Macrocyclic drug conjugate as well as preparation method and application thereof

Abstract

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

Description

Technical Field

[0001] This application relates to the field of targeted therapy, specifically to a macrocyclic drug conjugate. Background Technology

[0002] Antibody-drug conjugates (ADCs) for cancer treatment typically consist of a monoclonal antibody, a bioactive molecule (primarily a cytotoxic agent that kills tumor cells), and a linker. The bioactive molecule is covalently coupled to the antibody via the linker. The antibody recognizes specific targets on the surface of tumor cells, guiding the ADC to the tumor microenvironment and the surface of cancer cells. The ADC then enters the cancer cells via endocytosis. The bioactive molecule is then released within the cancer cells, killing them by inhibiting microtubules or damaging their DNA, thereby minimizing damage to normal tissue cells.

[0003] ErbB family receptor tyrosine kinases are important mediators of cell growth, differentiation, and survival. This family includes four members: epidermal growth factor receptor (EGFR or ErbB1), Her2 (ErbB2), Her3 (ErbB3), and Her4 (ErbB4). Clinically, the anti-ErbB2 antibody trastuzumab (trade name Herceptin) is commonly used to treat breast cancer with high ErbB2 expression, but the clinical response rate is low. To improve treatment efficacy, in recent years, conjugates of anti-ErbB2 antibodies with microtubule inhibitors such as maytansine (e.g., DM1), aurestatins (e.g., MMAE), or DNA topoisomerase I inhibitors (e.g., Dxd) have been used in clinical treatment (Trastuzumab bemtansine, Disitamab vedotin, Trastuzumab deruxtecan).

[0004] With the widespread clinical application of the aforementioned ADC drugs in treating Her2-expressing tumors, safety issues and drug resistance problems, including neurotoxicity, hematologic toxicity, hepatotoxicity, and interstitial pneumonia, have gradually emerged (Pharmacology & Therapeutics 2019, 200, 110-125; Breast Cancer Research and Treatment 2020, 183, 23-39; JAMA Oncol. 2021, 7, 1873-1881; Drug Deliv. 2022, 29, 1335-1344; Cancers 2023, 15, 1130; Cancers 2023, 15, 1278).

[0005] Specifically, both Disitamab vedotin and Trastuzumab deruxtecan use maleimide linkers (MC) as the linker portion. Literature reports that under physiological conditions, MC linkers are prone to reverse Michael reaction and thiol exchange, resulting in reduced efficacy and increased toxicity (Nat Biotechnol. 2012, 32, 184-189; Bioconjugate Chem. 2015, 26, 145-152). Regarding the hydrophilicity / hydrophobicity of linkers, Disitamab vedotin and Trastuzumab deruxtecan utilize the relatively hydrophobic valine-citrulline (Val-Cit) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) linkages, respectively. Literature reports that the hydrophilicity of linkers significantly affects the hydrophilicity of ADCs, thereby influencing ADC aggregation, pharmacokinetics, and toxicity (Chemical Linkers in Antibody-Drug Conjugates (ADCs), Drug Discovery Series No. 81, Chapter 3). For the bioactive molecule that exerts its cancer-killing effect, Trastuzumab emtansine uses the tubulin inhibitor DM1 as a cytotoxic agent, which, when combined with a non-cleavable linker, results in a weak bystander effect. Disitamab vedotin uses the auristatin toxin MMAE as the bioactive molecule, which is prone to neurotoxicity accumulation after repeated use. Furthermore, both of these ADC drugs are non-site-specific random conjugations, resulting in poor homogeneity. Summary of the Invention

[0006] The present invention aims to improve upon the aforementioned problems in existing pharmaceutical technologies, specifically by providing a class of antibody-drug conjugates for tumor treatment. These antibody-drug conjugates exhibit good stability, homogeneity, hydrophilicity, efficacy, and safety.

[0007] This application relates to an antibody-drug conjugate and exemplarily discloses a conjugate having the general formula Ab-[MLED]. x The antibody-drug conjugates with the structure shown are described. Results showed that the conjugates exhibit excellent binding activity and proliferation inhibition against tumor cells, for example, showing good targeted killing effects against Her2-positive tumors (e.g., breast cancer, lung cancer, or gastric cancer). Therefore, this application provides an antibody-drug conjugate for treating tumors, a pharmaceutical composition containing the antibody-drug conjugate, and their use in treating tumors.

[0008] The drug-linker is a crucial component of antibody-drug conjugates (ADCs), and the structures of both the drug and linker portions significantly influence the overall efficacy and safety of the ADC. This application also relates to a macrocyclic compound having the structure shown in the general formula DEL-M'. This macrocyclic compound can be used to prepare antibody-drug conjugates, and these conjugates exhibit excellent targeted killing effects against tumors.

[0009] compound

[0010] In one aspect, this application provides a compound or a pharmaceutically acceptable salt thereof having the structure shown in formula DEL-M', wherein:

[0011] M' is -M-Lg, where Lg is the leaving group of the nucleophilic substitution reaction, and M is the structural fragment that binds to the target site;

[0012] L is a structural segment connecting M and E;

[0013] E is a structural segment connecting L and D;

[0014] D is a cytotoxic drug fragment.

[0015] In some implementations, Lg is selected from halogens (e.g., F, Cl, Br, I) and halogenated C. 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, 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, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments, wherein the haloC 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, 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, halophenoxy, alkenyl, ynyl and ynyl-containing structural segments may optionally be substituted by one or more suitable substituents.

[0016] In some implementations, Lg is selected from halogens (e.g., F, Cl, Br, I) and halogenated C. 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C1-6 Alkyl sulfonyl, 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, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments.

[0017] In some implementations, Lg is selected from halogenated, substituted, or unsubstituted C. 1-6 Alkyl sulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH), or amino (-NH2).

[0018] In some embodiments, Lg is selected from halogen, substituted or unsubstituted methanesulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH) or amino (-NH2).

[0019] In some implementations, Lg is selected from C 1-6 Alkyl sulfonyl or halophenoxy.

[0020] In some implementations, Lg is selected from methanesulfonyl or pentafluorophenoxy.

[0021] In some implementations, Lg is a methanesulfonyl group.

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

[0023]

[0024] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0025]

[0026] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0027]

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

[0029]

[0030] In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

[0031]

[0032] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural segments: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, -N(R')-, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (e.g., Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-L ys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, G ly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Ala-Ala-Glu, Gly-Gly-Phe-Gly (GGFG,SEQ ID NO:41), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:42), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO:43), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO:44), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO:45)),

[0033]

[0034] R' is composed of one or more of the following groups, including but not limited to hydrogen and C. 1-6 Alkyl, C 1-6 Alkyl group, amino group, hydroxyl group, carboxyl group, acyl group, -O-, glucosyl group, galactosyl group, glucuronic acid group, galacturonic acid group, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl group, -(CH2N(Me)-C(=O))r -C 1-6 Alkyl groups, polyethylene glycol segments containing 1-10 EO units (i.e., -(CH2CH2O)). 1-10 -C 1-6 Alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA, where r is selected from an integer from 1 to 20 (e.g., an integer from 1 to 15, such as 1 to 12, 3 to 12, 1 to 10, 1 to 8, 3 to 8, 1 to 6, 1 to 4, an integer from 1 to 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); s is selected from an integer from 1 to 20 (e.g., an integer from 1 to 15, such as 1 to 12, 3 to 12, 5 to 10, an integer from 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

[0035] In some implementations, s is denoted as n.

[0036] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments: natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (e.g., Val-Ala, Val-Cit). R' consists of one or more of the following groups, including but not limited to -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C) 1-6Alkyl)-NOTA, wherein r is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); s is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, R' is -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA.

[0037] In some implementations, "-NOTA" refers to

[0038] In some implementations, "-DOTA" refers to

[0039] In some implementations, "-DOTAGA" refers to

[0040] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural segments:

[0041] R' consists of one or more of the following groups, including but not limited to hydrogen, C 1-6 Alkyl, C 1-6 Alkyl group, amino group, hydroxyl group, carboxyl group, acyl group, -O-, glucosyl group, galactosyl group, glucuronic acid group, galacturonic acid group, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, polyethylene glycol segments containing 1-10 EO units (i.e., -(CH2CH2O)). 1-10 -C 1-6 Alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA, where r is selected from an integer from 1 to 20 (e.g., an integer from 1 to 15, such as 1 to 12, 3 to 12, 1 to 10, 1 to 8, 3 to 8, 1 to 6, 1 to 4, an integer from 1 to 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); s is selected from an integer from 1 to 20 (e.g., an integer from 1 to 15, such as 1 to 12, 3 to 12, 5 to 10, an integer from 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

[0042] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural segments: R' consists of one or more of the following groups, including but not limited to -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA, wherein r is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); s is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, R' is -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA.

[0043] In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural segments: R' consists of one or more of the following groups, including but not limited to -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, or -C1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA, wherein r is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); s is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, R' is -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA.

[0044] In some implementations, L is selected from the following substituted or unsubstituted structural segments:

[0045] R' consists of one or more of the following groups, including but not limited to -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA, s is selected from integers from 1 to 20, preferably from integers from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, 8 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0046] In some implementations, L is selected from the following substituted or unsubstituted structural segments:

[0047]

[0048] Where s is selected from an integer from 1 to 20, preferably from an integer from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, 8 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

[0049] In some embodiments, E is a single bond, substituted or unsubstituted -NH-CH2-, or a structural fragment selected from the following substituted or unsubstituted segments:

[0050]

[0051] In some implementations, E is a single bond, substituted or unsubstituted -NH-CH2- or In some implementations, E is substituted or unsubstituted -NH-CH2- or In some implementations, E is a single bond or In some implementations, E is In some implementation schemes, Selected from the following substituted or unsubstituted structures:

[0052]

[0053]

[0054]

[0055]

[0056] s is an integer selected from 1 to 20.

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

[0058]

[0059]

[0060]

[0061] s is an integer selected from 1 to 20.

[0062] In some implementation schemes, Selected from the following substituted or unsubstituted structures:

[0063]

[0064]

[0065]

[0066]

[0067] In some implementation schemes, Selected from the following substituted or unsubstituted structures:

[0068]

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

[0070] The cytotoxic drugs disclosed in this application typically contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), secondary amino (-NR1H), and tertiary amino (-NR2R3), where R1, R2, and R3 represent only non-hydrogen substituents on N, or thiol (-SH). These functional groups can react with suitable functional groups in the remainder of the conjugate to achieve linkage.

[0071] In some embodiments, the cytotoxic drug is linked to the E in the antibody-drug conjugate via a -OH, primary amino, secondary or tertiary amino group, or -SH group. In some embodiments, D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amino group on the cytotoxic drug.

[0072] In some embodiments, the cytotoxic agent is selected from the following substituted or unsubstituted compounds or compounds labeled with their isotopes:

[0073]

[0074] In some implementations, D is selected from the following substituted or unsubstituted structures:

[0075]

[0076] In some implementations, D has the following structure:

[0077]

[0078] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is selected from I'-1 to I'-2 below:

[0079] I'-1:

[0080] I'-2:

[0081] Where s is selected from integers from 1 to 20 (e.g., integers from 1 to 15, such as 1 to 12, 3 to 12, 5 to 10, 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

[0082] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is selected from I-1 to I-2 below:

[0083] I-1:

[0084] I-2:

[0085] In some embodiments, the compound described above or a pharmaceutically acceptable salt thereof may optionally be replaced by one or more suitable substituents.

[0086] Coupled

[0087] A second aspect of the invention provides a coupling as shown in formula (II), wherein:

[0088] Ab-[MLED]x

[0089] Equation (II)

[0090] Ab is the target region; M, L, E, and D are as described above;

[0091] x is between 1 and 10.

[0092] In some preferred embodiments, the targets of Ab are selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylyl cyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, O772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, F cRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CD70, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2. BMPR1B, E16, STEAP1, Tyro7, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, c-Met, ApoE, CD1 lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL- 6. ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN , ANGPTL4, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR 1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and TENB2. .

[0093] In some preferred embodiments, Ab is a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an aryl sulfonamide derivative (e.g., a carbonic anhydrase IX inhibitor), a polyene linking two aliphatic indoles, an anthocyanin dye, or IR-783 or a derivative thereof.

[0094] In some preferred embodiments, Ab is an antibody, such as a monoclonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment includes Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody (e.g., scFv), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, probody, bispecific antibody, or multispecific antibody.

[0095] In some embodiments, Ab is an antibody or an antigen-binding fragment thereof. In some embodiments, Ab is an antibody or an antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0096] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0097] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0098] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0099] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0100] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0101] or,

[0102] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0103] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0104] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0105] Wherein, the variant described in any 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0106] or,

[0107] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0108] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0109] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0110] Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0111] or,

[0112] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0113] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0114] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0115] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions.

[0116] In some embodiments, the antibody or its antigen-binding fragment comprises

[0117] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0118] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0119] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0120] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0121] or,

[0122] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0123] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0124] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0125] Wherein, the variant described in any 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0126] or,

[0127] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0128] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0129] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0130] Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0131] or,

[0132] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0133] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0134] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0135] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions.

[0136] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0137] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0138] (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,

[0139] (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;

[0140] or,

[0141] (2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0142] (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,

[0143] (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;

[0144] or,

[0145] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0146] (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,

[0147] (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;

[0148] or,

[0149] (4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0150] (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,

[0151] (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.

[0152] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0153] (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

[0154] (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;

[0155] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0156] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0157] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0158] (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4.

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

[0160] (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and

[0161] (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

[0162] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

[0163] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35.

[0164] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36.

[0165] In some embodiments, the antibody or its antigen-binding fragment 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.

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

[0167] (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0168] (2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

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

[0170] (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38; or

[0171] (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40.

[0172] In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or lack a C-terminal lysine residue 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 may be cyclized to pyroglutamic acid.

[0173] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0174] In some embodiments, compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.

[0175] In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0176] In some embodiments, the N-terminal glutamine of the VH of the sequence shown in SEQ ID NO:1 or 3 or a variant thereof, or the N-terminal glutamine of the heavy chain of the sequence shown in SEQ ID NO:37 or 39 or a variant thereof, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0177] In some embodiments, the heavy chain constant region (CH) of the heavy chain as shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof, lacks a C-terminal lysine.

[0178] In some embodiments, the antibody or its antigen-binding fragment is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 97 and the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 80.

[0179] In some implementations, M is linked to a thiol group (-SH) or an amino group (-NH2) on Ab.

[0180] In some implementations, M is linked to a thiol group (-SH) on Ab.

[0181] In some implementations, when Ab-[MLED] is present x In the antibody-drug conjugate with the structure shown, MLED is formed from the compound shown in DEL-M', preferably by removing Lg from DEL-M', wherein the compound shown in DEL-M' is as defined above, Ab is as defined above, and x is 1-10.

[0182] In some implementations, when Ab-[MLED] is present x In the antibody-drug conjugates with the structures shown, MLED is formed from the compounds shown in I'-1 or I'-2, preferably by removing the -SO2Me from the compounds, Ab as defined above, and x is 1-10.

[0183] In some implementations, when Ab-[MLED] is present x In the antibody-drug conjugates with the structures shown, MLED is formed from the compounds shown in I-1 or I-2, preferably by removing the -SO2Me from the compounds, Ab as defined above, and x is 1-10.

[0184] In some implementations, when Ab-[MLED] is present x In the antibody-drug conjugate with the structure shown,

[0185] M is selected from the following substituted or unsubstituted structural segments:

[0186]

[0187] L is selected from one or more of the following substituted or unsubstituted structural segments: R' consists of one or more of the following groups, including but not limited to -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA, wherein r is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); s is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); preferably, R' is -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, or

[0188] L is selected from one or more of the following substituted or unsubstituted structural segments: R' consists of one or more of the following groups, including but not limited to -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C) 1-6Alkyl)-NOTA, wherein r is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); s is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); preferably, R' is -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA;

[0189] Preferably, L is selected from the following substituted or unsubstituted structural segments:

[0190]

[0191]

[0192] Where s is selected from integers from 1 to 20, preferably from integers from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, 8 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10; E is

[0193] D has the following structure:

[0194]

[0195] Ab is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0196] The antibody or its antigen-binding fragment is as defined above.

[0197] Preferably, the antibody or its antigen-binding fragment comprises:

[0198] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0199] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0200] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0201] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0202] or,

[0203] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0204] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0205] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0206] Wherein, the variant described in any 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0207] or,

[0208] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0209] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0210] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0211] Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0212] or,

[0213] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0214] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0215] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0216] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0217] Preferably, the antibody or its antigen-binding fragment comprises:

[0218] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0219] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0220] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0221] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0222] or,

[0223] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0224] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0225] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0226] Wherein, the variant described in any 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0227] or,

[0228] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0229] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0230] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0231] Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0232] or,

[0233] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0234] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0235] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0236] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0237] Preferably, the antibody or its antigen-binding fragment comprises:

[0238] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0239] (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,

[0240] (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;

[0241] or,

[0242] (2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0243] (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,

[0244] (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;

[0245] or,

[0246] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0247] (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,

[0248] (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;

[0249] or,

[0250] (4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0251] (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,

[0252] (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;

[0253] Preferably, the antibody or its antigen-binding fragment comprises:

[0254] (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

[0255] (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;

[0256] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with its source sequence, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to its source sequence; preferably, the substitutions are conservative substitutions;

[0257] Preferably, the antibody or its antigen-binding fragment comprises:

[0258] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0259] (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4;

[0260] Preferably, the antibody or its antigen-binding fragment is selected from Trastuzumab or Pertuzumab or its antigen-binding fragment.

[0261] In the antibody-drug conjugate, the cytotoxic drug can be linked to the antibody or its antigen-binding fragment via the “MLE” fragment shown in this application.

[0262] Those skilled in the art will understand that the antibody-drug conjugates described in this application can be prepared in a modular manner. For example, a compound in its free form as a "drug-linker" (which can be understood as M'-LED, where M' is the structural form of M before covalently linking it to an antibody or its antigen-binding fragment) can be obtained, and then covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugates described in this application. Accordingly, in the free form of the "drug-linker," M' is linked to one or more thiol (-SH) or amino (-NH2) groups on the antibody or its antigen-binding fragment through a substitution reaction (e.g., removal of the -SO2Me or pentafluorophenol structure) or an addition reaction.

[0263] In some embodiments, the antibody-drug conjugate is selected from ADC I'-1 to ADC I'-2 as shown below:

[0264] ADC I'-1:

[0265] ADC I'-2:

[0266] Where s is selected from integers from 1 to 20 (e.g., integers from 1 to 15, such as 1 to 12, 3 to 12, 5 to 10, 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

[0267] In some embodiments, the antibody-drug conjugate is selected from ADC I-1 to ADC I-2 as shown below:

[0268] ADC I-1:

[0269] ADC I-2:

[0270] In each antibody-drug conjugate, HA represents an antibody or its antigen-binding fragment, preferably an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase.

[0271] Or it may indicate the specific way in which the thiol group in the antibody or its antigen-binding fragment is linked to the M fragment.

[0272] In some implementations, the antibody or its antigen-binding fragment in each antibody-drug conjugate is as defined above.

[0273] In some implementations, the HA includes:

[0274] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0275] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0276] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0277] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0278] or,

[0279] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0280] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0281] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0282] Wherein, the variant described in any 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0283] or,

[0284] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0285] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0286] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0287] Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0288] or,

[0289] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL):

[0290] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0291] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0292] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions.

[0293] In some implementations, the HA includes

[0294] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0295] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0296] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0297] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0298] or,

[0299] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0300] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0301] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0302] Wherein, the variant described in any 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0303] or,

[0304] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0305] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0306] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0307] Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions;

[0308] or,

[0309] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0310] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0311] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0312] 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions.

[0313] In some implementations, the HA includes:

[0314] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0315] (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,

[0316] (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;

[0317] or,

[0318] (2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0319] (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,

[0320] (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;

[0321] or,

[0322] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0323] (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,

[0324] (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;

[0325] or,

[0326] (4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0327] (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,

[0328] (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.

[0329] In some implementations, the HA includes:

[0330] (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

[0331] (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;

[0332] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0333] In some implementations, the HA includes:

[0334] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0335] (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4.

[0336] In some implementations, the HA further includes:

[0337] (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and

[0338] (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

[0339] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

[0340] In some embodiments, the HA comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids).

[0341] In some embodiments, the HA comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions).

[0342] In some embodiments, the HA includes 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.

[0343] In some implementations, the HA includes:

[0344] (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0345] (2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0346] In some implementations, the HA includes:

[0347] (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38; or

[0348] (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40.

[0349] In some implementations, HA is selected from Trastuzumab or Pertuzumab, wherein the amino acid sequence of Trastuzumab has a lookup accession number (IMGT / mAb-DB ID) of 97 in the IMGT database, and the amino acid sequence of Pertuzumab has a lookup accession number (IMGT / mAb-DB ID) of 80 in the IMGT database.

[0350] In some implementations, the HA in each antibody-drug conjugate represents trastuzumab, pertuzumab, or their antigen-binding fragments.

[0351] In some implementations, the HA in each antibody-drug conjugate represents the following antibody or antigen-binding fragment:

[0352] (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0353] (2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0354] In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or lack a C-terminal lysine residue 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 may be cyclized to pyroglutamic acid.

[0355] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0356] In some embodiments, compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.

[0357] In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0358] In some embodiments, the N-terminal glutamine of the VH of the sequence shown in SEQ ID NO:1 or 3 or a variant thereof, or the N-terminal glutamine of the heavy chain of the sequence shown in SEQ ID NO:37 or 39 or a variant thereof, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0359] In some embodiments, the heavy chain constant region (CH) of the heavy chain as shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof, lacks a C-terminal lysine.

[0360] In some implementations, x in the coupling shown as Ab-[MLED]x is 1-10, for example 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, or 4-10.

[0361] In some implementations, x in the coupling shown as Ab-[MLED]x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0362] In some implementations, x in the coupling shown as Ab-[MLED]x is 1, 2, 3, or 4.

[0363] In some implementations, x in the coupling shown as Ab-[MLED]x is 3, 4, or 5.

[0364] In some implementations, x in the coupling shown as Ab-[MLED]x is 2 or 4.

[0365] In some embodiments, the conjugate described in this invention is an antibody-drug conjugate (ADC).

[0366] In some embodiments, the coupling compound of the present invention may optionally be replaced by one or more suitable substituents.

[0367] intermediate

[0368] In some embodiments, this application provides intermediate compounds having the following structures, or salts, stereoisomers, tautomers, or isotopically labeled compounds thereof:

[0369]

[0370]

[0371] In this configuration, each PG1 is independently an H or amino protecting group. The amino protecting group can be an alkoxycarbonyl group, such as benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), or methoxycarbonyl (or ethoxycarbonyl); or an acyl group, such as phthaloyl (Pht), p-toluenesulfonyl (Tos), or trifluoroacetyl (Tfa). , o-(p-)nitrobenzenesulfonyl (Ns), p-valeryl, benzoyl, tert-butoxycarbonyl, 9-fluorenmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilylethoxycarbonyl, benzyloxycarbonyl, p-methylbenzenesulfonyl, p-nitrobenzenesulfonyl, trifluoroacetyl, methoxycarbonyl, or ethoxycarbonyl; alkyl amino protecting groups, such as triphenylmethyl (Trt), 2,4-dimethoxybenzyl (Dmb), 4-methoxybenzyl (PMB), benzyl (Bn);

[0372] s is selected from integers from 1 to 20, such as integers from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

[0373] Lg is as defined above.

[0374] In some embodiments, this application provides intermediate compounds having the following structures, or salts, stereoisomers, tautomers, or isotopically labeled compounds thereof:

[0375]

[0376]

[0377] In another aspect, this application provides the use of the intermediate compound as described above, or its salt, stereoisomer, tautomer, or isotopically labeled compound, in the preparation of the compound of the present invention or its pharmaceutically acceptable salt.

[0378] Composition

[0379] On the other hand, this application provides compositions of antibody-drug conjugates (ADCs) as described herein. Such compositions may comprise a plurality of ADCs as described herein, wherein each ADC contains a drug-linker as described herein, wherein x independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug-linkers. Therefore, the compositions are characterized by a drug-antibody ratio (DAR) in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0380] For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 10 or any subrange therebetween, such as: 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.

[0381] In some 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 4.0 to 4.5, about about 4.5 to 5.0, about 5.0 to 5.5, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 6.5 to 8.5, about 7.0 to 7.5, about 7.0 to 8.0.

[0382] In some embodiments, the DAR of the ADC composition described herein is about 1 to 5, such as about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 3.5 to 4.5, about 4.0 to 4.5, or about about 4.5 to 5.0.

[0383] In some embodiments, the DAR of the ADC composition described herein is about 1 to 3, such as about 1.0 to 1.5, about 1.0 to 2.0, about 1.0 to 2.5, about 1.0 to 3.0, about 1.5 to 2.0, about 1.5 to 2.5, about 1.5 to 3.0, about 2.0 to 2.5, about 2.0 to 3.0, or about 2.5 to 3.0.

[0384] In some embodiments, the DAR of the ADC compositions described herein is about 1.0 to 6.0, for example about 1.0 to 5.5, about 1.0 to 5.0, about 1.5 to 6.0, about 1.5 to about 5.5, about 1.5 to 5.0, 2.0 to 5.5, about 2.0 to about 5.0, for example 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1 .25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.3, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.4, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1. 46, approximately 1.47, approximately 1.48, approximately 1.49, approximately 1.5, approximately 1.51, approximately 1.52, approximately 1.53, approximately 1.54, approximately 1.55, approximately 1.56, approximately 1.57, approximately 1.58, approximately 1.59, approximately 1.6, approximately 1.61, approximately 1.62, approximately 1.63, approximately 1.64, approximately 1.65, approximately 1.66, approximately 1.6 7, approximately 1.68, approximately 1.69, approximately 1.7, approximately 1.71, approximately 1.72, approximately 1.73, approximately 1.74, approximately 1.75, approximately 1.76, approximately 1.77, approximately 1.78, approximately 1.79, approximately 1.8, approximately 1.81, approximately 1.82, approximately 1.83, approximately 1.84, approximately 1.85, approximately 1.86, approximately 1.87, approximately 1.88 Approximately 1.89, approximately 1.9, approximately 1.91, approximately 1.92, approximately 1.93, approximately 1.94, approximately 1.95, approximately 1.96, approximately 1.97, approximately 1.98, approximately 1.99, approximately 2.0, approximately 2.01, approximately 2.02, approximately 2.03, approximately 2.04, approximately 2.05, approximately 2.06, approximately 2.07, approximately 2.08, approximately 2.09. Approximately 2.1, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.2, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 2.3, 2. .31, about 2.32, about 2.33, about 2.34, about 2.35, about 2.36, about 2.37, about 2.38, about 2.39, about 2.4, about 2.41, about 2.42, about 2.43, about 2.44, about 2.45, about 2.46, about 2.47, about 2.48, about 2.49, about 2.5, about 2.51, about 2.52, approximately 2.53, approximately 2.54, approximately 2.55, approximately 2.56, approximately 2.57, approximately 2.58, approximately 2.59, approximately 2.6, approximately 2.61, approximately 2.62, approximately 2.63, approximately 2.64, approximately 2.65, approximately 2.66, approximately 2.67, approximately 2.68, approximately 2.69, approximately 2.7, approximately 2.71, approximately 2.72, approximately 2. 73, approximately 2.74, approximately 2.75, approximately 2.76, approximately 2.77, approximately 2.78, approximately 2.79, approximately 2.8, approximately 2.81, approximately 2.82, approximately 2.83, approximately 2.84, approximately 2.85, approximately 2.86, approximately 2.87, approximately 2.88, approximately 2.89, approximately 2.9, approximately 2.91, approximately 2.92, approximately 2.93, approximately 2.9 4. Approximately 2.95, 2.96, 2.97, 2.98, 2.99, 3.0, 3.01, 3.02, 3.03, 3.04, 3.05, 3.06, 3.07, 3.08, 3.09, 3.1, 3.11, 3.12, 3.13, 3.14, 3.1 5. Approximately 3.16, 3.17, 3.18, 3.19, 3.2, 3.21, 3.22, 3.23, 3.24, 3.25, 3.26, 3.27, 3.28, 3.29, 3.3, 3.31, 3.32, 3.33, 3.34, 3.35, 3.36 Approximately 3.37, 3.38, 3.39, 3.4, 3.41, 3.42, 3.43, 3.44, 3.45, 3.46, 3.47, 3.48, 3.49, 3.5, 3.51, 3.52, 3.53, 3.54, 3.55, 3.56, 3.57 Approximately 3.58, 3.59, 3.6, 3.61, 3.62, 3.63, 3.64, 3.65, 3.66, 3.67, 3.68, 3.69, 3.7, 3.71, 3.72, 3.73, 3.74, 3.75, 3.76, 3.77, 3.78 Approximately 3.79, approximately 3.8, approximately 3.81, approximately 3.82, approximately 3.83, approximately 3.84, approximately 3.85, approximately 3.86, approximately 3.87, approximately 3.88, approximately 3.89, approximately 3.9, approximately 3.91, approximately 3.92, approximately 3.93, approximately 3.94, approximately 3.95, approximately 3.96, approximately 3.97, approximately 3.98, approximately 3.99, approximately 4.0, approximately 4.01, approximately 4.02, approximately 4.03, approximately 4.04, approximately 4.05, approximately 4.06, approximately 4.07, approximately 4.08, approximately 4.09, approximately 4.1, approximately 4.11, approximately 4.12, approximately 4.13, approximately 4.14, approximately 4.15, approximately 4.16, approximately 4.17, approximately 4.18, approximately 4.19, approximately 4.2, approximately 4.21, approximately 4.22, approximately 4.23, approximately 4.24, approximately 4.25, approximately 4.26, approximately 4.27, approximately 4.28, approximately 4.29, approximately 4.3, approximately 4.31, approximately 4.32, approximately 4.33, approximately 4.34, approximately 4.35, approximately 4.36, approximately 4.37, approximately 4.38, approximately 4.39, approximately 4.4, approximately 4.41, approximately 4.42, approximately 4.43, approximately 4.44, approximately 4.45, approximately 4.46, approximately 4.47, approximately 4.48, approximately 4.49, approximately 4.5, approximately 4.51, approximately 4.52, approximately 4.53, approximately 4.54, approximately 4.55, approximately 4.56, approximately 4.57, approximately 4.58, approximately 4.59, approximately 4.6, approximately 4.61, approximately 4.62, approximately 4.63, approximately 4.64, approximately 4.65, approximately 4.66, approximately 4.67, approximately 4.68, approximately 4.69, approximately 4.7, approximately 4.71, approximately 4.72, approximately 4.73, approximately 4.74, approximately 4.75, approximately 4.76, approximately 4.77, approximately 4.78, approximately 4.79, approximately 4.8, approximately 4.81, approximately 4.82, approximately 4.83, approximately 4.84, approximately 4.85, approximately 4.86, approximately 4.87, approximately 4.88, approximately 4.89, approximately 4.9, approximately 4.91, approximately 4.92, approximately 4.93, approximately 4.94, approximately 4.95, approximately 4.96, approximately 4.97, approximately 4.98, approximately 4.99, approximately 5.0.

[0385] In some embodiments, the DAR of the ADC composition described herein is about 4.23.

[0386] Pharmaceutical Composition

[0387] In another aspect, this application provides a pharmaceutical composition comprising any of the antibody-drug conjugates or drug-linkers described in any of the preceding claims, and one or more pharmaceutical excipients.

[0388] The antibody-drug conjugates, compounds, or drug-linkers described herein can be formulated in a single injectable form with a pharmaceutically acceptable parenteral medium for parenteral use, such as bolus injection, intravenous injection, or intratumoral injection. Optionally, antibody-drug conjugates of desired purity can be mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers in the form of lyophilized or solution formulations (Remington's Pharmaceutical Sciences (1980) 16). th (ed., Osol, A.Ed.). The antibody-drug conjugates described herein or pharmaceutical compositions containing the antibody-drug conjugates may be administered via any route appropriate for the individual to be treated.

[0389] application

[0390] The antibody-drug conjugates, drug-linkers, compositions, or drug compositions thereof described herein can be used to treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematologic malignancies such as urothelial carcinoma, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0391] Therefore, this application provides the use of any of the antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the thereof described in the foregoing in the preparation of medicaments for the prevention or treatment of Her2-expressing cancers.

[0392] Additionally, this application provides the use of any of the antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the thereof described above in a medicament for the prevention or treatment of Her2-expressing cancer.

[0393] Additionally, this application provides a method for preventing or treating Her2-expressing cancers, comprising administering an effective amount of any of the preceding antibody-drug conjugates, drug-linkers, compositions, or pharmaceutical compositions containing the conjugates to a subject in need of such treatment.

[0394] In some embodiments, the antibody-drug conjugate, drug-linker, combination thereof, or pharmaceutical composition is sufficient (e.g., in a subject):

[0395] (1) Inhibits cell (such as tumor cells) proliferation;

[0396] (2) Inhibits tumor growth;

[0397] (3) Inducing and / or increasing antibody-dependent cytotoxic activity;

[0398] (4) Inhibit HER2-mediated signal transduction;

[0399] (5) Prevention and / or treatment of HER2-mediated diseases / disorders; or

[0400] (6) Any combination of (1)-(5) above.

[0401] In some implementations, the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

[0402] All technical features disclosed in this specification, such as the definitions of various functional groups, except for mutually exclusive technical features, can be combined in any way to obtain different general formula ranges or specific solutions. These ranges and solutions are all within the scope of this invention.

[0403] definition

[0404] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques obvious to those skilled in the art. Furthermore, laboratory procedures used herein, such as those related to genomics, nucleic acid chemistry, and molecular biology, are standard procedures widely used in their respective fields. While it is believed that the following terms will be readily understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0405] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The amino acid assignment in each region or domain can follow various numbering systems known in the art. The term "antibody" also includes embodiments where the heavy chain constant region contains a C-terminal lysine, or lacks a C-terminal lysine, or is a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized into pyroglutamate. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise N-terminal glutamine or glutamic acid, or N-terminal amino acid cyclized into pyroglutamate.

[0406] The term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0407] In this invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.

[0408] The following general rules (published at www.bioinf.org.uk: Professor Andrew C. Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with the amino acids that form the antigenic epitope that the antibody binds to. In rare cases, these generally constant features may not appear; however, Cys residues are the most conserved feature.

[0409]

[0410]

[0411] V H The complete amino acid sequence is typically numbered according to Kabat, while the three CDRs within the variable region can be defined according to any of the aforementioned numbering systems. In some embodiments, V H The amino acid sites in the sequence can be numbered sequentially starting from amino acid site 1 until the end of the sequence, or they can be numbered according to Kabat. Unless otherwise stated, the V mentioned herein... H and V L The amino acid sites in the sequence are defined according to their sequential numbering.

[0412] The amino acid sites in the heavy chain constant region can be numbered sequentially from amino acid site 1 to the end of the sequence, or they can be numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu starts from site 118 and ends at site 447. Unless otherwise stated, the amino acid sites of the heavy and light chains described herein are defined according to sequential numbering.

[0413] The term "framework region" or "FR" residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0414] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0415] The term "antigen-binding fragment" in antibody refers to a fragment of the antibody polypeptide, such as a fragment of the full-length antibody polypeptide, which 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; it is also referred to as the "antigen-binding moiety". See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology 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 peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0416] The term "Fd" refers to an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

[0417] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0418] The term "Fc" refers to an antibody fragment formed by disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0419] The term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:46) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:47) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some implementations, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, domains containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH of scFv.

[0420] The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, referring to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0421] Each of the above antibody fragments 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.

[0422] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0423] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0424] The term "mouse antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies, and then screening, preparing and purifying the antibodies; or it refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells after the antigen enters the mouse body.

[0425] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance the immune response. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance the immune response).

[0426] The term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0427] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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).

[0428] The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented as A or Ala.

[0429] The term "linker" refers to a structural segment that connects a cytotoxic drug to an antibody or antigen-binding fragment. For example, the formula Ab-[MLED] x The -MLE- structure fragment in the text.

[0430] The term "drug-linker" refers to the structure of the cytotoxic drug and linker described in this invention before they are linked to an antibody or its antigen-binding fragment. For example, "drug-linker" refers to M'-LED, where M' is the structural form of M before it is covalently linked to an antibody or its antigen-binding fragment. The "drug-linker" is covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described in this application.

[0431] The "drug-linker" also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the "drug-linker" compounds of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g.) 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0432] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0433] As used in this article, This indicates the location where a structural segment connects to other parts of the molecule.

[0434] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, such as "C". 1-20 Alkyl", C 1-10 Alkyl", C 1-6 Alkyl", C 1-4 Alkyl", C 1-3Alkyl groups, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0435] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C..." 2-6 "alkenyl", "C" 2-4 Examples of these include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc.

[0436] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond. This includes, for example, "C..." 2-6 "Alkyne", "C" 4-6 Examples of "alkynyl" include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butyrynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentyrynyl, 1,4-pentyrynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexadiynyl, etc.

[0437] The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, including but not limited to monocycloalkyl and bicycloalkyl (such as spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl). The term "C" 3-6 "Cycloalkyl" refers to a cycloalkyl group having 3 to 6 cyclic carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc., which may optionally be substituted by one or more (such as 1, 2 or 3) suitable substituents, such as methyl-substituted cyclopropyl.

[0438] The term "heterocyclic group" or "heterocycle" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic cyclic structure whose ring atoms consist of carbon atoms and at least one (e.g., 1, 2, or 3) heteroatoms selected from nitrogen, oxygen, and sulfur. The heterocyclic group can be connected to the rest of the molecule through any one ring atom, provided that valence requirements are met. The heterocyclic group in this invention is preferably a 3-6 membered heterocyclic group. The term "3-6 membered heterocyclic group" as used in this invention refers to a heterocyclic group having 3 to 6 ring atoms, including 3-membered, 4-membered, 5-membered, and 6-membered heterocyclic groups, including nitrogen-containing heterocyclic groups and oxygen-containing heterocyclic groups, such as 4-6 membered heterocyclic groups, for example, 4-6 membered nitrogen-containing heterocyclic groups and 4-6 membered oxygen-containing heterocyclic groups. Common heterocyclic groups include (but are not limited to) azetidinyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, and morpholinyl. The heterocyclic groups in this invention may optionally be substituted with one or more of the substituents described herein. The heterocyclic groups in this invention may optionally be fused with one or more aromatic or non-aromatic rings.

[0439] The term "oxygen-containing heterocycle" refers to a heterocycle as described above, in which one or more (e.g., 1, 2, or 3) ring atoms are oxygen atoms, such as 5-6 membered oxygen-containing heterocycles. Specific examples include, but are not limited to, ethylene oxide rings, tetrahydrofuran rings, furan rings, tetrahydropyran rings, and pyran rings. The term "nitrogen-containing heterocycle" as used in this invention refers to a heterocycle as described above, in which one or more (e.g., 1, 2, or 3) ring atoms are nitrogen atoms.

[0440] The term "alkoxy" refers to a group having an "alkyl-O-" structure, where alkyl is defined as described above. For example, C 1-6 Alkoxy, C 1-4 Alkoxy, C 1-3 Alkoxy or C 1-2 Alkoxy groups, etc. Common alkoxy groups include (but are not limited to) methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups in this invention are optionally substituted by one or more substituents described in this invention.

[0441] The term "halogenated" or "halogenated" is defined as including F, Cl, Br, or I.

[0442] The term "isotopically labeled compound" means that the compound is structurally identical to the compound of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g.) 37 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); and isotopes of sulfur (e.g. 35 S).

[0443] As used herein, the term "suitable substituent" refers to modifications of a compound that can be made by those skilled in the art to suit the needs of the compound's substituents. "Suitable substituents" include oxo (=O), halogen, cyano, and NR. 8 R 9 Carboxyl, thiol, hydroxyl, ester group (e.g., -C) 1-6 Alkyl-C(=O)-OC 1-6 Alkyl), C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Alkyl-OC 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-10 membered heterocyclic, 5-10 membered heteroaryl, C 6-10 Aryl, benzyl, hydroxy-substituted benzyl, indolylmethylene and C 1-6 Haloalkoxy, R 8 R 9 Each is independently selected from H and C. 1-6 Alkyl, C 3-6 Cycloalkyl, 3-10 membered heterocyclic, 5-10 membered heteroaryl, C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy groups, halogens, hydroxyl groups, carboxyl groups, and ester groups (e.g., -C)1-6 Alkyl-C(=O)-OC 1-6 alkyl).

[0444] The term "substitution" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms on a specified compound or structural segment by a substituent, provided that the substitution does not exceed the normal valence of the specified atom in the present case and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound. In some embodiments, each substituent is independently composed of one or more of the following structures: -O-, -S-, -NR'-, halogen, -CN, -OH, -NH2, -NO2, -CN, =O, C1-C6 (alkylene) group, C1-C6 haloalkyl (alkylene) group, C1-C6 alkoxy group, C2-C6 (alkenyl) group, C2-C6 (alkynyl) group, C3-C8 (cycloalkyl) group, 3-8 membered (heterocyclic) group, C6-C 10 (A)aryl and 5-10 membered (A)heteroaryl, etc. In some embodiments, the substituents are each independently composed of one or more of the following structures: NR 8 R 9 -O-, -S-, -NR'-, halogen, -CN, -OH, -SH, -NH2, -NO2, -C(O)-, -CN, =O, C1-C6 (alkylene) group, C1-C6 haloalkylene group, C1-C6 alkoxy group, C2-C6 (alkenylene) group, C2-C6 (alkynylene) group, C3-C8 (cycloalkylene) group, 3-10 membered (heterocyclic) group, C6-C 10 (sub-)aryl and 5-10 quinone (sub-)heteroaryl, etc., among which R 8 R 9 R' is as defined above. For example, the substituent can be a suitable substituent as described above.

[0445] If a functional group or structural segment is described as “substituted or unsubstituted”, then the functional group or structural segment may be (1) unsubstituted or (2) substituted.

[0446] As used herein, the term “one or more” means one or more under reasonable conditions, such as two, three, four, five or ten.

[0447] Unless otherwise specified, as used herein, the connection point of a substituent may be located at any suitable position of the substituent.

[0448] Pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts. Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts, including aspartate, fumarate, glucohepanoate, glucuronate, glucuronate, hexafluorophosphate, etc. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts, including aluminum salts, arginine salts, choline salts, diethylamine salts, etc. A review of suitable salts can be found in Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

[0449] Whether explicitly stated or not, all numerical values ​​in this application are modified by the term "about". The term "about" means within ±20%, ±10%, preferably ±5%, and more preferably ±2% of the stated numerical value. Detailed Implementation

[0450] The present invention will be further described below through specific embodiments, but this is not intended to limit the 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 ideas and scope of the invention.

[0451] Information about the sequences involved in this invention is described in the table below:

[0452]

[0453]

[0454]

[0455]

[0456] The abbreviations used in this article have the following meanings:

[0457]

[0458] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0459] Nuclear magnetic resonance (¹H NMR) measurements were performed using a Bruker 400MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0460] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0461] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0462] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0463] Example 6: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

[0464]

[0465] Step 1: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamidyl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6-2)

[0466] (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (150.0 mg, 0.31 mmol) and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanine (115.1 mg, 0.37 mmol) were dissolved in DCM (8 mL) and MeOH (2 mL). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (229.7 mg, 0.93 mmol) was added, and the mixture was stirred at room temperature for 15 hours. The solution was concentrated under reduced pressure to obtain the crude product, which was then purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0–90%) and concentrated again under reduced pressure to obtain the title compound (194.2 mg, 0.25 mmol).

[0467] Its structural characterization data are as follows:

[0468] MS m / z (ESI): 782.2 [M+H] +

[0469] Step 2: Preparation of (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (INT-6-3)

[0470] Allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (194.2 mg, 0.25 mmol) was dissolved in DMF (5 mL), and pyridine hydrofluoric acid salt (390.2 mg, 3.93 mmol) was added. The mixture was stirred at room temperature for 15 hours. After the reaction was complete, 20 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL x 3), washed with 10 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound. The crude product was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to obtain the title compound (109.3 mg, 0.21 mmol).

[0471] Its structural characterization data are as follows:

[0472] MS m / z (ESI): 566.1 [M+Na] +

[0473] Step 3: Preparation of allyl (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (INT-6)

[0474] (S)-(5-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (109.3 mg, 0.21 mmol) was dissolved in DMF (5 mL), DIPEA (81.3 mg, 0.63 mmol) was added, and p-nitrophenyl chloroformate (50.8 mg, 0.25 mmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to give the title compound (134.50 mg, 0.19 mmol).

[0475] Its structural characterization data are as follows:

[0476] MS m / z (ESI): 731.2 [M+Na] +

[0477] Example 10: 2,2',2”-(10-(2-((5-((22S,25S)-1-(3,5-bis(2-((methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-(((((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S, Preparation of 28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctadecanohydro-25H-2,27:5,8:11,15-tricyclooxy-23,25-bridged ethylene-2,28-methylfuran[3,2-i]furan[2',3':5,6]pyrano[4,3-b][1,4]dioxane-18-yl)propyl)carbamoyl)oxy)methyl)benzyl))methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (I-1)

[0478]

[0479] Step 1: (5-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionamid)-2-(((((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl Preparation of -7,14-dimethylene-21-oxooctadecylhydro-25H-2,27:5,8:11,15-tricyclooxy-23,25-bridged ethylene-2,28-methylfuran[3,2-i]furan[2',3':5,6]pyrano[4,3-b][1,4]dioxane-18-yl)propyl)carbamoyl)oxy)methyl)benzyl)methyl)carbamate (methyl)carbamate (I-1-1)

[0480] INT-6 (422.38 mg, 595.98 μmol), eribulin (300 mg, 411.02 μmol), HOBt (66.64 mg, 493.22 μmol), and pyridine (162.35 mg, 2.06 mmol) were added sequentially to DMF (4 mL). After the addition was complete, the mixture was stirred at 30 °C for 6 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (485 mg, 324.70 μmol).

[0481] Its structural characterization data are as follows:

[0482] MS m / z(ESI): 1316.6 [M+18]+

[0483] Its preparation method is as follows:

[0484] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0485] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0486] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0.00 55 45 30 2.00 55 45 30 16.00 99 1 30

[0487] Step 2: (5-((S)-2-aminopropionamido)-2-(((((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethyl Preparation of -21-oxooctadecylhydro-25H-2,27:5,8:11,15-tricyclooxy-23,25-bridged ethylene-2,28-methylfuran[3,2-i]furan[2',3':5,6]pyran[4,3-b][1,4]dioxane-18-yl)propyl)carbamoyl)oxy)methyl)benzyl)methyl)carbamate (methyl)carbamate (I-1-2)

[0488] I-1-1 (400 mg, 307.81 μmol) and diethylamine (100 mg, 1.36 mmol) were added to DMF (5 mL). After the addition was complete, the mixture was stirred at 25 °C for 3 h. The reaction solution was purified by rapid column chromatography (C18, 0.05% sodium bicarbonate aqueous solution / acetonitrile = 0%–80%) and then freeze-dried to give the title compound (240 mg, 204.96 μmol).

[0489] Its structural characterization data are as follows:

[0490] MS m / z (ESI): 1077.2 [M+H] +

[0491] Step 3: (5-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutamido)propamido)-2-(((((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methyl Preparation of oxy-13-methyl-7,14-dimethylene-21-oxooctacosahedrohydro-25H-2,27:5,8:11,15-tricyclooxy-23,25-bridged ethylene-2,28-furanmethane[3,2-i]furan[2',3':5,6]pyran[4,3-b][1,4]dioxane-18-yl)propyl)carbamoyl)oxy)methyl)benzyl)methyl)carbamate (I-1-3)

[0492] I-1-2 (270 mg, 250.64 μmol), (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (89.32 mg, 263.17 μmol), DIPEA (129.57 mg, 1.00 mmol), and HATU (142.94 mg, 375.95 μmol) were added sequentially to DMF (3 mL). After the addition was complete, the mixture was stirred at 25 °C for 4 h. The reaction solution was extracted with water (60 mL) and ethyl acetate (40 mL * 3). The organic phase was washed once with water and once with saturated brine, and dried by rotary evaporation to obtain the crude product of the title compound (411 mg).

[0493] Its structural characterization data are as follows:

[0494] MS m / z (ESI): 1416.7 [M+18] +

[0495] Step 4: (5-((S)-2-((S)-2-amino-3-methylbutamido)propamido)-2-(((((S)-2-hydroxy-3-((2S,5S,8S),11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7 Preparation of ,14-dimethylene-21-oxooctadecylhydro-25H-2,27:5,8:11,15-tricyclooxy-23,25-bridged ethylene-2,28-furanmethane[3,2-i]furan[2',3':5,6]pyran[4,3-b][1,4]dioxane-18-yl)propyl)carbamoyl)oxy)methyl)benzyl))methyl)carbamate(methyl)carbamate (I-1-4)

[0496] Crude I-1-3 (350 mg) and diethylamine (54.91 mg, 750.74 μmol) were added to DMF (4 mL). After the addition was complete, the mixture was stirred at 25 °C for 3 h. The reaction solution was purified by rapid column chromatography (C18, 0.05% sodium bicarbonate aqueous solution / acetonitrile = 0%–80%) and then freeze-dried to give the title compound (250 mg, 195.51 μmol).

[0497] Its structural characterization data are as follows:

[0498] MS m / z (ESI): 1177.76 [M+H] +

[0499] Step 5: (5-((22S,25S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amino)-2-(((((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aS) Preparation of R,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctadecylhydro-25H-2,27:5,8:11,15-tricyclooxy-23,25-bridged ethylene-2,28-furanmethane[3,2-i]furan[2',3':5,6]pyrano[4,3-b][1,4]dioxane-18-yl)propyl)carbamoyl)oxy)methyl)benzyl)methyl)carbamate(methyl)carbamate (I-1-5)

[0500] I-1-4 (240 mg, 204.01 μmol), INT-5 (253.83 mg, 244.82 μmol), and DIPEA (131.83 mg, 1.02 mmol) were added sequentially to DMF (2 mL). After stirring for 5 min, HATU (116.35 mg, 306.02 μmol) was added. After the addition was complete, the mixture was stirred at 25 °C for 2 h. The reaction solution was purified by rapid column chromatography (C18, 0.05% formic acid aqueous solution / acetonitrile = 0%–65%) and then freeze-dried to obtain the title compound (330 mg, 161.13 μmol).

[0501] Its structural characterization data are as follows:

[0502] MS m / z(ESI): 1884.2 [M+H] +

[0503] Step Six: 4-((22S,25S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-((methylamino)methyl)benzyl((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS, Preparation of 23R,24aS,25S,26aR,27S,28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctadecylhydro-25H-2,27:5,8:11,15-tricyclooxy-23,25-bridged ethylene-2,28-furanmethane[3,2-i]furan[2',3':5,6]pyran[4,3-b][1,4]dioxane-18-yl)propyl)carbamate (I-1-6)

[0504] I-1-5 (110 mg, 58.38 μmol) was added to DCM (5 mL), and after purging with nitrogen three times, tetrakis(triphenylphosphine)palladium (20.24 mg, 17.51 ​​μmol), formic acid (50 μL), and N-methylmorpholine (100 μL) were added sequentially. After the addition was complete, the mixture was stirred at 25 °C for 3 h. The reaction solution was purified by rapid column chromatography (C18, 0.05% formic acid aqueous solution / acetonitrile = 0%–43%) and then freeze-dried to obtain the formate salt of the title compound (77 mg, 38.37 μmol).

[0505] Its structural characterization data are as follows:

[0506] MS m / z (ESI): 1799.9 [M+H] +

[0507] Step 7: 2,2',2”-(10-(2-((5-((22S,25S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-22-isopropyl-25-methyl-1,20,23-trioxo-5,8,11,14,17-pentaoxa-2,21,24-triazahexacosane-26-amido)-2-(((((S)-2-hydroxy-3-((2S,5S,8S,11S,13R,15R,16aS,18R,19R,19aS,23R,24aS,25S,26aR,27S, Preparation of 28R,29aR,29bS)-19-methoxy-13-methyl-7,14-dimethylene-21-oxooctadecanohydro-25H-2,27:5,8:11,15-tricyclooxy-23,25-bridged ethylene-2,28-methylfuran[3,2-i]furan[2',3':5,6]pyrano[4,3-b][1,4]dioxane-18-yl)propyl)carbamoyl)oxy)methyl)benzyl))methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (I-1)

[0508] I-1-6 (77 mg, 38.37 μmol), 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (104.58 mg, 208.55 μmol), and DIPEA (86 mg, 667.2 μmol) were added sequentially to DMF (3 mL). After the addition was complete, the mixture was stirred at 25 °C for 6 h. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the formate salt of the title compound (40.21 mg, 17.11 μmol).

[0509] Its structural characterization data are as follows:

[0510] MS m / z(ESI): 1094.0 [1 / 2M+H] +

[0511] Its preparation method is as follows:

[0512] Column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

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

[0514] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0.00 25 75 28 2.00 25 75 28 24.00 90 10 28

[0515] III. Conjugation of antibodies with compounds containing cellular bioactive molecules and linkers

[0516] 1. Example: Preparation of Trastuzumab-I-1

[0517]

[0518] Take 0.27 mL of Trastuzumab antibody (14.8 mg / mL), dilute with 14 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.60 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 15.2 μL, pH 7.60) solution and mix well. Incubate at room temperature for 1.5 h. Then add 6 times the amount of antibody dissolved in dimethyl sulfoxide I-1 (17.4 μL, 10 mM) solution and mix well. Incubate at room temperature for 18 h. After completion, replace the buffer with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., ADC Trastuzumab-I-1). The DAR value was 4.23 as determined by mass spectrometry.

[0519] Table 9: Measured molecular weight and DAR calculation of Trastuzumab-I-1

[0520]

[0521] IV. Evaluation of the inhibitory effect of antibody-drug conjugates on tumor growth in a mouse subcutaneous xenograft model

[0522] The formulation containing the ADC of the present invention was administered to a subcutaneously transplanted human gastric cancer cell line NCI-N87 mouse CDX model via tail vein injection. Tumor volume and animal weight changes were measured twice a week to calculate the tumor-suppressing efficacy of the ADC of the present invention in tumor-bearing mice.

[0523] test drug

[0524] Drug name, source, and preparation method: Take an appropriate amount of the ADC of this invention (Sichuan Kelun Biotech Co., Ltd.), and dilute the stock solution to the dosing solution with 0.9% NaCl injection at a dosage volume of 1 mg / kg. Use 0.9% NaCl injection as a solvent control (Vehicle).

[0525] Laboratory animals and cell lines

[0526] Balb / c Nude mice (Chengdu Yaokang Biotechnology Co., Ltd.)

[0527] Human gastric cancer cells NCI-N87 (ATCC)

[0528] Experimental grouping and evaluation methods

[0529] Select tumors with an average volume of approximately 150 mm. 3 Tumor-bearing mice were randomly divided into groups (the number of groups was determined based on the sample size). Each group was administered either 0.9% NaCl injection (hereinafter referred to as the solvent control, Vehicle) or the ADC of this invention. The dosing frequency is detailed in the specific embodiments. The administration method was tail vein injection, with a volume of 10 ml / kg. After administration, the tumor diameter was measured twice weekly using calipers, and the tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

[0530] The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the tumor-suppressing efficacy of the ADC of this invention:

[0531] V T末 >V T0 TGI(%) = [1 - (V) T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 TGI(%) = [1-(V T末 -

[0532] VT0) / VT0]*100%.

[0533] Where V T末 Mean tumor volume at the end of the experiment in the treatment group

[0534] V T0 Mean tumor volume at the start of treatment in the treatment group

[0535] V C末 Mean tumor volume at the end of the experiment in the negative control group

[0536] V C0 Mean tumor volume at the start of drug administration in the negative control group

[0537] The tumor relative proliferation rate (T / C%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of the ADC of this invention:

[0538] T / C = (V T末 / V T0 ) / (V C末 / V C0 ).

[0539] V. Evaluation of the tumor growth inhibitory effect of the antibody-drug conjugate on a mouse subcutaneous xenograft tumor model

[0540] The preparation containing the ADC of the present invention was respectively administered by tail vein injection to a CDX model of mice subcutaneously transplanted with human breast cancer cells JIMT-1. The tumor volume and the change in animal body weight were measured twice a week, and the antitumor efficacy of the ADC of the present invention on tumor-bearing mice was calculated.

[0541] Test drug

[0542] Drug name, source, and preparation method: Take an appropriate amount of the ADC of the present invention (Sichuan Kelunbo Pharma Co., Ltd.), and dilute the mother liquor to the administration solution with 0.9% NaCl injection according to the administration volume of 3 mg / kg. 0.9% NaCl injection was used as the vehicle control (Vehicle).

[0543] Experimental animals and cell lines

[0544] NOD SCID mice (Chengdu Yakang Biotechnology Co., Ltd., production license number: SCXK (Sichuan) 2020-0034, animal certificate number: 511214900024561)

[0545] Human breast cancer cell line JIMT-1 (Nanjing Kebai)

[0546] Experimental grouping and evaluation method

[0547] Tumor-bearing mice with an average tumor volume of approximately 150 mm 3 were randomly grouped (the number of groups was determined according to the number of samples). According to the group, 0.9% NaCl injection (hereinafter referred to as vehicle control, Vehicle) and the ADC of the present invention were respectively administered. The administration frequency is shown in the specific examples. The administration method was tail vein injection, and the administration volume was 10 ml / kg. After administration, the tumor diameter was measured twice a week with a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. The death of animals was observed and recorded every day.

[0548] The following formula was used to calculate the tumor growth inhibition rate TGI (%) to evaluate the antitumor efficacy of the ADC of the present invention:

[0549] V T末 >V T0 , TGI (%) = [1 - (V T末 -V T0 ) / (V C末 -V C0 )] * 100% or V T末 ≤V T0TGI(%) = [1 - (V) T末 -

[0550] VT0) / VT0]*100%.

[0551] Where V T末 Mean tumor volume at the end of the experiment in the treatment group

[0552] V T0 Mean tumor volume at the start of treatment in the treatment group

[0553] V C末 Mean tumor volume at the end of the experiment in the negative control group

[0554] V C0 Mean tumor volume at the start of drug administration in the negative control group

[0555] The tumor relative proliferation rate (T / C%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of the ADC of this invention:

[0556] T / C = (V T末 / V T0 ) / (V C末 / V C0 ).

[0557] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound or a pharmaceutically acceptable salt thereof having the structure shown in formula M'-LED, wherein: M' is -M-Lg, where Lg is the leaving group of the nucleophilic substitution reaction, and M is the structural fragment that binds to the target site; L is a structural segment connecting M and E; E is a structural segment connecting L and D; D is a cytotoxic drug fragment.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, Lg is selected from halogens (e.g., F, Cl, Br, I) and halogenated C. 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, 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, halophenoxy, hydroxy (-OH), mercapto (-SH), amino (-NH2), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments, wherein the haloC 1-6 Alkyl, C 1-6 Alkyl sulfonyl, halogenated C 1-6 Alkyl sulfonyl, 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, halophenoxy, alkenyl, ynyl and ynyl-containing structural segments may optionally be substituted by one or more suitable substituents; Preferably, Lg is selected from halogens, substituted or unsubstituted C. 1-6 Alkyl sulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH), or amino (-NH2); Preferably, Lg is selected from C 1-6 Alkyl sulfonyl group, for example, methyl sulfonyl group.

3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein, The M structure can be substituted or unsubstituted.

4. The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein, L is selected from one or more of the following divalent or unsubstituted structural segments: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, -N(R')-, carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)rOCH3)), and short peptides composed of amino acids (e.g., Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys). Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit , Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, Ala-Ala-Glu), R' is composed of one or more of the following groups, including but not limited to hydrogen and C. 1-6 Alkyl, C 1-6 Alkyl group, amino group, hydroxyl group, carboxyl group, acyl group, -O-, glucosyl group, galactosyl group, glucuronic acid group, galacturonic acid group, -CH2N(C 1-6 Alkyl)-C(=O)-(CH2CH2O) r -C 1-6 Alkyl group, -(CH2N(Me)-C(=O)) r -C 1-6 Alkyl groups, polyethylene glycol segments containing 1-10 EO units (i.e., -(CH2CH2O)). 1-10 -C 1-6 Alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA, wherein r is selected from integers 1-20, such as integers 1-15, such as 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; s is selected from integers 1-20, such as integers 1-15, such as 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

5. The compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, wherein L is selected from one or more of the following substituted or unsubstituted structural fragments: R' consists of one or more of the following groups, including but not limited to -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA, wherein r is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); s is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); preferably, R' is -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA; or L is selected from one or more of the following substituted or unsubstituted structural segments: R' consists of one or more of the following groups, including but not limited to -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA, -C 1-6 Alkyl-N(C) 1-6 Alkyl)-DOTAGA, or -C 1-6 Alkyl-N(C) 1-6 Alkyl)-NOTA, wherein r is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 1-10, 1-8, 3-8, 1-6, 1-4, 1-2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); s is selected from integers 1-20 (e.g., integers 1-15, such as 1-12, 3-12, 5-10, 8-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); preferably, R' is -C 1-6 Alkylene-N(C) 1-6 Alkyl)-DOTA.

6. The compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein L is selected from the following substituted or unsubstituted structural fragments: Where s is selected from an integer from 1 to 20, preferably from an integer from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, 8 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10.

7. The compound according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein, E is a single bond, substituted or unsubstituted -NH-CH2-, or selected from the following substituted or unsubstituted structural segments:

8. The compound according to any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein, E is a single bond, substituted or unsubstituted -NH-CH2-, or For example, 9. The compound according to any one of claims 1-8 or a pharmaceutically acceptable salt thereof, wherein, Selected from the following substituted or unsubstituted structures: Where s is selected from an integer from 1 to 20, preferably selected from an integer from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, 8 to 10, for example, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

10. The compound of any one of claims 1-9 or a pharmaceutically acceptable salt thereof, wherein, The cytotoxic drug is an eribulin-like compound; Preferably, the cytotoxic drug is selected from the following compounds or compounds labeled with their isotopes: Preferably, the cytotoxic drug is linked to the E in the antibody-drug conjugate via a -OH, primary amino, secondary amino, or tertiary amino group thereon.

11. The compound according to any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein, The compound is selected from I'-1 to I'-2 or I-1 to I-2: Where s is selected from integers from 1 to 20, such as integers from 1 to 15, such as integers from 1 to 12, 3 to 12, 5 to 10, 8 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

12. A coupling having the structure shown in formula Ab-[MLED]x, Ab is the target region; M, L, E and D are as described in any one of claims 1-11; x is between 1 and 10.

13. The coupling of claim 12, wherein, Ab is an antibody or its antigen-binding fragment; preferably, Ab is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase. Preferably, the antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL): (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions; or, (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL): (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or, (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions; or, (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL): (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions; or, (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL): (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or, (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions; Preferably, the antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system: (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions; or, (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system: (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or, (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions; or, (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system: (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (3a), (3b), and (3c) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions; or, (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system: (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or, (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence 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 respect to the sequence from which it originates, 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 originates; preferably, the substitutions are conservative substitutions; Preferably, the antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is 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 regions (VH) and light chain variable regions (VL), wherein the CDR is 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 regions (VH) and light chain variable regions (VL), where CDR is 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 regions (VH) and light chain variable regions (VL), wherein the CDR is 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 its antigen-binding fragment 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; The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with its source sequence, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to its source sequence; preferably, the substitutions are conservative substitutions; Preferably, the antibody or its antigen-binding fragment 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 its antigen-binding fragment further comprises: (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids). Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region. Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35; Preferably, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment 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 its antigen-binding fragment comprises: (1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or (2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO:

36.

14. The coupling according to any one of claims 12-13, wherein, x is 3 to 6, more preferably x is about 4; or x is 1 to 4, more preferably x is about 2.

15. The coupling according to any one of claims 12-14, wherein, MLED can be obtained from M'-LED by a substitution reaction (e.g., removal of the -SO2Me structure), wherein M', L, E, and D are as described in any one of claims 1-11.

16. The coupling according to any one of claims 12-15, wherein, M is attached to a thiol group (-SH) or an amino group (-NH2) on Ab.

17. The coupling agent according to any one of claims 12-16, wherein the coupling agent is selected from: in, HA is an antibody or its antigen-binding fragment, preferably an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase group, and the antibody or its antigen-binding fragment is preferably as defined in claim 13; s is an integer selected from 1 to 20, preferably an integer selected from 1 to 15, such as integers 1 to 12, 3 to 12, 5 to 10, 8 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, preferably 5, 8, 10; This indicates the specific way in which the thiol group in the antibody or its antigen-binding fragment is linked to the linker.

18. A composition comprising one or more conjugates according to any one of claims 13-17, wherein the DAR value (drug-antibody conjugate ratio) of the composition is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5- 7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-8, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.0, 6.5-8.5, 7.0-7.5, 7.0-8.0, or 7.5-8.

0. Alternatively, the composition has a DAR value of about 1.0 to 6.0, for example, about 1.0 to 5.5, about 1.0 to 5.0, about 1.5 to 6.0, about 1.5 to about 5.5, about 1.5 to 5.0, 2.0 to 5.5, or about 2.0 to about 5.0, for example, about 1.0, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.2, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, or about 1.

26. Approximately 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47 Approximately 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.

68. Approximately 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89 Approximately 1.9, approximately 1.91, approximately 1.92, approximately 1.93, approximately 1.94, approximately 1.95, approximately 1.96, approximately 1.97, approximately 1.98, approximately 1.99, approximately 2.0, approximately 2.01, approximately 2.02, approximately 2.03, approximately 2.04, approximately 2.05, approximately 2.06, approximately 2.07, approximately 2.08, approximately 2.09, approximately 2.1, approximately 2 .11, about 2.12, about 2.13, about 2.14, about 2.15, about 2.16, about 2.17, about 2.18, about 2.19, about 2.2, about 2.21, about 2.22, about 2.23, about 2.24, about 2.25, about 2.26, about 2.27, about 2.28, about 2.29, about 2.3, about 2.31, about 2 .32, about 2.33, about 2.34, about 2.35, about 2.36, about 2.37, about 2.38, about 2.39, about 2.4, about 2.41, about 2.42, about 2.43, about 2.44, about 2.45, about 2.46, about 2.47, about 2.48, about 2.49, about 2.5, about 2.51, about 2.52, about 2.53, approximately 2.54, approximately 2.55, approximately 2.56, approximately 2.57, approximately 2.58, approximately 2.59, approximately 2.6, approximately 2.61, approximately 2.62, approximately 2.63, approximately 2.64, approximately 2.65, approximately 2.66, approximately 2.67, approximately 2.68, approximately 2.69, approximately 2.7, approximately 2.71, approximately 2.72, approximately 2.73, approximately 2. 74, approximately 2.75, approximately 2.76, approximately 2.77, approximately 2.78, approximately 2.79, approximately 2.8, approximately 2.81, approximately 2.82, approximately 2.83, approximately 2.84, approximately 2.85, approximately 2.86, approximately 2.87, approximately 2.88, approximately 2.89, approximately 2.9, approximately 2.91, approximately 2.92, approximately 2.93, approximately 2.94, approximately 2.9 5. Approximately 2.96, 2.97, 2.98, 2.99, 3.0, 3.01, 3.02, 3.03, 3.04, 3.05, 3.06, 3.07, 3.08, 3.09, 3.1, 3.11, 3.12, 3.13, 3.14, 3.15, 3.1 6, approximately 3.17, approximately 3.18, approximately 3.19, approximately 3.2, approximately 3.21, approximately 3.22, approximately 3.23, approximately 3.24, approximately 3.25, approximately 3.26, approximately 3.27, approximately 3.28, approximately 3.29, approximately 3.3, approximately 3.31, approximately 3.32, approximately 3.33, approximately 3.34, approximately 3.35, approximately 3.36, approximately 3.37 Approximately 3.38, 3.39, 3.4, 3.41, 3.42, 3.43, 3.44, 3.45, 3.46, 3.47, 3.48, 3.49, 3.5, 3.51, 3.52, 3.53, 3.54, 3.55, 3.56, 3.57, 3.58 Approximately 3.59, approximately 3.6, approximately 3.61, approximately 3.62, approximately 3.63, approximately 3.64, approximately 3.65, approximately 3.66, approximately 3.67, approximately 3.68, approximately 3.69, approximately 3.7, approximately 3.71, approximately 3.72, approximately 3.73, approximately 3.74, approximately 3.75, approximately 3.76, approximately 3.77, approximately 3.78, approximately 3.79 Approximately 3.8, approximately 3.81, approximately 3.82, approximately 3.83, approximately 3.84, approximately 3.85, approximately 3.86, approximately 3.87, approximately 3.88, approximately 3.89, approximately 3.9, approximately 3.91, approximately 3.92, approximately 3.93, approximately 3.94, approximately 3.95, approximately 3.96, approximately 3.97, approximately 3.98, approximately 3.99, approximately 4.0, approximately 4 .01, about 4.02, about 4.03, about 4.04, about 4.05, about 4.06, about 4.07, about 4.08, about 4.09, about 4.1, about 4.11, about 4.12, about 4.13, about 4.14, about 4.15, about 4.16, about 4.17, about 4.18, about 4.19, about 4.2, about 4.21, about 4.22, approximately 4.23, approximately 4.24, approximately 4.25, approximately 4.26, approximately 4.27, approximately 4.28, approximately 4.29, approximately 4.3, approximately 4.31, approximately 4.32, approximately 4.33, approximately 4.34, approximately 4.35, approximately 4.36, approximately 4.37, approximately 4.38, approximately 4.39, approximately 4.4, approximately 4.41, approximately 4.42, approximately 4.43, approximately 4.44, approximately 4.45, approximately 4.46, approximately 4.47, approximately 4.48, approximately 4.49, approximately 4.5, approximately 4.51, approximately 4.52, approximately 4.53, approximately 4.54, approximately 4.55, approximately 4.56, approximately 4.57, approximately 4.58, approximately 4.59, approximately 4.6, approximately 4.6 1. Approximately 4.62, 4.63, 4.64, 4.65, 4.66, 4.67, 4.68, 4.69, 4.7, 4.71, 4.72, 4.73, 4.74, 4.75, 4.76, 4.77, 4.78, 4.79, 4.8, 4.81, 4.82, 4.83, 4.84, 4.85, 4.86, 4.87, 4.88, 4.89, 4.9, 4.91, 4.92, 4.93, 4.94, 4.95, 4.96, 4.97, 4.98, 4.99, 5.

0.

19. A pharmaceutical composition comprising the conjugate of any one of claims 12-17, or the composition of claim 18, and one or more pharmaceutical excipients.

20. Use of the conjugate of any one of claims 12-17, or the composition of claim 18, or the pharmaceutical composition of claim 19 in the preparation of a medicament for treating cancer.

21. The use of claim 20, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

22. The conjugate of any one of claims 12-17, or the composition of claim 18, or the pharmaceutical composition of claim 19, for the treatment of cancer.

23. The conjugate of any one of claims 12-17, or the composition of claim 18, or the pharmaceutical composition of claim 19, for the treatment of solid tumors or hematologic malignancies, for example selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), and urothelial carcinoma.

24. A method for the prevention or treatment of cancer, the method comprising a preventive or therapeutically effective amount of the conjugate of any one of claims 12-17, or the composition of claim 18, or the pharmaceutical composition of claim 19, based on an individual in need.

25. The method of claim 24, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

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