodiments, L is selected from:

[0054] In some embodiments, L is selected from:

[0055] In some embodiments, L is selected from:

[0056] In some embodiments, L is selected from:

[0057] In some embodiments, L is selected from:

[0058] In some preferred embodiments, the imino end of each L above and Q are connected.

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

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

[0061] In some embodiments, E is -NH-CH2-, In some preferred embodiments, the left end of E is connected to L and the right end of E is connected to D.

[0062] In some embodiments, E is a single bond, -NH-CH2-, or In some preferred embodiments, the left end of E is connected to L and the right end of E is connected to D.

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

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

[0065] In some embodiments, E is

[0066] In some embodiments, the active molecule is an antimetabolite antineoplastic drug.

[0067] In some embodiments, the antimetabolite antineoplastic drug is selected from the group consisting of PARP inhibitor antineoplastic active compounds.

[0068] In some embodiments, the PARP inhibitor class anti-neoplastic active compound is Olaparib, Niraparib, Rucaparib, Talazoparib, Pamiparib, Fuzoloparib, Veliparib, Stenoparib, Senaparib, Venadaparib, AZ9482, AZD-2461, Amelparib, Saruparib (AZD5305), AZD9574, A-966492, Nesuparib, Simmiparib, AG-14361, SC10914, AMXI-5001, Mefuparib, Iniparib (BSI-201), Atamparib (RBN-2397), RBN-2397, OUL232, Basroparib, WXFL10040340, TSL-1502, MP-124, NMS-P293, and pharmaceutically acceptable salts, esters, and analogs thereof.

[0069] The drugs or active molecules disclosed in this application typically contain one or more functional groups, such as hydroxyl (-OH), primary amino (-NH2), secondary amine (-NR1H), where R1 here represents only a non-hydrogen substituent on N, which can react with suitable functional groups in the rest of the conjugate to effect attachment to the drug molecule.

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

[0071] In some embodiments, D is selected from

[0072] In some embodiments, D is

[0073] In some embodiments, -L-E- is selected from the following structures:

[0074] In some embodiments, -L-E- is selected from the following structures:

[0075] In some embodiments, -L-E- is

[0076] In some embodiments, -L-E- is

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

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

[0079] In some embodiments, -E-D is

[0080] In some embodiments, -E-D is

[0081] In some embodiments, -E-D is

[0082] In some embodiments, -L-E-D is selected from the following structures:

[0083] In some embodiments, the free form of "Drug-Linker" is selected from the following A-1 ~ A-8, B-1 ~ B-3, C-1 ~ C-4:

[0084] In some embodiments, the aforementioned Drug-Linker can be optionally substituted with one or more suitable substituents.

[0085] Intermediate

[0086] In some embodiments, the present application provides an intermediate compound having the structure shown below or a salt, stereoisomer, tautomer, or isotopically labeled compound thereof:

[0087] wherein D is an active molecular moiety, as defined previously;

[0088] LG represents a leaving group, as defined previously;

[0089] PG1and PG2are each independently H or an amino protecting group, preferably an alkoxycarbonyl amino protecting group such as carbobenzyloxy (Cbz), tert-butoxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), meth(o / eth)oxycarbonyl; an acyl amino protecting group such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p)nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, carbobenzyloxy, p-methylbenzenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; an alkyl amino protecting group such as trityl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn).

[0090] In some embodiments, the present application provides an intermediate compound having the structure shown below:

[0091] wherein LG represents a leaving group, as defined previously;

[0092] PG1and PG2are each independently H or an amino protecting group, preferably an alkoxycarbonyl amino protecting group such as carbobenzyloxy (Cbz), tert-butoxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), meth(o / eth)oxycarbonyl; an acyl amino protecting group such as phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), o-(p)nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, carbobenzyloxy, p-methylbenzenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; an alkyl amino protecting group such as trityl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn).

[0093] In some embodiments, the present application provides an intermediate compound having the structure shown below:

[0094] In some embodiments, the intermediate compounds described supra can be optionally substituted with one or more suitable substituents.

[0095] In another aspect, the present application provides use of an intermediate compound as described supra, or a salt, stereoisomer, tautomer, or isotopically-labeled compound thereof, in the manufacture of a compound of Formula (I), or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically-labeled compound, metabolite, or prodrug thereof.

[0096] Antibody drug conjugate

[0097] In another aspect, the present application provides an antibody drug conjugate having a structure of Formula (II),

[0098] wherein L, E and D are as described supra,

[0099] M is a linker structure attached to an antibody or an antigen binding fragment thereof;

[0100] Ab is an antibody or an antigen binding fragment thereof;

[0101] x is selected from 1 to 10.

[0102] In some embodiments, M is selected from

[0103] wherein the mark 1 in the structure of M indicates the connection of M to Ab, and the mark 2 in the structure of M indicates the connection of M to L; p is selected from an integer from 1 to 12.

[0104] In some embodiments, M is selected from

[0105] wherein the mark 1 in the structure of M indicates the connection of M to Ab, and the mark 2 in the structure of M indicates the connection of M to L;

[0106] p is selected from an integer from 1 to 12.

[0107] In some embodiments, p is selected from an integer from 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, for example, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0108] In the antibody drug conjugate, D can be connected to the antibody or the antigen binding fragment thereof through a linker (such as the structure of “M-L-E” as described in the present application).

[0109] In some embodiments, M is selected from the following structures:

[0110] In some embodiments, M is

[0111] In some 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).

[0112] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0113] (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:

[0114] (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,

[0115] (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;

[0116] wherein the variant of 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 (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] (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:

[0119] (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,

[0120] (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;

[0121] 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 substitution is a conservative substitution;

[0122] or,

[0123] (3) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined by the Kabat numbering system:

[0124] (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,

[0125] (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;

[0126] 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;

[0127] or,

[0128] (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:

[0129] (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,

[0130] (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;

[0131] 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.

[0132] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0133] (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:

[0134] (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,

[0135] (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;

[0136] or,

[0137] (2) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system:

[0138] (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,

[0139] (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;

[0140] or,

[0141] (3) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the Kabat numbering system:

[0142] (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,

[0143] (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;

[0144] or,

[0145] (4) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system:

[0146] (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,

[0147] (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.

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

[0149] (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

[0150] (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;

[0151] 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.

[0152] In some embodiments, said antibody or antigen binding fragment thereof comprises:

[0153] (a) a VH as depicted in SEQ ID NO: 1, and, a VL as depicted in SEQ ID NO: 2; or

[0154] (b) a VH as depicted in SEQ ID NO: 3, and, a VL as depicted in SEQ ID NO: 4.

[0155] In some embodiments, said antibody or antigen binding fragment thereof further comprises:

[0156] (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

[0157] (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.

[0158] 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.

[0159] 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 compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative substitutions; e.g., 1, 2, 3, 4, or 5 conservative substitutions).

[0160] 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 compared to SEQ ID NO: 41 (e.g., up to 15, up to 10, or up to 5 conservative substitutions; e.g., 1, 2, 3, 4, or 5 conservative substitutions).

[0161] 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 compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 conservative substitutions; e.g., 1, 2, 3, 4, or 5 conservative substitutions).

[0162] 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.

[0163] 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.

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

[0165] (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;

[0166] (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

[0167] (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

[0168] (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.

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

[0170] (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;

[0171] (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;

[0172] (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

[0173] (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.

[0174] 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 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.

[0175] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0176] In certain embodiments, provided herein are compositions comprising the antibodies or antigen binding fragments disclosed herein, where 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] In some embodiments, the Ab is selected from Trastuzumab, Pertuzumab, a Trastuzumab mutant, a Pertuzumab mutant, or a biparatopic antibody constructed from Trastuzumab and Pertuzumab, or an antigen binding fragment thereof.

[0181] In some embodiments, the antibody or antigen binding fragment thereof is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which is query accession number (IMGT / mAb-DB ID) 97 in the IMGT database, or the amino acid sequence of which is query accession number (IMGT / mAb-DB ID) 80 in the IMGT database, respectively.

[0182] Those skilled in the art will appreciate that the antibody drug conjugates described herein can be prepared modularly. For example, a "drug-linker" in a pre-conjugated form (which can be understood as Q-L-E-D, wherein Q is a structure before M is covalently linked to an antibody or an antigen binding fragment thereof) can be first obtained, and then covalently linked to an antibody or an antigen binding fragment thereof to obtain the antibody drug conjugates described herein. The "drug-linker" in the pre-conjugated form can be in a free form. Accordingly, Q in the "drug-linker" in the pre-conjugated form is linked to one or more thiol (-SH) or amino (-NH2) groups on the antibody or the antigen binding fragment thereof by substitution reaction (e.g., removal of a structure such as -SO2Me or -Br thereon) or by addition reaction, etc.

[0183] In some embodiments, the antibody drug conjugate is selected from the group consisting of ADC A-1 to ADC A-8, ADC B-1 to ADC B-3, ADC C-1 to ADC C-4:

[0184] wherein Ab' in each antibody drug conjugate represents an antibody or an antigen binding fragment thereof;

[0185] represents a specific linkage mode of a thiol group in the antibody or the antigen binding fragment thereof to M; x is selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0186] In some embodiments, the antibody or the antigen binding fragment thereof in each antibody drug conjugate is as defined in any one of the preceding embodiments.

[0187] In some embodiments, Ab' in each antibody drug conjugate represents an antibody or an antigen binding fragment thereof that specifically binds to a member of the ErbB family of receptor tyrosine kinases, epidermal growth factor receptor 2 (Her2).

[0188] In some embodiments, the Ab' comprises

[0189] (1) a heavy chain variable region (VH) and / or a light chain variable region (VL) as defined below, wherein the CDRs are defined according to the Chothia numbering system:

[0190] (1a) a heavy chain variable region (VH) comprising 3 CDRs as follows: CDR-H1 of SEQ ID NO:5 or a variant thereof, CDR-H2 of SEQ ID NO:6 or a variant thereof, CDR-H3 of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs as follows: 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,

[0191] (1b) a heavy chain variable region (VH) comprising 3 CDRs as follows: CDR-H1 of SEQ ID NO:20 or a variant thereof, CDR-H2 of SEQ ID NO:21 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 as follows: 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;

[0192] wherein the variant of 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 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;

[0193] or,

[0194] (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:

[0195] (2a) a heavy chain variable region (VH) comprising 3 CDRs as follows: 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 3 CDRs as follows: 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] (2b) a heavy chain variable region (VH) comprising 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 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 (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 with the sequence from which it is derived, or said 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 said substitutions are conservative substitutions;

[0198] or,

[0199] (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:

[0200] (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,

[0201] (3b) a heavy chain variable region (VH) comprising three 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 three 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;

[0202] 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;

[0203] or,

[0204] (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:

[0205] (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, 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, CDR-L3 of SEQ ID NO: 10 or a variant thereof; or,

[0206] (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;

[0207] 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 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.

[0208] In some embodiments, the Ab' comprises

[0209] (1) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the Chothia numbering system:

[0210] (1a) a heavy chain variable region (VH) comprising 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 three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or,

[0211] (1b) a heavy chain variable region (VH) comprising 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 three CDRs: CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0212] or,

[0213] (2) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system:

[0214] (2a) a heavy chain variable region (VH) comprising 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 CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or,

[0215] (2b) a heavy chain variable region (VH) comprising 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 CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25;

[0216] or,

[0217] (3) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the Kabat numbering system:

[0218] (3a) a heavy chain variable region (VH) comprising 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 CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or,

[0219] (3b) a heavy chain variable region (VH) comprising 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 CDR-L1 of SEQ ID NO: 23, CDR-L2 of SEQ ID NO: 24, and CDR-L3 of SEQ ID NO: 25.

[0220] or,

[0221] (4) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0222] (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,

[0223] (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.

[0224] In some embodiments, the Ab’ comprises:

[0225] (a) a VH of SEQ ID NO: 1 or a variant thereof, and / or, a VL of SEQ ID NO: 2 or a variant thereof; or

[0226] (b) a VH of SEQ ID NO: 3 or a variant thereof, and / or, a VL of SEQ ID NO: 4 or a variant thereof;

[0227] wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or the variant has one or 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.

[0228] In some embodiments, the Ab’ comprises:

[0229] (a) VH as depicted in SEQ ID NO: 1, and, VL as depicted in SEQ ID NO: 2; or

[0230] (b) VH as depicted in SEQ ID NO: 3, and, VL as depicted in SEQ ID NO: 4.

[0231] In some embodiments, the Ab’ further comprises:

[0232] (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more substitutions, deletions, or additions of amino acids as compared to the wild type sequence from which it is derived (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); and

[0233] (b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more substitutions, deletions, or additions of amino acids as compared to the wild type sequence from which it is derived (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).

[0234] 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.

[0235] 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 of amino acids as compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids).

[0236] 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 of amino acids as compared to SEQ ID NO: 41 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids).

[0237] 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.

[0238] 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.

[0239] 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.

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

[0241] (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;

[0242] (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

[0243] (3) 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: 41, 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; or

[0244] (4) 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: 41, 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.

[0245] In some embodiments, the Ab' comprises:

[0246] (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;

[0247] (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;

[0248] (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

[0249] (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.

[0250] 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 a C-terminal glycine-lysine dipeptide. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof can be cyclized to pyroglutamic acid.

[0251] As known to those of skill in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0252] In certain embodiments, provided herein are compositions comprising the antibodies or antigen-binding fragments disclosed herein, where 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.

[0253] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein comprise 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.

[0254] In certain embodiments, the N-terminal glutamine of the VH set forth in SEQ ID NO: 1 or 3 or a variant thereof or the heavy chain set forth in SEQ ID NO: 37, 39, 42, or 43 or a variant thereof is cyclized to form pyroglutamic acid or pyroglutamate.

[0255] 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 the C-terminal lysine.

[0256] In some embodiments, Ab is selected from Trastuzumab, Pertuzumab, a Trastuzumab mutant, a Pertuzumab mutant, or a biparatopic antibody or antigen binding fragment thereof constructed from Trastuzumab and Pertuzumab.

[0257] In some embodiments, Ab' is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which has the query accession number (IMGT / mAb-DB ID) of 97 in the IMGT database, or the amino acid sequence of which has the query accession number (IMGT / mAb-DB ID) of 80 in the IMGT database.

[0258] In some embodiments, Ab' in each antibody drug conjugate represents Trastuzumab, Pertuzumab, or an antigen binding fragment thereof.

[0259] In some embodiments, Ab' in each antibody drug conjugate represents the following antibody or antigen binding fragment:

[0260] (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;

[0261] (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

[0262] (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

[0263] (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.

[0264] 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 pyroglutamate.

[0265] 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 lacks a C-terminal lysine.

[0266] In some embodiments, x in the conjugate of Ab-[M-L-E-D]x is 1-10, for example: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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.

[0267] In some embodiments, x in the conjugate of 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.

[0268] In some embodiments, x in the conjugate of Ab-[M-L-E-D]x is 4.0-8.0.

[0269] In some embodiments, x in the conjugate of Ab-[M-L-E-D]x is 6.0-8.0.

[0270] In some embodiments, x in the conjugate of Ab-[M-L-E-D]xis from 7.0 to 7.5.

[0271] In some embodiments, x in the conjugate of Ab-[M-L-E-D]xis from 7.0 to 8.0.

[0272] In some embodiments, x in the conjugate of Ab-[M-L-E-D]xis from 7.0 to 9.0.

[0273] In some embodiments, x in the conjugate of Ab-[M-L-E-D]xis from 7.5 to 8.5.

[0274] In some embodiments, x in the conjugate of Ab-[M-L-E-D]xis from 7.5 to 8.0.

[0275] In some embodiments, the conjugate described herein is an antibody drug conjugate (ADC).

[0276] In some embodiments, the conjugate described herein is optionally substituted with one or more suitable substituents.

[0277] Linking unit

[0278] In some embodiments, the present application provides a linking unit of the structure -M-L-E-, wherein M, L and E are as described in any one of the above. In some embodiments, the linking unit is used to link an active molecule and an antibody or antigen binding fragment thereof to obtain an antibody drug conjugate. In some embodiments, in the linking unit, E is a linking moiety for linking an active molecule, and M is a linker for linking to an antibody or antigen binding fragment thereof. In some embodiments, in the linking unit, E is for linking to D as described in any one of the present application, and M is for linking to an antibody or antigen binding fragment thereof as described in any one of the present application.

[0279] In some embodiments, the linking unit of the structure -M-L-E- is selected from the following structures:

[0280] In some embodiments, 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 -M-L-E- is provided.

[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 -M-L-E- and Ab and / or D, said linking unit being attached to Ab and / or D; wherein Ab or D is as defined in any of the preceding embodiments.

[0282] In some embodiments, the present application provides a linking unit of the structure Q-L-E-, wherein Q, L and E are as defined in any of the preceding embodiments. In some embodiments, the linking unit is used to link an active molecule and an antibody or antigen binding fragment thereof to obtain an antibody drug conjugate. In some embodiments, in the linking unit, E is a linking moiety for linking an active molecule, and Q is a structure capable of reacting with and being attached to an antibody or antigen binding fragment thereof. In some embodiments, in the linking unit, E is used to link to D as defined in any of the preceding embodiments, and Q is used to react with and be attached to an antibody or antigen binding fragment thereof as defined in any of the preceding embodiments.

[0283] 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 Q-L-E-.

[0284] 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 Q-L-E- and D, said linking unit being attached to D; wherein D is as defined in any of the preceding embodiments.

[0285] In some embodiments, the present application provides a linking unit of the structure -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 antigen binding fragment thereof through a linker to obtain an antibody drug conjugate.

[0286] In some embodiments, the present application provides a linking unit of the structure -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 antigen binding fragment thereof to obtain an antibody drug conjugate.

[0287] In some embodiments, provided is 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.

[0288] In some embodiments, the linking units described supra can be optionally substituted with one or more suitable substituents.

[0289] All of the technical features disclosed in the specification can be combined in any manner, except for mutually exclusive technical features. The present application encompasses compounds, conjugates, and linking units resulting from any combination of the various embodiments.

[0290] Compositions

[0291] 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, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug-linkers. Thus, the composition is characterized by a drug-antibody ratio (DAR) in the range of about 1 to about 10. Methods for determining DAR are well known to the skilled person, including methods using reverse phase chromatography or HPLC-MS.

[0292] 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: 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.

[0293] 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, or about 7.5 to 8.0.

[0294] 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.

[0295] In certain embodiments, the DAR of the ADC compositions described herein is about 7.0 to 9.0.

[0296] In certain embodiments, the DAR of the ADC compositions described herein is about 7.5 to 8.5.

[0297] In certain embodiments, the DAR of the ADC compositions described herein is about 7.99, about 7.98, about 8, about 8.0, or about 7.94.

[0298] In certain embodiments, the DAR of the ADC compositions described herein is 7.99, 7.98, 8, 8.0, or 7.94.

[0299] Pharmaceutical composition or drug combination

[0300] In another aspect, the present application provides a pharmaceutical composition comprising the antibody drug conjugate of any one of the preceding, the drug-linker compound of any one of the preceding, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, or the composition of any one of the preceding, and one or more pharmaceutical excipients.

[0301] The antibody drug conjugates, drug-linker compounds, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, or prodrugs thereof, or compositions described herein are typically formulated in unit injectable form in a parenterally acceptable vehicle for parenteral administration, e.g., a push-fit injection, an injection with an needleless syringe, etc. Optionally, the antibody drug conjugate with the desired degree of purity can be mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.) in the form of a lyophilized or aqueous solution. The antibody drug conjugates described herein, or pharmaceutical compositions containing such antibody drug conjugates, can be administered by any route appropriate to the condition to be treated. th edition,Osol,A.Ed.) can be used. The antibody drug conjugates described herein, or pharmaceutical compositions containing such antibody drug conjugates, can be administered by any route appropriate to the condition to be treated.

[0302] In some embodiments, the pharmaceutical compositions of the present disclosure further comprise, optionally, one or more other therapeutic agents. In some embodiments, the other therapeutic agent is an antibody drug conjugate. In some embodiments, the other therapeutic agent is an antibody drug conjugate targeting Her2, such as DS8201 or Trastuzumab-Z-1.

[0303] In another aspect, the present application provides a pharmaceutical combination comprising:

[0304] Component a: a compound of any of the preceding, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, an antibody drug conjugate, or a composition, and one or more pharmaceutical excipients; and

[0305] 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.

[0306] In some embodiments, the pharmaceutical combination is a kit.

[0307] In some embodiments, component a and component b are in separate containers.

[0308] In some embodiments, the antibody drug conjugate, drug-linker compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, composition, pharmaceutical 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 this 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.

[0309] The above pharmaceutical composition or pharmaceutical combination can comprise 0.01 mg to 1000 mg of at least one antibody drug conjugate, drug-linker compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, or composition of the present application.

[0310] Applications

[0311] The antibody drug conjugate, drug-linker compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, composition, pharmaceutical combination or pharmaceutical composition described herein can be used for treating a variety of diseases or disorders, such as Her2 expressing (Her2 positive) cancer, including solid tumors or hematological malignancies, such as urothelial cancer, ovarian cancer, pancreatic cancer, nasopharyngeal cancer, head and neck cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, in particular lung adenocarcinoma), or lymphoma.

[0312] Therefore, the present application provides use of the antibody drug conjugate, drug-linker compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, composition, pharmaceutical combination or pharmaceutical composition of any one of the preceding items in the manufacture of a medicament, in particular in the manufacture of a medicament for treating Her2 expressing cancer.

[0313] Meanwhile, the present application provides the antibody drug conjugate, drug-linker compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, composition, pharmaceutical combination or pharmaceutical composition of any one of the preceding items for use in treating Her2 expressing cancer.

[0314] Meanwhile, the present application also provides a method of treating Her2 expressing cancer, comprising the step of administering to a subject in need thereof an effective amount of the antibody drug conjugate, drug-linker compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, composition, pharmaceutical combination or pharmaceutical composition of any one of the preceding items.

[0315] The antibody drug conjugate, drug-linker compound, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, composition described herein can be used in combination with one or more other therapeutic agents for treating a variety of diseases or disorders, such as Her2 expressing cancer, including solid tumors or hematological malignancies, such as urothelial cancer, ovarian cancer, pancreatic cancer, nasopharyngeal cancer, head and neck cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, in particular lung adenocarcinoma), or lymphoma.

[0316] In some embodiments, the "in combination with one or more other therapeutic agents" includes administration or application simultaneously (concurrently) or sequentially or in any order, or in a combination of the foregoing, of the aforementioned pharmaceutical composition or pharmaceutical combination. In some embodiments, the other therapeutic agent is an antibody drug conjugate.

[0317] In some embodiments, the other therapeutic agent is an antibody drug conjugate targeting Her2.

[0318] In some embodiments, the other therapeutic agent is an anti-human HER2 antibody drug conjugate, the anti-human HER2 antibody is Trastuzumab.

[0319] In some embodiments, the other therapeutic agent is an antibody drug conjugate, the drug is a topoisomerase inhibitor.

[0320] In some embodiments, the topoisomerase inhibitor is a topoisomerase I inhibitor.

[0321] In some embodiments, the topoisomerase I inhibitor is a camptothecin compound, such as Topotecan, Irinotecan, Belotecan, Dxd, SN38 or

[0322] In some embodiments, the other therapeutic agent is, for example, DS8201.

[0323] In some embodiments, the other therapeutic agent is, for example, Trastuzumab-Z-1.

[0324] In some embodiments, the antibody drug conjugate, drug-linker compound, or pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug, composition or pharmaceutical composition thereof, is sufficient to (e.g., in a subject):

[0325] (1) inhibit proliferation of a cell (such as a tumor cell);

[0326] (2) inhibit tumor growth;

[0327] (3) induce and / or increase antibody-dependent cellular cytotoxicity activity;

[0328] (4) inhibit Her2-mediated signal transduction;

[0329] (5) prevent and / or treat a Her2-mediated disease / disorder; or

[0330] (6) any combination of (1)-(5) above.

[0331] In some embodiments, the cancer is selected from a solid tumor or a hematological malignancy; for example from urothelial cancer, ovarian cancer, pancreatic cancer, nasopharyngeal cancer, head and neck cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, in particular lung adenocarcinoma), and lymphoma.

[0332] In some embodiments, the cancer is from breast cancer.

[0333] Definitions

[0334] Unless otherwise defined, 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 by those in the art, including variations or replacements of those technical terms that are obvious to those skilled in the art or equivalent technical terms. 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 those skilled in the art, the following definitions are set forth to better explain the present application.

[0335] 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.

[0336] 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 numbering system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), IMGT (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or AbM (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 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 (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0337] 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 Kabat, 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.

[0338] 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 occur; but Cys residues are the most conserved feature.

[0339] 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

[0340] The amino acid positions in the heavy chain constant region can be sequentially numbered starting with amino acid position 1 and continuing through the end of the sequence, or can be numbered 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 begins with position 118 and ends with position 447. Unless otherwise specified, the amino acid positions of the heavy and light chains described herein are defined according to sequential numbering.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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).

[0345] 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.

[0346] 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.

[0347] 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- scFv comprising NH2-VL-VL-COOH.

[0348] 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 to which a full-length antibody binds (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0349] 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.

[0350] 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.

[0351] 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 procedures) from a given antibody (e.g., an antibody provided herein), and screened for specificity in the same manner as is used for intact antibodies.

[0352] The term "murine antibody" refers to an antibody obtained by fusing B cells from 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.

[0353] 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, a humanized antibody has all or a portion of the CDR regions 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) from a human immunoglobulin (acceptor 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).

[0354] 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).

[0355] 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 for 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).

[0356] 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.

[0357] The term "linker" refers to the structure that links the cytotoxic drug to the antibody or antigen binding fragment. For example, to the -M-L-E- structure in the formula Ab-[M-L-E-] x .

[0358] The term "drug-linker" refers to the structure of a cytotoxic drug and linker of the present application prior to attachment to an antibody or antigen binding fragment thereof. For example, "drug-linker" refers to Q-L-E-D, wherein Q is the structure of M prior to covalent attachment to an antibody or antigen binding fragment thereof. Covalent attachment of "drug-linker" to an antibody or antigen binding fragment thereof results in an antibody drug conjugate of the present application. In some embodiments, L is preferably attached to Q through an imino terminus. In some embodiments, -L-E- is preferably attached to Q through an imino terminus.

[0359] The "drug-linker" also includes all pharmaceutically acceptable isotopically-labeled compounds thereof which are identical to those recited in "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).

[0360] 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.

[0361] The term "alkyl" denotes a straight or branched chain hydrocarbon group, for example "C 1-20 alkyl", "C 1- 10 alkyl", "C 1-6 alkyl", "C 1-4 alkyl", "C 1-3The term "alkyl" denotes a branched or straight-chain saturated hydrocarbon group, which can be substituted or unsubstituted, having the number of carbon atoms specified, n, i.e., (Cn). Examples of alkyl groups 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.

[0362] The term "haloalkyl" denotes a group in which the alkyl moiety is substituted with one or more halogen atoms (e.g., F, Cl, Br, I), including mono-substituted, poly-substituted, or per-substituted forms, e.g., "haloC 1-6 The term "alkyl," "C 1-6 The term "haloalkyl" denotes a group in which the alkyl moiety is substituted with one or more halogen atoms (e.g., F, Cl, Br, I), including mono-substituted, poly-substituted, or per-substituted forms, e.g., "haloC

[0363] The term "alkylsulfonyl" denotes a group in which the hydrogen atoms of a sulfonyl group are replaced by alkyl groups, of the formula "alkyl-S(O)2-", e.g., "C 1-6 The term "alkylsulfonyl," "C 1-4 The term "alkylsulfonyl" denotes a group in which the hydrogen atoms of a sulfonyl group are replaced by alkyl groups, of the formula "alkyl-S(O)2-", e.g., "C

[0364] The term "haloalkylsulfonyl" denotes a group in which the hydrogen atoms of a sulfonyl group are replaced by haloalkyl groups, of the formula "haloalkyl-S(O)2-", e.g., "C 1-6 The term "haloalkylsulfonyl" denotes a group in which the hydrogen atoms of a sulfonyl group are replaced by haloalkyl groups, of the formula "haloalkyl-S(O)2-", e.g., "C

[0365] The term "halosulfonyl" denotes a group in which the hydrogen atoms of a sulfonyl group are replaced by halogen atoms (e.g., F, Cl, Br, I), of the formula "-SO2X", including but not limited to: sulfonyl chloride (-SO2Cl), sulfonyl fluoride (-SO2F), and the like.

[0366] The term "alkylsulfonate" denotes a group in which the hydrogen atoms of a sulfonate group are replaced by alkyl groups, of the formula "alkyl-S(O)2-O-", e.g., "C 1-6 The term "alkylsulfonate" denotes a group in which the hydrogen atoms of a sulfonate group are replaced by alkyl groups, of the formula "alkyl-S(O)2-O-", e.g., "C

[0367] 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 "haloC 1-6 alkylsulfonate", specific examples include, but are not limited to: triflate (CF3-S(O)2-O-), 2-chloroethanesulfonate, 1,1-difluoropropanesulfonate, and the like.

[0368] The term "alkylsulfinate" denotes a group in which the hydrogen atom of a sulfinate group is replaced by an alkyl group, of the formula "alkyl-S(O)-O-", for example "C 1-6 alkylsulfinate", specific examples include, but are not limited to: methylsulfinate (CH3-S(O)-O-), ethylsulfinate, isopropylsulfinate, and the like.

[0369] The term "alkylsulfoxide" denotes a group in which the hydrogen atom of a sulfoxide group is replaced by an alkyl group, of the formula "alkyl-S(O)-", for example "C 1-6 alkylsulfoxide", specific examples include, but are not limited to: methylsulfoxide (CH3-S(O)-), ethylsulfoxide, isopropylsulfoxide, tert-butylsulfoxide, and the like.

[0370] 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 include, but are not limited to: 4-fluorophenoxy, 2,6-dichlorophenoxy, 3-bromo-5-iodophenoxy, pentafluorophenoxy, and the like.

[0371] The term "alkenyl" denotes a straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond, for example "C 2-20 alkenyl", "C 2-6 alkenyl", and the like, specific examples include, but are not limited to: ethenyl (CH2=CH-), 1-propenyl, 2-butenyl, isoprenyl, 1,3-pentadienyl, and the like.

[0372] The term "alkynyl" denotes a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond, for example "C 2-20 alkynyl", "C 2-6 alkynyl", and the like, specific examples include, but are not limited to: ethynyl (HC≡C-), 1-propynyl, 2-butynyl, 3-methyl-1-pentynyl, and the like.

[0373] The term "aryl" denotes a monocyclic or bicyclic aromatic hydrocarbon group, for example "6-10 membered aryl" and the like, specific examples include, but are not limited to: phenyl, 1-naphthyl, 2-naphthyl, 3-naphthyl, and the like.

[0374] 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.

[0375] 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.

[0376] 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 D, 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).

[0377] A solid wedge may be used herein to indicate a preferred or a non-preferred Chemical bonds of the compounds of the invention are depicted. The use of a solid line to depict a bond to an asymmetric carbon atom is intended to indicate all possible stereoisomers at that carbon atom (e.g., a particular enantiomer, a racemic mixture, etc.). 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 thereof (e.g., racemic mixtures and diastereomeric pairs).

[0378] As used herein, the term "suitable substituents" refers to modifications that one of skill in the art can make to a compound according to the needs of the substituents of the compound. "Suitable substituents" include oxo (=0), halogen, cyano, NR 8 R 9 , carboxyl, thiol, hydroxyl, ester (e.g., -C 1-6 alkyl-C(=0)-OC 1-6 alkyl), C 1-6 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 are 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 (e.g., -C 1-6 alkyl-C(=0)-OC 1-6 alkyl).

[0379] The term "substituted" means that one or more (e.g., 1, 2, 3, 4, or 5) hydrogens on the designated compound or structure are replaced with a substituent, provided that the designated atom's normal valence is not exceeded, and that 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, the substituents each independently are constructed from one or more of the following structures: NR 8 R 9 , -O-, -S-, -NR'-, halogen, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -CN, =O, C1-C6(alkyl)ene, C1-C6halo(alkyl)ene, C1-C6alkoxy, C2-C6(alkenyl)ene, C2-C6(alkynyl)ene, 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 defined above. For example, the substituents can be suitable substituents as described above.

[0380] If a functional group or structure is described as "substituted or unsubstituted," that functional group or structure can be (1) unsubstituted or (2) substituted.

[0381] As used herein, the term "DAR" or "drug 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 0-10, e.g., an integer from 1-10; 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 0-10, e.g., an integer or decimal number from 1-10.

[0382] Unless explicitly stated otherwise, numerical values in the present application are modified by the term "about." The term "about" means within ±10% of the stated value, preferably within ±5%, and more preferably within ±2%. BRIEF DESCRIPTION OF DRAWINGS

[0383] The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and serve to explain the principles of the application, and are not intended to limit the application. In the drawings:

[0384] 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.

[0385] Figure 2 shows the changes in body weight of mice in each treatment group in the human breast cancer cell JIMT-1 CDX model. DETAILED DESCRIPTION

[0386] The present invention will be further described below by describing specific embodiments, but this is not intended to limit the present invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic idea and scope of the present invention.

[0387] The information of the sequences involved in the present invention is described in the following table:

[0388] The abbreviations used in this document have the following meanings:

[0389] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0390] Nuclear magnetic resonance (1H NMR) measurements were performed using a Bruker 400 MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0391] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0392] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0393] Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0394] Intermediate Preparation Example 1: Preparation of 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosane-24-ynoic acid (INT-1)

[0395] Step 1: Preparation of 1-amino-3,6,9,12,15-pentaoxoctadecane-18-oic acid (INT-1-2)

[0396] Dissolve 2,2-dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azatricosane-23- acid (0.50 g, 1.22 mmol) in dichloromethane (3 mL), add trifluoroacetic acid (3 mL), stir the reaction at room temperature for 3 hours. After the reaction is completed, the reaction solution is directly concentrated under reduced pressure to obtain the crude trifluoroacetate salt of the title compound (650 mg, 1.21 mmol).

[0397] The structural characterization data thereof are as follows:

[0398] MS m / z (ESI): 310.2 [M+H] +

[0399] Step two: Preparation of 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16- pentaaoxa-19-azapentacosan-24-ynoic acid (INT-1)

[0400] 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), and then add 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (441.91 mg, 1.21 mmol) portionwise, stir the reaction at room temperature for 1 hour. After the reaction is completed, the reaction solution is directly purified by reverse-phase column chromatography (acetonitrile / 0.05% aqueous sodium bicarbonate solution = 0-60%) and freeze-dried to obtain the title compound (646 mg, 1.15 mmol).

[0401] The structural characterization data thereof are as follows:

[0402] MS m / z (ESI): 560.3 [M+H] +

[0403] Intermediate Preparation Example Two: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-alanyl-L-alanine (INT-2)

[0404] Step one: Preparation of L-alanyl-L-alanine (INT-2-1)

[0405] 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 completed, the reaction solution is concentrated under reduced pressure to remove the solvent to obtain the title compound (160 mg, 370.93 μmol) in light yellow.

[0406] The structural characterization data thereof are as follows:

[0407] MS m / z (ESI): 161.1 [M+H] +

[0408] Step two: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-alanyl-L-alanine (INT-2)

[0409] Dissolve 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) in N,N-dimethylacetamide (2 mL), add N,N-diisopropylethylamine (266.33 mg, 2.06 mmol), and react at 20 °C for 1 hour. After the reaction is completed, freeze-drying after column chromatography (C18, water / acetonitrile = 2 / 1) to obtain the title compound (125 mg, 304.55 μmol).

[0410] The structural characterization data thereof are as follows:

[0411] MS m / z (ESI): 428.1 [M+H2O] +

[0412] Intermediate Preparation Example Three: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)glycylglycine (INT-3)

[0413] Dissolve 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) in dimethyl sulfoxide (10 mL), and stir at room temperature for 2 hours. After the reaction is completed, freeze-drying after column chromatography (C18, water / acetonitrile = 2 / 1) to obtain the title compound (2.52 g, 6.59 mmol).

[0414] The structural characterization data thereof are as follows:

[0415] MS m / z (ESI): 383.2 [M+H]+

[0416] Preparation Example Four: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoyl)glycylglycyl-L-phenylalanine (INT-4)

[0417] Step One: Preparation of glycylglycyl-L-phenylalanine (INT-4-2)

[0418] ((Benzoyl)oxy)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).

[0419] The structural characterization data thereof are as follows:

[0420] MS m / z (ESI): 280.2 [M+H] +

[0421] Step Two: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)glycylglycyl-L-phenylalanine (INT-4)

[0422] 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 rapid column chromatography (C18, water / acetonitrile = 2 / 1).

[0423] The structural characterization data thereof are as follows:

[0424] MS m / z (ESI): 530.3 [M+H] +

[0425] Preparation Example Five: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (INT-5)

[0426] Step one: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino) propanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5)

[0427] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-2- (((tert-butyldimethylsilyl)oxy)methyl)benzyl)(methyl)carbamate (6.45 g, 8.25 mmol) was dissolved in DMF (60 mL), hydrofluoric acid pyridine salt (15.40 g, 155.39 mmol) was added dropwise, and the reaction was stirred at room temperature for 24 hours. After the reaction was completed, the reaction solution was added to water and extracted with ethyl acetate. The organic phase was washed with brine, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10-100%) and concentrated under reduced pressure again to obtain the title compound (4.4 g, 8.09 mmol).

[0428] The structural characterization data thereof are as follows:

[0429] MS m / z (ESI): 561.4 [M+H2O] + ; 526.4 [M-H2O+H] +

[0430] Step two: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino) propanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-5)

[0431] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-2- (((tert-butyldimethylsilyl)oxy)methyl)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, and then bis(4-nitrophenyl) carbonate (671.54 mg, 2.21 mmol) was added, and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was added to water, extracted with ethyl acetate, and the organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The title compound (1.3 g, 1.83 mmol) was obtained by purifying the crude product by silica gel column chromatography (petroleum ether / ethyl acetate = 0-80%) and concentrating under reduced pressure.

[0432] The structural characterization data thereof are as follows:

[0433] MS m / z (ESI): 726.1 [M+H2O] + ; 731.3 [M+Na] +

[0434] Example 1: Preparation of 2,2',2"-(10-(2-((2-((((2-(dimethylamino)ethyl))(((8S,9R)-5- fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)aminocarbonyl)oxy)methyl)-5-((2S,5S)-5- isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22- pentoaoxa-3,6,25-triazatritriacontan-30-ynamido)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7- tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-1)

[0435] Step 1: Preparation of (8S,9R)-2-(((2-(dimethylamino)ethyl)amino)methyl)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazine- 3-one (A-1-2)

[0436] (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H- pyrido[4,3,2-de]phthalazine-3-one (300 mg, 788.75 µmol) and N',N'-dimethylethane-1,2-diamine (695.29 mg, 7.89 mmol, 861.57 µL) were dissolved in ethanol (15 mL), then 37% formaldehyde (640.08 mg, 7.89 mmol) was added, and the reaction was stirred at 80 ℃ for 4 hours. After the reaction was completed, the reaction solution was reduced to room temperature and concentrated under reduced pressure. The crude product was added with ethyl acetate, then washed with water for 10 times, washed with brine for 5 times, dried with anhydrous sodium sulfate, and concentrated under reduced pressure at room temperature to obtain the crude product of the title compound (380 mg, 608.93 µmol).

[0437] The structural characterization data thereof are as follows:

[0438] MS m / z (ESI): 481.3 [M+H] +

[0439] Step 2: Preparation of (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)- 2-((((2-(dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl) aminoformyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (A-1-3)

[0440] To a solution of (8S,9R)-2-(((2-(dimethylamino)ethyl)amino)methyl)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrrolo[4,3,2- de]phthalazine-3-one crude (380.00 mg, 474.49 µmol) and (S)-(5-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl) benzyl)(methyl)carbamic acid allyl ester (380 mg, 536.18 µmol) and HOBt (83.35 mg, 616.84 µmol) in DMF (5 mL), DIPEA (122.65 mg, 948.99 µmol) was added and stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the formate salt of the title compound (170 mg, 155.09 µmol).

[0441] The structural characterization data thereof are as follows:

[0442] MS m / z (ESI): 1050.7 [M+H] + ; 526.0 [M / 2+H] +

[0443] The preparation method thereof is as follows:

[0444] Column: Waters Sunfire Prep C18 OBD (5 µm*19 mm*150 mm)

[0445] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0446] Step 3: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3- methylbutanamido)propanamido)-2-((((2-(dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2- de]phthalazine-2(7H)-yl)methyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (A-1-5)

[0447] Allyl (5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-2-((((2- (dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5- yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl)carbamoyl)oxy)methyl) benzyl)(methyl)carbamate (200 mg, 190.46 μmol) was dissolved in DMF (4 mL), diethylamine (282.80 mg, 3.87 mmol, 0.4 mL) was added, and the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the solvent was removed by lyophilization to obtain the crude product of the title compound (157 mg, 189.64 μmol), which was then directly used in the next reaction.

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

[0449] MS m / z (ESI): 829.3 [M+H]+; 414.8 [M / 2+H] +

[0450] Step 4: Preparation of (5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3- methylbutanamido)propanamido)-2-((((2-(dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2- de]phthalazine-2(7H)-yl)methyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (A-1-5)

[0451] ((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (128.72 mg, 379.28 umol) was dissolved in DMF (1 mL), DIPEA (73.53 mg, 568.93 umol) and HATU (144.21 mg, 379.28 umol) were added, the reaction was stirred at room temperature for 0.5 hours. Then to the reaction solution was added a solution of crude (5-((S)-2-aminopropanamido)-2-((((2-(dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl)aminocarbonyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (157 mg, 189.64 umol) in DMF (1 mL), the reaction was continued to stir for 0.5 hours. After the reaction was completed, the reaction solution was directly used in the next step reaction without treatment in the form of a solution.

[0452] The structural characterization data thereof are as follows:

[0453] MS m / z (ESI): 1150.6 [M+H] + ; 575.5 [M / 2+H] +

[0454] Step five: preparation of (5-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-((((2-(dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl)aminocarbonyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (A-1-6)

[0455] To the reaction mixture of Step 1 containing (5-((S)-2-((S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-methylbutyramido)propionamido)-2-((((2- (dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl) carbamoyl)oxy)methyl)benzyl)(methyl)carbamic acid allyl ester (217 mg, 188.82 μmol) and DMF (4 mL) was added diethylamine (707.00 mg, 9.67 mmol) and the reaction was stirred at room temperature for 1 hour. The reaction mixture was filtered and then purified directly by reverse phase column chromatography (acetonitrile / 0.05% aqueous ammonium bicarbonate = 5-90%) and lyophilized to give the title compound (175 mg, 188.78 μmol).

[0456] The structural characterization data thereof are as follows:

[0457] MS m / z (ESI): 927.5 [M+H]+; 464.3 [M / 2+H] +

[0458] Step six: Preparation of 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((2S,5S)-5- isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22- pentoaoxa-3,6,25-triazatritriacontan-30-ynoylamido)benzyl(2-(dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9- dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl)carbamic acid ester (A-1-7)

[0459] Allyl (5-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)-2-((((2- (dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl) carbamimidoyl)oxy)methyl)benzyl)(methyl)carbamate (30 mg, 32.36 μmol) was added, and the reaction was stirred at room temperature for 100 min. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography to give the formate salt of the title compound (46 mg, 30.37 μmol) after lyophilization.

[0460] The structural characterization data thereof are as follows:

[0461] MS m / z (ESI): 1470.7 [M+H] + ; 735.0 [M / 2+H] +

[0462] The preparation method thereof is as follows:

[0463] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0464] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0465] Step seven: Preparation of (2-(dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2- de]phthalazine-2(7H)-yl)methyl)(4-((2S,5S)-5-isopropyl-2-methyl-31-(2- (methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25- triazatriacontan-30-ynoylamido)-2-((methylamino)methyl)benzyl)carbamic acid allyl ester (A-1-8)

[0466] Formic acid (24.40 mg, 530.14 umol) and N-methylmorpholine (36.80 mg, 363.82 umol) were dissolved in DMF (1.5 mL), 2-((((allyloxy)carbonyl)(methyl)amino)methyl)-4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatriacontan-30-ynoyl)benzyl(2-(dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl)carbamate (26 mg, 17.70 umol) was added, then tetrakis(triphenylphosphine)palladium (20.46 mg, 17.70 umol) was added, and the reaction was stirred at room temperature for 3 hours under nitrogen. 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 (10 mg, 7.22 umol).

[0467] The structural characterization data thereof are as follows:

[0468] MS m / z (ESI): 1385.6 [M+H] + ; 693.0 [M / 2+H] +

[0469] The preparation method thereof is as follows:

[0470] Column: Waters Sunfire Prep C18 OBD (5 um*19 mm*150 mm)

[0471] Mobile phase A: acetonitrile; mobile phase B: water (0.05% ammonium bicarbonate)

[0472] Step eight: preparation of 2,2',2”-(10-(2-((2-((((2-(dimethylamino)ethyl))(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl)aminoformyl)oxy)methyl)-5-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatritriacontan-30-ynamido)benzyl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-1)

[0473] A solution of 4-((2S,5S)-5-isopropyl-2-methyl-31-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,26-trioxo-10,13,16,19,22-pentaoxa-3,6,25-triazatritriacontan-30-ynamido)-2-((methylamino)methyl)benzyl (2-(dimethylamino)ethyl)(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine-2(7H)-yl)methyl)aminoformate (5 mg, 3.61 µmol) in DMF (1 mL) was added DIPEA (16 mg, 123.80 µmol), followed by 2,2',2'-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (7.24 mg, 14.45 µmol), and stirred at 25 °C for 6 h. 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 (2.97 mg, 1.34 µmol).

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

[0475] MS m / z (ESI): 1772.2 [M+H] +

[0476] The preparation method thereof is as follows:

[0477] Column: Waters Sunfire Prep C18 OBD (5 µm*19 mm*150 mm)

[0478] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0479] Example Two: Preparation of N-((S)-1-(((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)- 1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (B-1)

[0480] Step One: Preparation of (9H-fluoren-9-yl)methyl (S)-(1-((chloromethyl)amino)- 1-oxopropan-2-yl)carbamate (B-1-2)

[0481] To 1,2-dichloroethane (4 mL) was added methyl (S)-(2-((((9H-fluoren-9-yl)methoxy) carbonyl)amino)propanamido)acetate (50 mg, 130.75 μmol), then TMSCl (142.05 mg, 1.31 mmol) was added dropwise. After addition, the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to give the crude product of the title compound (46 mg, 128.20 μmol), which was used directly in the next step without purification.

[0482] Step Two: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)carbamate (B-1-3)

[0483] (8S,9R)-5-Fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)- 2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazine-3-one (30 mg, 78.87 μmol) was dissolved in THF (1.5 mL) and cooled to -78 °C under nitrogen atmosphere. Then NaHMDS (78.87 μmol, 2M in THF) was added dropwise and the system turned yellow. The reaction was stirred for 20 minutes at -78 °C and then (9H-fluoren-9-yl)methyl (S)-(1- ((chloromethyl)amino)-1-oxopropan-2-yl)carbamate (42.45 mg, 118.31 μmol) in THF (1.5 mL) was added dropwise. After 20 minutes at -78 °C, the reaction was stirred for 1 hour at -10 °C. After the reaction was completed, water was added to the reaction mixture 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 title compound (70 mg, 69.73 μmol). The crude product was used directly in the next step without purification.

[0484] The structural characterization data thereof are as follows:

[0485] MS m / z (ESI): 703.3 [M+H] +

[0486] Step three: Preparation of (S)-2-amino-N-(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1- methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazin-2(7H)- yl)methyl)propionamide (B-1-4)

[0487] (9H-fluoren-9-yl)methyl ((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl- 1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazin-2(7H)-yl)methyl) amino)-1-oxopropan-2-yl)carbamate (70 mg, 69.73 μmol) was dissolved in DMF (1 mL) and diethylamine (0.2 mL) was added. The reaction was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction mixture was directly purified by reverse phase column chromatography (acetonitrile / 0.05% aqueous ammonium bicarbonate solution = 0-95%) and freeze-dried to obtain the title compound (20 mg, 41.63 μmol).

[0488] The structural characterization data thereof are as follows:

[0489] MS m / z (ESI): 481.2 [M+H] +

[0490] Step four: Preparation of N-((S)-1-(((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4- fluorophenyl))-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)- 1-oxopropan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (B-1)

[0491] (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-alanine-L-alanine (17.09 mg, 41.63 µmol) was dissolved in DMF (1 mL), HATU (17.41 mg, 45.79 µmol) and DIPEA (16.14 mg, 124.88 µmol) were added, the reaction was stirred at room temperature for 0.5 hours, then (S)-2-amino-N-(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)propanamide (20 mg, 41.63 µmol) was added, the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (11.37 mg, 12.19 µmol).

[0492] The structural characterization data thereof are as follows:

[0493] MS m / z (ESI): 873.7 [M+H] + ; 437.5 [M / 2+H] +

[0494] The preparation method thereof is as follows:

[0495] Column: Waters Sunfire Prep C18 OBD (5 µm*19 mm*150 mm)

[0496] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0497] Example 3: Preparation of N-((S)-7-benzyl-l-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l- methyl-lH-l,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)- 3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex- 5-ynamide (B-2)

[0498] Step one: Preparation of (9H-fluoren-9-yl)methyl (2-((chloromethyl)amino)-2- oxoethyl)carbamate (B-2-2) To 1,2-dichloroethane (10 mL) was added methyl (2-((((9H-fluoren-9-yl)methoxy) carbonyl)amino)acetamido)acetate (410 mg, 1.11 mmol), then TMSCl (852.29 mg, 11.13 mmol) was added dropwise. The reaction was stirred at room temperature for 1 hour, and the system was gradually dissolved. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to obtain the crude product of the title compound (383 mg, 1.11 mmol), which was used directly in the next step without purification.

[0499] Step two: Preparation of (9H-fluoren-9-yl)methyl (2-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine- 2(7H)-yl)methyl)amino)-2-oxoethyl)carbamate (B-2-3)

[0500] (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-2,7,8,9-tetrahydro- 3H-pyrido[4,3,2-de]phthalazine-3-one (230 mg, 604.71 μmol) was dissolved in THF (8 mL) and cooled to -78 °C under nitrogen protection. Then NaHMDS (604.71 μmol, 2M in THF) was added dropwise, and the system turned yellow. After stirring for 20 minutes, (9H-fluoren-9-yl)methyl (S)-(l- ((chloromethyl)amino)-l-oxopropan-2-yl)carbamate (312.75 mg, 907.06 μmol) in THF (8 mL) was added dropwise. After the addition was completed, the system was warmed to 0 °C and stirred for 60 minutes. After the reaction was completed, water was added to the reaction solution, and ethyl acetate was extracted. The organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (416 mg, 604.05 μmol), which was used directly in the next step without purification.

[0501] The structural characterization data thereof are as follows:

[0502] MS m / z (ESI): 689.2 [M+H] +

[0503] Step three: Preparation of 2-amino-N-(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1- methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine- 2(7H)-yl)methyl)acetamide (B-2-4)

[0504] The crude (9H-fluoren-9-yl)methyl (2-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1- methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine- 2(7H)-yl)methyl)amino)-2-oxoethyl)carbamate (416 mg, 604.05 μmol) was dissolved in DMF (4 mL), diethylamine (1 mL) was added, and the reaction was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was directly purified by reverse-phase column chromatography (acetonitrile / 0.05% aqueous ammonium bicarbonate solution = 0~95%) and freeze-dried to obtain the title compound (112 mg, 168.08 μmol).

[0505] The structural characterization data thereof are as follows:

[0506] MS m / z (ESI): 467.1 [M+H] +

[0507] Step four: Preparation of N-((S)-7-benzyl-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1- methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrrolo[4,3,2-de]phthalazine- 2(7H)-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-6-(2-(methylsulfonyl) pyrimidin-5-yl)hex-5-ynamide (B-2)

[0508] (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)glycylglycyl-L-phenylalanine (25 mg, 47.21 μmol) was dissolved in DMF (1.5 mL), DIPEA (16 mg, 123.80 μmol) was added, then HATU (16.98 mg, 47.17 μmol) was added, stirred at room temperature for 10 minutes. 2-amino-N-(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)acetamide (28.57 mg, 42.88 μmol) was added, and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (20.40 mg, 19.52 μmol).

[0509] The structural characterization data thereof are as follows:

[0510] MS m / z (ESI): 978.5 [M+H] + ; 489.6 [M / 2+H] +

[0511] The preparation method thereof is as follows:

[0512] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

[0513] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0514] Example Four: Preparation of N-((4S,7S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl))-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-7-isopropyl-4-methyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)amidine (B-3)

[0515] Step 1: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)- 3-methyl-1-oxobutan-2-yl)carbamate (B-3-1)

[0516] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine (38.57 mg, 113.65 μmol) was dissolved in DMF (1 mL), HATU (41.21 mg, 114.47 μmol) and DIPEA (26.90 mg, 208.13 μmol) were added, the reaction was stirred at room temperature for 10 minutes, then (S)-2-amino-N-(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl) propanamide (50 mg, 104.06 μmol) was added, the reaction was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was directly used for the next step reaction without any treatment.

[0517] The structural characterization data thereof are as follows:

[0518] MS m / z (ESI): 802.6 [M+H] +

[0519] Step 2: Preparation of (S)-2-amino-N-((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1- methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine- 2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (B-3-2)

[0520] To the reaction solution of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((((8S,9R)-5- fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)-3- methyl-1-oxobutan-2-yl)carbamate (83 mg, 103.51 μmol) and DMF (1.5 mL) was added diethylamine (0.5 mL) and stirred at room temperature for 20 min. After the reaction was completed, the reaction solution was freeze-dried to remove the solvent to obtain the crude product of the title compound (59 mg, 101.79 μmol), which was used directly in the next reaction without purification.

[0521] The structural characterization data thereof are as follows:

[0522] MS m / z (ESI): 580.3 [M+H] +

[0523] Step three: preparation of N-((4S,7S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1- methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)- yl)-7-isopropyl-4-methyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide (B-3)

[0524] (6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)glycylglycine (39.58 mg, 103.52 μmol) was dissolved in DMF (2 mL), and DIPEA (26.76 mg, 207.04 μmol) and HATU (37.27 mg, 103.52 μmol) were added, and the reaction was stirred at room temperature for 15 min. Then the solution was added to a reaction solution containing (S)-2-amino-N-((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (59.00 mg, 101.79 μmol), and the reaction was stirred at room temperature for 0.5 h. 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 (12.39 mg, 12.26 μmol).

[0525] The structural characterization data thereof are as follows:

[0526] MS m / z (ESI): 944.5 [M+H] + ; 472.9 [M / 2+H] +

[0527] The preparation method thereof is as follows:

[0528] Column: Waters Sunfire Prep C18 OBD (5 pm*19 mm*150 mm)

[0529] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0530] Example Five: Preparation of N-((S)-1-(((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4- fluorophenyl))-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)- 1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-1-(6-(2-(methylsulfonyl)pyrimidin- 5-yl)hex-5-ynoylamino)-3,6,9,12,15-pentaoxaoctadecan-18-amide (C-1)

[0531] Step One: Preparation of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((S)-1-((((8S,9R)-5- fluoro-8-(4-fluorophenyl))-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9- dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan- 2-yl)amino)-1-oxopropan-2-yl)amino)methyl)carbamate (C-1-1)

[0532] (S)-2-amino-N-(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl) methyl) propanamide (15 mg, 31.22 μmol) and ((9H-fluoren-9-yl)methoxy)carbonyl)-L- alanine-L-alanine (11.94 mg, 31.22 μmol) were dissolved in DMF (1 mL), DIPEA (16.14 mg, 124.88 μmol) was added, then PyBOP (16.25 mg, 31.22 μmol) was added, the reaction was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was directly used for the next step reaction without any treatment.

[0533] The structural characterization data thereof are as follows:

[0534] MS m / z (ESI): 845.5 [M+H] +

[0535] Step two: preparation of (S)-2-amino-N-((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2- de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2- yl)propanamide (C-1-2)

[0536] Directly to the next step containing (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(((S)-1- (((8S,9R)-5-fluoro-8-(4)-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9- dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl) amino)-1-oxopropan-2-yl)carbamate (26 mg, 30.77 μmol) and DMF (1 mL) of the previous reaction solution, drop diethylamine (0.2 mL), stir the reaction at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was directly purified by reverse phase column chromatography (acetonitrile / 0.05% aqueous ammonium bicarbonate solution = 0~90%) and freeze-dried to obtain the title compound (22 mg, 28.27 μmol).

[0537] The structural characterization data thereof are as follows:

[0538] MS m / z (ESI): 623.3 [M+H]+

[0539] Step 3: Preparation of N-((S)-1-(((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4- fluorophenyl))-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)- 1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-1-(6-(2-(methylsulfonyl)pyrimidin- 5-yl)hex-5-ynoyl)-3,6,9,12,15-pentaoxaoctadecan-18-amide (C-1)

[0540] (S)-2-amino-N-((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl))-9-(1- methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine- 2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)propanamide (15.00 mg, 24.09 µmol) and 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16- pentaoxa-19-azapentacosan-24-yne acid (13.48 mg, 24.09 µmol) were dissolved in DMF (1 mL), DIPEA (6.23 mg, 48.18 µmol) and PyBOP (13.79 mg, 26.50 µmol) were added, and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (12.39 mg, 12.26 µmol).

[0541] The structural characterization data thereof are as follows:

[0542] MS m / z (ESI): 1164.6 [M+H] + ; 583.0 [M / 2+H] +

[0543] The preparation method thereof is as follows:

[0544] Column: Waters Sunfire Prep C18 OBD (5 µm*19 mm*150 mm)

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

[0546] Example Six: Preparation of N-((S)-1-(((S)-1-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxopropan-2-yl)amino)- 3-methyl-1-oxobutan-2-yl)-1-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5- ynamido)-3,6,9,12,15-pentaoxaoctadecan-18-amide (C-2)

[0547] (S)-2-amino-N-((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H- 1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)- yl)methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (20.00 mg, 34.51 μmol) and 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16- pentaaoxa-19-azapentacosan-24-ynamide (19.31 mg, 34.51 μmol) were dissolved in DMF (1 mL), DIPEA (8.92 mg, 69.01 μmol) and PyBOP (17.96 mg, 34.51 μmol) were added, and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (17.97 mg, 15.24 μmol).

[0548] The structural characterization data thereof are as follows:

[0549] MS m / z (ESI): 1126.6 [M+H] + ; 561.4 [M / 2+H] +

[0550] The preparation method thereof is as follows:

[0551] Column: Waters Sunfire Prep C18 OBD (5 μm*19 mm*150 mm)

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

[0553] Example Seven: Preparation of N-((4S,7S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1- methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine- 2(7H)-yl)-7-isopropyl-4-methyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)- 1-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl-amido)-3,6,9,12,15- pentaaoxaoctadecan-18-amide (C-3)

[0554] Step One: Preparation of (9H-fluoren-9-yl)methyl ((4S,7S)-1-((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2- de]phthalazine-2(7H)-yl)-7-isopropyl-4-methyl-3,6,9,12-tetraoxo-2,5,8,11- tetraazatridecan-13-yl)carbamate (C-3-1)

[0555] (S)-2-amino-N-((S)-1-((((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4- triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)- 1-oxopropan-2-yl)-3-methylbutanamide (22 mg, 37.96 µmol) and (((9H-fluoren-9- yl)methoxy)carbonyl)glycylglycine (13.45 mg, 37.96 µmol) were dissolved in DMF (1 mL), DIPEA (4.91 mg, 37.96 µmol) and PyBOP (19.75 mg, 37.96 µmol) were added, and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was used directly in the next step without any treatment.

[0556] The structural characterization data thereof are as follows:

[0557] MS m / z (ESI): 916.5 [M+H] +

[0558] Step two: Preparation of (S)-2-(2-(2-aminoacetamido)acetamido)-N-((S)-1-((((8S,9R)- 5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxoprop-2-yl)-3-methylbutanamide (C-3-2)

[0559] Directly to the previous step containing (9H-fluoren-9-yl)methyl ((4S,7S)-1-((8S,9R)- 5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)-7-isopropyl-4-methyl-3,6,9,12-tetraoxo-2,5,8,11- tetraazatridecan-13-yl)carbamate (34 mg, 37.12 μmol) and DMF (1 mL) drop diethylamine (0.5 mL), after adding, stirring the reaction at room temperature for 0.5 hours. After the reaction was completed, the reaction liquid was directly purified by reverse phase column chromatography (acetonitrile-0.05% aqueous ammonium bicarbonate solution = 0-90%) and freeze-dried to obtain the title compound (23 mg, 33.16 μmol).

[0560] The structural characterization data thereof are as follows:

[0561] MS m / z (ESI): 694.3 [M+H] + ; 1389.4 [M+H] +

[0562] Step three: Preparation of N-((4S,7S)-1-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl- 1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)-7- isopropyl-4-methyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-1-(6-(2- (methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamino)-3,6,9,12,15-pentaoxaoctadecan-18- amide (C-3)

[0563] (S)-2-(2-(2-aminoacetamido)acetamido)-N-((S)-1-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-1-oxoprop-2-yl)-3- methylbutanamide (23.00 mg, 33.16 μmol) and 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosan-24-yne acid (18.55 mg, 33.16 μmol) were dissolved in DMF (1 mL), PyBOP (17.25 mg, 33.16 μmol) and DIPEA (4.29 mg, 33.16 μmol) were added, and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (21.36 mg, 16.34 μmol).

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

[0565] MS m / z (ESI): 1236.7 [M+H] + ; 618.5 [M / 2+H] +

[0566] The preparation method thereof is as follows:

[0567] Column: Waters Xbridge Prep C18 OBD (5 μm*19 mm*150 mm)

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

[0569] Example Eight: Preparation of N-((S)-7-benzyl-1-((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)-3,6,9,12-tetraoxo-2,5,8,11- tetraazatridecan-13-yl)-1-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5- ynylcarbamoyl)-3,6,9,12,15-pentaoxaoctadecan-18-amide (C-4)

[0570] Step one: Preparation of (9H-fluoren-9-yl)methyl ((S)-7-benzyl-l-((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2- de]phthalazine-2(7H)-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)carbamate (C-4-1)

[0571] Dissolve 2-amino-N-(((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5- yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)acetamide (25 mg, 53.60 μmol) and (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (26.88 mg, 53.60 μmol) in DMF (2 mL), add PyBOP (27.89 mg, 53.60 μmol) and DIPEA (16 mg, 123.80 μmol), stir the reaction at room temperature for 1 hour. After the reaction is completed, the reaction solution is directly used for the next step reaction without any treatment.

[0572] The structural characterization data thereof are as follows:

[0573] MS m / z (ESI): 950.5 [M+H] +

[0574] Step two: Preparation of (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2- de]phthalazine-2(7H)-yl)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (C-4-2)

[0575] To the reaction solution of (9H-fluoren-9-yl)methyl ((S)-7-benzyl-l-((8S,9R)-5- fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan- 13-yl)carbamate (50 mg, 52.63 μmol) and DMF (1 mL) in the last step, dropwise added diethylamine (0.5 mL), after addition, stirred the reaction at room temperature for 0.5 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove diethylamine to obtain a crude product, which was purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (20 mg, 27.48 μmol).

[0576] The structural characterization data thereof are as follows:

[0577] MS m / z (ESI): 728.3 [M+H] +

[0578] The preparation method thereof is as follows:

[0579] Column: Waters Xbridge Prep C18 OBD (5 μm*19 mm*150 mm)

[0580] Mobile phase A: acetonitrile; mobile phase B: water (0.05% ammonium bicarbonate)

[0581] Step three: preparation of N-((S)-7-benzyl-l-((8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l- methyl-lH-l,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H-pyrido[4,3,2-de]phthalazine-2(7H)- yl)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)-l-(6-(2-(methylsulfonyl)pyrimidin- 5-yl)hex-5-ynoylaminol-3,6,9,12,15-pentaoxaoctadecan-18-amide (C-4)

[0582] (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((((8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-3-oxo-8,9-dihydro-3H- pyrido[4,3,2-de]phthalazine-2(7H)-yl)methyl)amino)-2-oxoethyl)-3- phenylpropanamide (20.00 mg, 27.48 μmol) and 25-(2-(methylsulfonyl)pyrimidin-5-yl)-20-oxo-4,7,10,13,16-pentaoxa-19-azapentacosan-24-yne acid (15.38 mg, 27.48 μmol) were dissolved in DMF (1.5 mL), DIPEA (7.10 mg, 54.97 μmol) and PyBOP (14.30 mg, 27.48 μmol) were added, and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (21.36 mg, 16.34 μmol).

[0583] The structural characterization data thereof are as follows:

[0584] MS m / z (ESI): 1270.7 [M+H] + ; 635.5 [M / 2+H] +

[0585] The preparation method thereof is as follows:

[0586] Column: Kinetex@5 μm XB-C18 100A (5 μm*21.2 mm*150 mm)

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

[0588] Example Nine: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinoline-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide (Z-1)

[0589] Step One:

[0590] 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.

[0591] The preparation method is as follows:

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

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

[0594] Step two:

[0595] 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 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.

[0596] The preparation method is as follows:

[0597] Column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

[0598] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0599] Step three:

[0600] 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.

[0601] The structural characterization data are as follows:

[0602] MS m / z (ESI): 1002.4 [M+H]+.

[0603] The isolation and purification method is as follows:

[0604] Column: Waters SunFire Prep C18 OBD (5 pm*19 mm*150 mm)

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

[0606] 1H 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).

[0607] Preparation of antibody drug conjugate

[0608] 1. Preparation of Trastuzumab-A-1

[0609] Take 1.088 mL of trastuzumab (14.7 mg / mL), dilute with 54 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.60 with 1 M Na2HPO4solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 60.6 μL, pH 7.60) solution and mix well, and stand at room temperature for 1.5 h. Then add a drug-linker A-1 (163 μL, 10 mM, 12-fold equivalent of antibody) solution dissolved in dimethyl sulfoxide and mix well, 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-1). The DAR value is 7.99 as determined by mass spectrometry.

[0610] 2. Preparation of Trastuzumab-B-1

[0611] 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 B-1 (252 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 a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain an antibody drug conjugate (i.e., Trastuzumab-B-1). Mass spectrometry determines the DAR value to be 7.98.

[0612] 3. Preparation of Trastuzumab-B-2

[0613] 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 B-2 (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 a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain an antibody drug conjugate (i.e., Trastuzumab-B-2). Mass spectrometry determines the DAR value to be 8.

[0614] 4. Preparation of Trastuzumab-B-3

[0615] 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 B-3 (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 a 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain an antibody drug conjugate (i.e., Trastuzumab-B-3). Mass spectrometry determines the DAR value to be 7.98.

[0616] 5. Preparation of Trastuzumab-C-1

[0617] 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 the drug-linker C-1 (21.13 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 the antibody drug conjugate (i.e. Trastuzumab-C-1). The DAR value is 8.0 determined by mass spectrometry.

[0618] 6. Preparation of Trastuzumab-C-2

[0619] 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 the drug-linker C-2 (20.88 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 the antibody drug conjugate (i.e. Trastuzumab-C-2). The DAR value is 8.0 determined by mass spectrometry.

[0620] 7. Preparation of Trastuzumab-C-3

[0621] 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, mix with 10 mM TCEP (tris(2-carboxyethyl)phosphine, 11.36 μL, pH 7.60) solution, and let stand at room temperature for 1.5 h. Then add the drug-linker C-3 (21.1 μL, 10 mM, 10-fold equivalent of antibody) solution dissolved in dimethyl sulfoxide, mix well, and let 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 the antibody drug conjugate (i.e., Trastuzumab-C-3). The DAR value is determined to be 8.0 by mass spectrometry.

[0622] 8. Preparation of Trastuzumab-C-4

[0623] 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 Na2HPO4solution, mix with 10 mM TCEP (tris(2-carboxyethyl)phosphine, 18.9 μL, pH 7.60) solution, and let stand at room temperature for 1.5 h. Then add the drug-linker C-4 (36.8 μL, 10 mM, 10-fold equivalent of antibody) solution dissolved in dimethyl sulfoxide, mix well, and let 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 the antibody drug conjugate (i.e., Trastuzumab-C-4). The DAR value is determined to be 7.94 by mass spectrometry.

[0624] 9. Preparation of Trastuzumab-Z-1

[0625] Take 0.957 mL Trastuzumab antibody (20.9 mg / mL), dilute with 52.0 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, 75.8 μL, pH 7.60) solution, and let stand at room temperature for 1.5 h. Then add the Z-1 (143.5 uL, 10 mM, 10-fold equivalent of antibody) solution dissolved in dimethyl sulfoxide, mix well, and let 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 the antibody drug conjugate (i.e., Trastuzumab-Z-1). The DAR value is determined to be 8.0 by mass spectrometry.

[0626] Biological evaluation

[0627] I. Evaluation of the tumor growth inhibition effect of antibody drug conjugate on subcutaneously transplanted tumor model in mice

[0628] The ADC of the present application was administered to the CDX model of subcutaneously transplanted human breast cancer cells JIMT-1 in mice by tail vein injection, respectively, and the tumor volume and animal body weight change were measured twice a week to calculate the tumor inhibition effect of the ADC of the present application on tumor-bearing mice.

[0629] Test drug

[0630] An appropriate amount of ADC was administered at 3 mg / kg and 10 mg / kg, and the specific administration dose is shown below. The mother solution (20 mM histidine buffer solution at pH 6.0 of each antibody drug conjugate obtained in the preparation example of the antibody drug conjugate) was diluted with 0.9% NaCl injection solution to the administration solution. 0.9% NaCl injection solution was used as a vehicle control (Vehicle).

[0631] Experimental animals and cell lines

[0632] NOD SCID mice (Jiangsu Jicui Yekang Biotechnology Co., Ltd.)

[0633] Human breast cancer cells JIMT-1 (Nanjing Kebai)

[0634] Experimental grouping and evaluation method

[0635] The tumor-bearing mice with an average tumor volume of about 150 mm 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 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 twice a week with a vernier caliper after administration, and the tumor volume was calculated according to the following calculation formula: V = 0.5a x b 2 , wherein a and b represent the long diameter and short diameter of the tumor, respectively. The animal death was observed and recorded every day.

[0636] The tumor growth inhibition rate TGI (%) was calculated using the following formula to evaluate the tumor inhibition effect of the ADC:

[0637] V T末 >V T0 , TGI (%) = [1-(V T末 -V T0 ) / (V C末 -V C0 )]*100%, or

[0638] V T末 ≤V T0 , TGI(%) = [1-(V T末 -V T0 ) / V T0 ]*100%.

[0639] wherein V T末 : mean tumor volume at the end of the experiment in the treatment group

[0640] V T0 : mean tumor volume at the beginning of the administration in the treatment group

[0641] V C末 : mean tumor volume at the end of the experiment in the negative control group

[0642] V C0 : mean tumor volume at the beginning of the administration in the negative control group

[0643] The relative proliferation rate T / C (%) of the tumor was calculated using the following formula, which was used to evaluate the anti-tumor effect of the ADC: T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 )*100%.

[0644] (1) The pharmacodynamic detection of the anti-human Her2 antibody drug conjugate in JIMT-1 model

[0645] The JIMT-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37℃ in 5% CO2. The JIMT-1 cells in the exponential growth phase were collected, resuspended to a suitable concentration with PBS containing 50% Matrigel, and inoculated subcutaneously into female NOD SCID mice to establish a breast cancer model. When the average volume of the tumors was about 150mm 3When the tumor size was appropriate, the mice were randomly divided into groups according to the tumor size, in turn: the vehicle control group (i.e. negative control, Vehicle group), the Trastuzumab-A-1 (D0: 3 mg / kg; D7 / D14: 10 mg / kg) group, the Trastuzumab-B-1 (D0: 3 mg / kg; D7 / D14: 10 mg / kg) group, the DS8201 (D0: 3 mg / kg) + Trastuzumab-B-1 (D0: 3 mg / kg; D7 / D14: 10 mg / kg) combination group, and the control DS8201 3 mg / kg (Note: DS8201, i.e. ENHERTU, is an ADC targeting human Her2 developed by First Three Company, and the sample used in this test is prepared by Kolon Biotech). Each group was injected intravenously (i.v.), and the control DS8201 was administered once on Day 0. The Trastuzumab-A-1 and Trastuzumab-B-1 were administered three times on Day 0, Day 7, and Day 14.

[0646] The ADC of the present application significantly inhibited the tumor growth of the JIMT-1 breast cancer xenograft model. Compared with the Vehicle group, the tumor growth inhibition rates (TGI) of the Trastuzumab-A-1, Trastuzumab-B-1, DS8201 + Trastuzumab-B-1, and DS8201 groups were 73.03%, 59.64%, 158.14%, and 58.90%, respectively. On Day 25, there was no animal death or significant animal 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 1, Figure 1, and Figure 2.

[0647] Table 1 Human breast cancer cell JIMT-1 CDX model

[0648] II. Evaluation of the tumor growth inhibition effect of the antibody-drug conjugate on a mouse subcutaneous xenograft model

[0649] The ADC of the present application was administered to the mouse CDX model of subcutaneously transplanted human breast cancer cells HCC1954 by tail vein injection, and the tumor volume and animal weight changes were measured once a week to calculate the tumor inhibition effect of the ADC of the present application on tumor-bearing mice.

[0650] Test drug

[0651] An appropriate amount of ADC was administered at 3 mg / kg, and the specific administration dose is shown below. The mother solution (20 mM histidine buffer solution at pH 6.0 of each antibody drug conjugate obtained in the preparation example of the antibody drug conjugate) was diluted with 0.9% NaCl injection solution to the administration solution. 0.9% NaCl injection solution was used as a vehicle control (Vehicle).

[0652] Experimental animals and cell lines

[0653] NOD SCID mice (Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd.)

[0654] Human breast cancer cells HCC1954 (Nanjing Kebai)

[0655] Experimental grouping and evaluation method

[0656] The tumor-bearing mice were randomly grouped (the number of groups was determined according to the number of samples) with an average tumor volume of about 150 mm 3 According to the groups, 0.9% NaCl injection solution (hereinafter referred to as vehicle control, Vehicle), ADC were administered, 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 short diameter of the tumor, respectively. The animal death was observed and recorded every day.

[0657] The tumor growth inhibition rate TGI (%) was calculated using the following formula to evaluate the tumor inhibition effect of ADC:

[0658] V T末 >V T0 , TGI (%) = [1-(V T末 -V T0 ) / (V C末 -V C0 )]*100%, or

[0659] V T末 ≤V T0 , TGI (%) = [1-(V T末 -V T0 ) / V T0 ]*100%.

[0660] wherein V T末 : the mean tumor volume at the end of the experiment of the treatment group;

[0661] V T0 : the mean tumor volume at the beginning of administration of the treatment group;

[0662] VC末 : mean tumor volume at the end of the negative control experiment

[0663] V C0 : mean tumor volume at the beginning of the negative control experiment

[0664] The relative proliferation rate T / C (%) of the tumor was calculated using the following formula to evaluate the anti-tumor efficacy of the ADC:

[0665] T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 )*100%.

[0666] (1) Anti-human Her2 antibody drug conjugate in HCC1954 model for efficacy detection

[0667] HCC1954 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in 5% CO2. Exponential growth phase HCC1954 cells were collected and resuspended to an appropriate concentration with PBS containing 50% Matrigel for subcutaneous inoculation in female BALB / c Nude mice to establish a breast cancer model. When the average tumor volume was about 150mm 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 3mpk, Trastuzumab-B-1 3mpk, Trastuzumab-Z-1 + Trastuzumab-B-1 3+3mpk combination, dosed at Day 0, and dosed once.

[0668] The ADC of the present application significantly inhibited the growth of HCC1954 breast cancer xenograft tumor model. Compared with the Vehicle group, the tumor growth inhibition rates (TGI) of the Trastuzumab-Z-1 3mpk, Trastuzumab-B-1 3mpk, and Trastuzumab-Z-1 + Trastuzumab-B-1 3+3mpk groups were 97.27%, 16.49%, and 151.58%, respectively. No animal deaths and no significant weight loss were observed in each treatment group on Day 41, 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 2.

[0669] Table 2 Human breast cancer cell HCC1954 CDX model

[0670] III. Evaluation of the tumor growth inhibition effect of antibody drug conjugate on a mouse subcutaneous xenograft tumor model

[0671] The CDX model of the present application is administered to the subcutaneously transplanted human breast cancer cell JIMT-1 mouse by tail vein injection, and the tumor volume and animal weight change are measured once a week to calculate the tumor inhibition effect of the ADC of the present application on tumor-bearing mice.

[0672] Test drug

[0673] An appropriate amount of DS8201 ADC was administered twice at a dose of 1.5 mg / kg, and then Trastuzumab-B was administered at a dose of 110 mg / kg. The specific dosages are shown below. The mother solution (20 mM histidine buffer solution at pH 6.0 obtained in the preparation example of the antibody drug conjugate) was diluted with 0.9% NaCl injection solution to obtain the administration solution. 0.9% NaCl injection solution was used as a vehicle control (Vehicle).

[0674] Experimental animals and cell lines

[0675] NOD SCID mice (Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd.)

[0676] Human breast cancer cell JIMT-1 (Nanjing Kebai)

[0677] Experimental grouping and evaluation method

[0678] The tumor-bearing mice were randomly divided into groups (the number of groups was determined according to the number of samples) with an average tumor volume of about 150 mm 3 According to the group, 0.9% NaCl injection solution (hereinafter referred to as vehicle control, Vehicle) and ADC were administered by tail vein injection at a dose of 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 short diameter of the tumor, respectively. The animal death was observed and recorded every day.

[0679] The tumor growth inhibition rate TGI (%) was calculated according to the following formula to evaluate the tumor inhibition effect of the ADC:

[0680] V T末 >V T0 , TGI (%) = [1-(V T末 -V T0 ) / (V C末 -V C0 )]*100%, or

[0681] V T末 ≤V T0, TGI (%) = [1 - (V T末 -V T0 ) / V T0 ]*100%.

[0682] wherein V T末 : mean tumor volume at the end of the experiment in the treatment group;

[0683] V T0 : mean tumor volume at the beginning of the administration in the treatment group;

[0684] V C末 : mean tumor volume at the end of the experiment in the negative control group;

[0685] V C0 : mean tumor volume at the beginning of the administration in the negative control group;

[0686] The relative proliferation rate T / C (%) of the tumor was calculated using the following formula to evaluate the anti-tumor efficacy of the ADC: T / C (%) = (V T末 / V T0 ) / (V C末 / V C0 )*100%.

[0687] (1) Pharmacodynamic detection of anti-human Her2 antibody drug conjugate in JIMT-1 model

[0688] JIMT-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2. Exponential growth phase 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 150mm 3 around, the mice were randomly divided into groups according to the tumor size, and sequentially administered with: vehicle control (i.e. negative control, Vehicle group), DS8201 1.5mg / kg twice on Day 0 and Day 7, followed by Trastuzumab-B-1 10mg / kg once on Day 14.

[0689] The ADC of the present application has a significant tumor growth inhibition effect on JIMT-1 breast cancer xenograft model. Compared with the Vehicle group, after administration of DS8201 1.5 mg / kg on D0 and D7, the tumor growth inhibition rate (TGI) on D13 was 35.61%, after administration of Trastuzumab-B-1 110 mg / kg on D14, the tumor growth inhibition rate (TGI) on D20 increased to 81.63%, the tumor growth inhibition rate (TGI) on D27 increased to 84.95%, the tumor growth inhibition rate (TGI) on D34 was 72.27%, and the tumor growth inhibition rate (TGI) on D39 was still 66.56%. No animal death and no significant animal weight loss were observed in the Day39 treatment group, and no obvious drug toxicity reaction was observed, and the mice were well tolerated during the treatment. The specific results are shown in Table 3.

[0690] Table 3 Human breast cancer cell JIMT-1 CDX model

[0691] 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 in accordance with all the teachings disclosed herein, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, QLED Formula (I) in: Q is the structure before covalent attachment to the antibody or its antigen-binding fragment; L is the connecting structure connecting Q and E; E is the structure connecting L and D; D is an active molecular moiety; preferably, D is a pharmaceutically active molecular moiety.

2. The compound of claim 1 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, wherein Q is selected from the following structures: in, p is an integer selected from 1 to 12; LG represents a leaving group, preferably each independently selected from halogen (such as F, Cl, Br, I), halogenated C 1-6 Alkyl, C 1-6 Alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 Alkyl sulfoxide, halogenated phenoxy, hydroxyl, thiol, amino, nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structures, the halogenated C 1-6 Alkyl, C 1- 6-alkylsulfonyl, halo C 1-6 Alkylsulfonyl, halosulfonyl, C 1-6 Alkyl sulfonate group, halogenated C 1-6 Alkyl sulfonate group, C 1-6 Alkyl sulfinate group, C 1-6 The alkylsulfoxide, halophenoxy, alkenyl, alkynyl, and alkynyl-containing structures are optionally substituted with one or more suitable substituents; 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 3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein L is selected from one or more of the following substituted or unsubstituted structures: C 1-6 Alkylene, 6-10 membered aryl, 5-6 membered heteroaryl, -N(R')-, 9-12 membered nitrogen-containing heterocyclic group, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-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), 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 ethoxy (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 structures 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 the group consisting of one or more of the following substituted or unsubstituted structures: 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 of 1-20, preferably, s is selected from an integer of 1-15, such as an integer of 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; R' represents hydrogen, glucosyl, galactosyl, glucuronyl, galacturonyl, -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 1-4 alkyl; Preferably, L is selected from substituted or unsubstituted structures consisting of one or more of the following: That Where s is an integer selected from 1 to 20; Preferably, L is -L1-L2-L3-, wherein L1 is absent or selected from wherein s is selected from an integer of 1-20, preferably, s is selected from an integer of 1-15, such as an integer of 1-10, 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; preferably, L1 is absent or selected from L2 is selected from amino acids and short peptides composed of amino acids, such as 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-Va l-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-Al a-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu; Preferably, L2 is selected from Val-Ala, Val-Cit, Ala-Ala-Ala, Gly-Gly-Phe-Gly and Gly-Gly-Val-Ala; L3 does not exist or is selected wherein 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, L3 is absent or selected from Preferably, L is selected from: Preferably, the imino terminus of L is linked to Q.

4. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein E is selected from a single bond, -NH-CH2-, or selected from the following structures: Preferably, E is -NH-CH2-, Preferably, the left end of E is connected to L, and the right end is connected to D.

5. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein the active molecule is selected from antimetabolite antitumor drugs; Preferably, the antimetabolite antitumor drug is selected from PARP inhibitor antitumor active compounds; Preferably, the PARP inhibitor anti-tumor active compound is Olaparib, Niraparib, Rucaparib, Talazoparib, Pamiparib, Fluzoparib, Veliparib, Stenoparib, Senaparib, Venadaparib, AZ9482, AZD-2461, Amelparib, Saruparib (AZD5305), AZD9574, A-966492, Nesuparib, Simmiparib, AG-14361, SC10914, AMXI-5001, Mefuparib, Iniparib (BSI-201), Atamparib (RBN-2397), RBN-2397, OUL232, Basroparib, WXFL10040340, TSL-1502, MP-124, NMS-P293 and pharmaceutically acceptable salts, esters and analogs thereof; Preferably, the antimetabolite antitumor drug is connected to the E via the -OH, primary amino, or secondary amine group thereon; Preferably, D is selected from:

6. The compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein -LE- is selected from the following structures: Or selected from the following structures:

7. The compound of any one of claims 1-6, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein -LED is selected from the following structures:

8. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the QLED has the following structure:

9. 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 independently selected from 1 to 10; L, E, and D are as described in any one of claims 1 to 8.

10. The antibody-drug conjugate according to claim 9, 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 / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5 or a variant thereof, CDR-H2 of SEQ ID NO: 6 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof, CDR-H2 of SEQ ID NO: 21 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof; wherein the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; or, (2) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof, CDR-H2 of SEQ ID NO: 19 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof, CDR-H2 of SEQ ID NO: 34 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof; wherein the variant described in any one of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; or, (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof, CDR-H2 of SEQ ID NO: 12 or a variant thereof, and CDR-H3 of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof, CDR-L2 of SEQ ID NO: 9 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof, CDR-H2 of SEQ ID NO: 27 or a variant thereof, and CDR-H3 of SEQ ID NO: 22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof, CDR-L2 of SEQ ID NO: 24 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof; wherein the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; or, (4) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof, CDR-H2 of SEQ ID NO: 14 or a variant thereof, and CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof, CDR-L2 of SEQ ID NO: 17 or a variant thereof, and CDR-L3 of SEQ ID NO: 10 or a variant thereof; or (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof, CDR-H2 of SEQ ID NO: 29 or a variant thereof, and CDR-H3 of SEQ ID NO: 30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 31 or a variant thereof, CDR-L2 of SEQ ID NO: 32 or a variant thereof, and CDR-L3 of SEQ ID NO: 25 or a variant thereof; wherein the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; 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 or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4; wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; 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 further comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., 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 (b) a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, such as a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 35 or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions); Preferably, 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, wherein the variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 41 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions); Preferably, 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, said variant having up to 20 conservative amino acid substitutions compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 and a light chain constant region (CL) as shown in SEQ ID NO: 36; 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, the 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.

11. The antibody-drug conjugate according to claim 9 or 10, 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; Preferably, M is selected from the following structures: Preferably, M is 12. The antibody-drug conjugate according to any one of claims 9 to 11, wherein: MLED can be obtained from QLED through a substitution reaction (eg, removal of -SO2Me structure), wherein Q, L, E, and D are as described in any one of claims 1-8.

13. The antibody-drug conjugate according to any one of claims 9 to 12, wherein: M is linked to the sulfhydryl (-SH) or amino (-NH2) group on Ab.

14. The antibody drug conjugate according to any one of claims 9 to 13, which is selected from the group consisting of: ADC A-1 to ADC A-8, ADC B-1 to ADC B-3, and ADC C-1 to ADC C-4; in, Ab' represents an antibody or its antigen-binding fragment; represents the specific connection mode between the thiol group in the antibody or antigen-binding fragment thereof and M; x is selected from 1-10.

15. A composition comprising the antibody drug conjugate of any one of claims 9 to 14, wherein the DAR value (drug-antibody conjugate ratio) of the composition is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10; Preferably, the composition has a DAR value of about 1-8, 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, or about 7.5 to 8.0, preferably 6 to 8, for example, 6.0 to 6.5, 6.0 to 7.0, 6.0 to 7.5, 6.0 to 8.0, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.0, 7.0 to 7.5, 7.0 to 8.0, or 7.5 to 8.0; Preferably, the composition has a DAR of about 6.0 to 10.0, for example, 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 7.8 5, 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 8.9 1. 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 8.0-8.5, about 8.0~9.0, about 8.0~9.5, about 8.0~10.

0.

16. A pharmaceutical composition comprising the compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, the antibody-drug conjugate of any one of claims 9 to 14, or the composition of claim 15, and one or more pharmaceutical excipients.

17. The pharmaceutical composition of claim 16, further comprising one or more other therapeutic agents; Preferably, the other therapeutic agent is an antibody-drug conjugate, for example, an antibody-drug conjugate targeting Her2, such as DS8201 or Trastuzumab-Z-1.

18. A pharmaceutical combination comprising: Component a: a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, an antibody-drug conjugate according to any one of claims 9 to 14, or a composition according to claim 15, 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.

19. Use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, the antibody drug conjugate according to any one of claims 9 to 14, the composition according to claim 15, the pharmaceutical composition according to claim 16 or 17 or the pharmaceutical combination according to claim 18 in the preparation of a medicament, in particular in the preparation of a medicament for treating Her2-expressing cancer; preferably, the cancer is selected from solid tumors or hematological malignancies; for example, selected from urothelial carcinoma, ovarian cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), and lymphoma.

20. A compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, the antibody drug conjugate according to any one of claims 9 to 14, the composition according to claim 15, the pharmaceutical composition according to claim 16 or 17, or the pharmaceutical combination according to claim 18, for treating Her2-expressing cancer; preferably, the cancer is selected from a solid tumor or a hematological malignancy; for example, selected from urothelial carcinoma, ovarian cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), and lymphoma.

21. A method for treating Her2-expressing cancer, comprising administering to a subject in need thereof an effective amount of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, the antibody-drug conjugate according to any one of claims 9 to 14, the composition according to claim 15, the pharmaceutical composition according to claim 16 or 17, or the pharmaceutical combination according to claim 18; Preferably, the cancer is selected from solid tumors or hematological malignancies; for example, selected from urothelial carcinoma, ovarian cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, in particular lung adenocarcinoma), and lymphoma.

22. The compound shown below, or a salt, stereoisomer, tautomer, or isotope-labeled compound thereof: Preferably, the compound is selected from: in, D and LG are as defined in any one of claims 1 to 8; PG1 and PG2 are each independently H or an amino protecting group, wherein 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-)nitrobenzenesulfonyl (Ns), pivaloyl, benzoyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; alkyl amino protecting groups, such as trityl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn).

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