Method for preparing antibody-drug conjugate

Abstract

The present invention relates to a method for preparing an antibody-drug conjugate. According to the method, a highly homogeneous antibody conjugate molecule is obtained by means of a specific chemical site-directed conjugation method, and the proportion of a DAR2 main peak thereof reaches 99% or above. The preparation method of the present invention has few steps, and is easy to operate, and is thus beneficial for industrial scaled-up production. In addition, the prepared product has few impurities and a high purity, the medication safety is obviously improved, and the production cost is also reduced.

Description

A method for preparing antibody drug conjugates Technical Field

[0001] The present invention belongs to the field of biomedicine, and more specifically, relates to a new method for preparing antibody-drug conjugates.

[0002] Background Overview

[0003] Antibody-drug conjugates (ADCs) are a new drug modality that links small-molecule drugs to monoclonal antibodies. Leveraging the targeting capabilities of monoclonal antibodies, they deliver small-molecule drugs to target sites for efficacy, minimizing toxic side effects and increasing the therapeutic window. In recent years, ADCs have become a hot topic in precision cancer therapy, offering hope for cancer treatment. ADCs are composed of an antibody drug targeting a specific antigen and a small-molecule cytotoxic drug linked via a linker. They combine the powerful anti-tumor effects of traditional small-molecule chemotherapy with the tumor-targeting properties of antibody drugs. To date, more than ten ADCs have been approved for marketing worldwide.

[0004] Antibody-drug conjugates consist of several components, including an antibody, a linker, and a payload. The antibody and drug-linker can be connected using various coupling methods. Coupling methods are primarily categorized as non-site-specific and site-specific. Early non-site-specific coupling methods, primarily lysine and cysteine ​​coupling, directly couple the drug to amino acid residues on the antibody using chemical methods without involving antibody modification or alteration. The number of toxin molecules coupled and the coupling site cannot be determined, resulting in poor uniformity. Currently, the most commonly used site-specific coupling method involves specific coupling through genetically engineered sites or special linkers, enabling more uniform coupling and enabling attachment of cytotoxins at specific sites. Antibody-drug conjugates produced using site-specific coupling can reduce fluctuations in efficacy, pharmacokinetic properties, and quality control caused by differences in the number of coupling sites and number of couplings. Common site-specific coupling methods include THIOMAB technology, non-natural amino acid coupling technology, glutamine enzymatic coupling technology, Sortase transpeptidase coupling technology, and Thiobridge technology. Among them, antibody modification through antibody engineering or enzymatic coupling may have a certain impact on the structural stability of the antibody and also have certain requirements for CMC. In addition, the Thiobridge technology using chemical coupling also has certain drawbacks. For example, DBM (dibromomaleimides)-type linkers undergo displacement reactions with other sulfhydryl-containing biological groups, which are unstable in plasma, resulting in reduced efficacy and increased toxic side effects (Chem.-Eur.J, 2019, 25, 43-59.).

[0005] The drug-to-antibody ratio (DAR) is a unique and important quality attribute of ADCs. It represents the average number of small molecule drugs conjugated to an antibody. The degree of drug conjugation affects the stability and aggregation tendency of ADCs. Therefore, the DAR value is a key quality attribute for ADC analysis and a crucial quality control step in ADC drug development. Site-specific conjugation technology enables the targeted and quantitative conjugation of antibodies and small molecule toxins. ADCs obtained using this technology exhibit appropriate DARs, high uniformity, excellent stability, and high batch-to-batch reproducibility, resulting in improved activity and pharmacokinetic properties. These ADCs are also more suitable for large-scale ADC production. The current trend in ADC drug development is to use site-specific conjugation methods to conjugate highly active small molecule drugs to antibodies, creating DAR2-targeted ADCs. For example, the approved CD19-targeted ADC (loncastuximab tesirine, Zynlonta) uses glycosylation-specific site-specific conjugation technology to conjugate a PBD small molecule to an antibody, achieving DAR2 targeting. The HER2-targeted ADC (ARX788) and the FRα-targeted ADC (MORAb-202) also utilize unnatural amino acid mutations to achieve DAR2 targeting. However, these methods require either the use of enzymes or modification of the antibody sequence, making the manufacturing process complex.

[0006] WO2022253033A1 discloses the following method for preparing an ADC conjugate: dilute the antibody with a solution of disodium edetate, then adjust the pH with a Na2HPO4 solution, add a TCEP (tris(2-carboxyethyl)phosphine) solution, mix well, and let stand at room temperature for 2 hours. To the above solution system, add a drug-linker dissolved in DMSO at an amount 5-10 times that of the antibody, mix well, let stand at room temperature for 20 hours, and then use a NAP-5 gel column (Cytiva) to replace the buffer with a 10mM histidine buffer solution at pH 6.0 to obtain an ADC product. Sample measurement shows that the DAR of the ADC conjugate prepared by the above non-site-specific conjugation method in this patent is 4, and there is no record of a DAR of 2.

[0007] WO2022078524A2 discloses a method for site-specific conjugation of antibody conjugates. After incubating the antibody with a variety of zinc complexes and TCEP for reduction, a payload / linker complex is introduced. Cysteine ​​is then added to consume excess TCEP and excess payload. Free thiol groups in the protein are oxidized. Finally, the reaction mixture is purified using a desalting column, UF / DF, or ion exchange chromatography. The results of HIC-HPLC analysis of the conjugate DAR showed that the proportion of conjugates with a DAR of 4 was about 60-70%, while the proportion of conjugates with a DAR of 2 was only 5-10%. It can be seen that the DAR of the antibody conjugates prepared using this patented method is mainly concentrated in 4, while the conjugate products with a DAR of 2 account for only a minority.

[0008] WO2020164561A1 prepares an antibody-drug conjugate (ADC) with improved homogeneity, and the coupling method used includes the following steps:

[0009] (a) Incubating a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)) and the antibody to be conjugated in a buffer system (e.g., Hepes, histidine buffer, PBS, MES, etc.) in the presence of an effective amount of a transition metal ion (e.g., Zn2+, etc.) to selectively reduce intrachain disulfide bonds within the antibody; (b) reacting an excess of a payload carrying a reactive group (e.g., a maleimide-linked drug) with the reduced sulfhydryl groups obtained in step (a); and (c) adding an effective amount of an oxidizing agent (e.g., dehydroascorbic acid (DHAA)) to reoxidize the unreacted sulfhydryl groups, and then recovering the resulting antibody-drug conjugate. Although the homogeneity of the antibody-drug conjugate (ADC) produced by this coupling method can be significantly improved, the weight ratio of DAR2 of its ADC product is only 10-28%, which is much lower than the weight ratio of DAR4.

[0010] For ADCs, the DAR value is a key quality attribute. While a high DAR can deliver more small molecule drugs to the target site, potentially leading to better therapeutic effects, it can compromise the stability and homogeneity of the ADC drug, potentially causing antibody aggregation, which can affect the ADC's efficacy and lead to significant side effects. Preparing ADCs with high homogeneity and / or purity and a DAR of 2 can avoid the adverse effects of a high DAR and improve the stability and efficacy of the ADC drug. However, as mentioned above, existing technologies can achieve a site-specific DAR of 2 through glycosylation-directed conjugation techniques. However, these methods either require the use of enzymes or antibody sequence modification, resulting in complex manufacturing processes. Currently reported chemical site-directed conjugation methods for preparing ADCs, while relatively simple, can only produce ADC products with a primary DAR of 4.

[0011] Therefore, there is an urgent need in this field for a simpler and more effective method for preparing site-directed DAR2-ADC.

[0012] Summary of the Invention

[0013] The present invention provides a novel method for preparing antibody-drug conjugates. Through a specific chemical site-directed conjugation method, highly homogeneous antibody conjugate molecules are obtained, with the DAR2 main peak accounting for over 99%. This method has few steps and is simple to operate, facilitating industrial scale-up. Furthermore, the product produced has low impurities and high purity, significantly improving drug safety and reducing production costs. The anti-tumor drug prepared using this conjugation method exhibits excellent anti-tumor efficacy and safety, including high stability and minimal side effects.

[0014] First aspect

[0015] The present invention provides a method for preparing an antibody-drug conjugate (ADC), the method comprising the following steps:

[0016] (a) adding a metal salt or metal complex and a reducing agent to a buffer solution containing an antibody, followed by incubation;

[0017] (b) adding a drug-containing linker to the reaction solution in (a) for coupling; and

[0018] (c) Purify the conjugate product using hydrophobic interaction chromatography (HICP) with a hydrophobic gel-type filler and a salt solution to obtain a high-purity DAR2-ADC conjugate.

[0019] In some embodiments, the metal in the metal salt or metal complex in step (a) can be selected from Zn, Cd, Hg, and the like.

[0020] In some embodiments, the metal salt or metal complex in step (a) is selected from one or more of the following: hydrochloride or sulfate of Zn, Cd and Hg, The Zn salts or Zn complexes disclosed in WO2022078524A2 include but are not limited to

[0021] Zn(NH2CH3)2 2+ , Zn(NH2CH2CH3)2 2+ , Zn(NH2CH2CH2CH3)2 2+ , Zn(NH2CH(CH3)2)2 2+ ,

[0022] Zn(NH2C(CH3)3)2 2+ , Zn(NH2CH2C(CH3)3)2 2+,Zn(NH(CH3)2)2 2+ ,Zn(NH(CH2CH3)2)2 2+ ,

[0023] Zn(NH(CH(CH3)2)2)2 2+ ,Zn(NH(C(CH3)3)2)2 2+ ,Zn(NH(CH(CH2CH3)2)2)2 2+ ,

[0024] Zn(NH(CH2C(CH3)3)2)2 2+ ,Zn(NH(CH2C(CH2CH3)3)2)2 2+ ,Zn(NH(CH2CH2C(CH3)3)2)2 2+ ,

[0025] Zn(NH2CH2CH2OH)2 2+ ,Zn(NH(CH2CH2OH)2)2 2+ ,Zn(N(CH2CH2OH)3)2 2+ ,

[0026] Zn(NH2CH2COOH)2 2+ ,Zn(NH2CH2CONH2)2 2+ ,Zn(NH2CH2COOCH3)2 2+ ,

[0027] Zn(NH2CH2COOCH2CH3)2 2+ ,Zn(NH2CH2COOC(CH3)3)2 2+ ,Zn(NH2CH2COOCH(CH3)2)2 2+ ,

[0028] Zn(NH2CH2CH2COOH)2 2+ ,Zn(NH(CH2COOH)2)2 2+ ,Zn(N(CH2CH2COOH)3)2 2+ ,Zn(NH2CH3)4 2+ ,

[0029] Zn(NH2CH2CH3)4 2+ ,Zn(NH2CH2CH2CH3)4 2+ ,Zn(NH2CH(CH3)2)4 2+ ,Zn(NH2C(CH3)3)4 2+ ,

[0030] Zn(NH2CH2C(CH3)3)4 2+ , Zn(NH(CH3)2)4 2+ , Zn(NH(CH2CH3)2)4 2+ ,Zn(NH(CH(CH3)2)2)4 2+ ,

[0031] Zn(NH(C(CH3)3)2)4 2+ , Zn(NH(CH(CH2CH3)2)2)4 2+ , Zn(NH(CH2C(CH3)3)2)4 2+ ,

[0032] Zn(NH(CH2C(CH2CH3)3)2)4 2+ , Zn(NH(CH2CH2C(CH3)3)2)4 2+ , Zn(NH2CH2CH2OH)4 2+ ,.

[0033] In some embodiments, the metal salt or metal complex in step (a) is selected from ZnCl2, CdCl2, HgCl2, One or more of .

[0034] In some embodiments, the reducing agent in step (a) is selected from tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), mercaptoethylamine and mercaptoethanol, preferably TCEP.

[0035] In some embodiments, the concentration of the antibody in step (a) is 0.5-150 mg / mL, e.g., 1-75 mg / mL, 1-50 mg / mL, 2-40 mg / mL, 3-30 mg / mL, 4-30 mg / mL, or 5-15 mg / mL, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, or 150 mg / mL.

[0036] In some embodiments, the amount of the reducing agent used in step (a) is 1-6 times the molar equivalent of the antibody, such as 2-6, 3-5, 3.5-5.0 or 4.0-4.5 times the molar equivalent of the antibody, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 times the molar equivalent of the antibody.

[0037] In some embodiments, the amount of the metal salt or metal complex used in step (a) is 1-5 times the molar equivalent of the antibody, such as 1-4, 1-3 or 1.5-2.5 times the molar equivalent of the antibody, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 times the molar equivalent of the antibody.

[0038] In some embodiments, the buffer solution in step (a) has a pH of 5-8, such as a pH of 5.5-7.5, 6.5-7.5 or 6.5-7.4, such as a pH of 5.0, 5.5, 6.0, 6.5, 6.9, 7.0, 7.5 or 8.0, preferably a pH of 6.9.

[0039] In some embodiments, the buffer solution in step (a) is selected from phosphate buffer, acetate buffer, citrate buffer, succinate buffer, preferably sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer. Preferably, the buffer concentration in the buffer solution is 5-100mM, such as 10-90mM, 20-80mM, 30-70mM, 40-60mM, 10-30 or mM, such as 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100mM.

[0040] In some embodiments, the incubation in step (a) is performed at 0-30°C, such as 0-15°C or 5-15°C.

[0041] It should be understood that those skilled in the art can easily determine the endpoint of each reaction of the present invention, thereby determining the time for the reaction to proceed.

[0042] In some embodiments, the incubation in step (a) is performed for 1-48 hours, such as 2-24 hours, 2-12 hours, 3-10 hours, 4-8 hours or 5-24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 35 hours.

[0043] In some embodiments, the amount of the drug-containing linker in step (b) is 1-8 times the molar equivalent of the antibody, such as 2-6, 2.5-5.5, 3-5 or 3.5-4.5 times the molar equivalent of the antibody, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 times the molar equivalent of the antibody.

[0044] In some embodiments, the coupling reaction temperature in step (b) is 0-30°C, such as 0-15°C or 3-15°C.

[0045] In some embodiments, the reaction solution in step (b) has a pH of 5-8, such as a pH of 5.5-7.5, 6.5-7.5, or 6.5-7.4, such as a pH of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0.

[0046] In some embodiments, the drug-containing linker is provided as a solution in DMA.

[0047] In some embodiments, the coupling reaction of step (b) is carried out for 0.2-24 hours, such as 0.5-12 hours, 1-10 hours, 1.5-5 hours, 1-3 hours or 1-4 hours, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 hours.

[0048] In some embodiments, the hydrophobic gel filler in step (c) is selected from Butyl-650M, Butyl-4FF, Butyl-650S, Butyl-ImpRes and Phenyl Sepharose HP.

[0049] In some embodiments, the type of salt in step (c) is selected from sulfates, chlorides, and the like, such as alkali metal (e.g., lithium, sodium, potassium) salts of sulfuric acid or hydrochloric acid, alkaline earth metal (e.g., calcium, magnesium) salts, salts formed by amines or ammonia with sulfuric acid or hydrochloric acid, preferably ammonium sulfate or sodium chloride; preferably, the salt concentration is 0.1-5 mol / L, such as 0.2-4 mol / L, 0.3-3 mol / L, 0.5-2 mol / L, 0.5-1.5 mol / L, 0.6-1.2 mol / L or 0.6-1.0 mol / L, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5 or 2.0 mol / L.

[0050] In some embodiments, the salt solution of step (c) is used as mobile phase A for hydrophobic interaction chromatography.

[0051] In some embodiments, the hydrophobic interaction chromatography in step (c) uses phosphate buffer as mobile phase B, preferably, the phosphate buffer has a phosphate concentration of 5-200 mM and a pH of 7.0-7.6, such as 7.1, 7.2, 7.3, 7.4 or 7.5.

[0052] In some embodiments, mobile phase A and / or mobile phase B may further contain other solvents such as isopropanol.

[0053] In some embodiments, step (b) is followed by step (b'): adding a metal ion chelating agent such as ethylenediaminetetraacetic acid (EDTA) to carry out the reaction.

[0054] In some embodiments, step (b') is performed at room temperature, eg, for 5 min-5 hours, 10 min-45 min, or 30 min.

[0055] In some embodiments, the amount of metal ion chelator used is 1-8 times the molar equivalent of antibody, such as 2-6, 2.5-5.5, 3-5 or 3.5-4.5 times the molar equivalent of antibody, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 times the molar equivalent of antibody.

[0056] In some embodiments, step (b') is followed by step (b"): adding dehydroascorbic acid (DHAA) to carry out the reaction.

[0057] In some embodiments, step (b") is performed at 25-35°C, for example, for 0.2-24 hours, such as 0.5-12 hours, 1-10 hours, 1.5-5 hours, 1-3 hours or 1-4 hours, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 hours.

[0058] In some embodiments, the amount of DHAA used is 1-16 times the molar equivalent of antibody, such as 2-10, 2-6, 6-16 or 6-12 times the molar equivalent of antibody, such as 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14 or 16 times the molar equivalent of antibody.

[0059] In some embodiments, the DAR2-ADC obtained by the methods of the present invention has a purity greater than 90%, such as greater than 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, as measured, for example, by HIC-HPLC.

[0060] In some embodiments, the antibody is selected from the group consisting of a humanized antibody, a murine antibody, a human antibody, a chimeric antibody, a single-chain antibody, and a bispecific antibody.

[0061] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

[0062] In some embodiments, the antibody is an antibody that binds to the following antigens: HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, C), Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2.

[0063] In some embodiments, the antibody is an anti-HER2 antibody.

[0064] In some embodiments, the antibody comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H, and HCDR3; or LCDR1, LCDR2, and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12, or hH2L1, or comprises the VH and / or VL of the antibody, or comprises the heavy chain and / or light chain of the antibody.

[0065] In some embodiments, the antibody is selected from adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12, and hH2L1.

[0066] In some embodiments, the antibody comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H, and HCDR3; or LCDR1, LCDR2, and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of trastuzumab, Pertuzumab, h23-12, or hH2L1, or comprises the VH and / or VL of trastuzumab, Pertuzumab, h23-12, or hH2L1, or comprises the heavy chain and / or light chain of the antibody.

[0067] In some embodiments, the antibody is selected from trastuzumab, pertuzumab, h23-12, and hH2L1.

[0068] In some embodiments, the drug-containing linker in step (b) is a compound represented by "L1-A-L2-D" or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,

[0069] Wherein L1 represents a linker unit for connecting to an antibody,

[0070] A represents one or more (e.g. 1, 2, 3 or 4) amino acids,

[0071] L2 represents a linker unit for connecting to a small molecule drug, and

[0072] D is a small molecule drug.

[0073] In some embodiments, the drug-containing linker in step (b) is a compound represented by Formula AI or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,

[0074] In the structural formula AI, and in the context of the present invention, unless otherwise specified, E is selected from the following groups, wherein The bond indicates the attachment site to M:

[0075] n is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0076] R6 and R7 are independently halogen or Ar'S-, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group. Preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-formylmorpholine substituted phenyl

[0077] M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in the substituted phenylene group, the substituent is selected from an alkyl group (e.g., a C1-C6 alkyl group, preferably a C1-C4 alkyl group), a haloalkyl group (e.g., a halo-C1-C6 alkyl group, preferably a halo-C1-C4 alkyl group, such as a trifluoromethyl group), an alkoxy group (e.g., a C1-C6 alkoxy group, preferably a C1-C4 alkoxy group, preferably a methoxy group), a halogen, an ester group, an amide group, and a cyano group; preferably, M is a halogen-substituted phenylene group.

[0078] SP1 is selected from C1-8 alkylene, C1-8 alkylenephenylene-, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11) straight-chain heteroalkylene, wherein the C1-21 straight-chain heteroalkylene contains 1-11 (preferably 1-6) heteroatoms selected from N, O or S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight-chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxy, oxo, amino, sulfonic acid and cyano;

[0079] SP2 is selected from -NH(CH2CH2O)aCH2CH2CO-, -NH(CH2CH2O)aCH2CO-, -S(CH2)aCO- or a chemical bond, wherein a is an integer of 1-20, preferably an integer of 1-10, more preferably an integer of 1-6;

[0080] A represents 2-4 amino acids, wherein when A represents 2 amino acids, it can be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO or NH-Val-Cit-CO. t-CO; when A represents 3 amino acids, it can be NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO; when A represents 4 amino acids, it can be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO. Preferably, A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO or NH-Ala-Ala-Ala-CO;

[0081] Preferably, A is selected from the group consisting of NH-Val-Cit-CO, NH-Val-Ala-CO, and NH-Gly-Gly-Phe-Gly-CO;

[0082] It should be understood that the NH on the left side of A is the amino group in the left amino acid of A, and the CO on the right side of A is the carbonyl group in the right amino acid of A.

[0083] L2 is selected from In some embodiments, M is selected from

[0084] In some embodiments, Selected from

[0085] wherein q is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0086] p is an integer selected from 1-20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

[0087] In some embodiments, Selected from

[0088] In some embodiments, D is selected from the group consisting of camptothecins, calicheamicins, maytansinoids, dolastatins, auristatins, and trichothecenes.

[0089] In some embodiments, the maytansinoid derivative is selected from DM1, DM3, and DM4; the auristatin compound is selected from MMAE and MMAF; and the camptothecin compound is selected from camptothecin, 10-hydroxycamptothecin, exatecan, SN-38, topotecan, and camptothecin derivatives.

[0090] In some embodiments, the drug-containing linker is a compound represented by Formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,

[0091] wherein E, M, SP1, SP2 and A are as defined above, and CPT is a camptothecin compound.

[0092] In some embodiments, the drug-containing linker is a compound represented by Formula III' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,

[0093] wherein E, M, SP1, SP2 and A are as defined above, and D' is an auristatin compound.

[0094] In some embodiments, the compound of formula III is further a compound of formula IIIA:

[0095] Wherein, R6 and R7 are independently halogen or Ar'S-, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group. Preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl wherein M, SP1, SP2, A and CPT are as defined above.

[0096] In some embodiments, the compound of formula III' is further a compound of formula IIIA':

[0097] wherein R6, R7, M, SP1, SP2, A and D' are as defined above.

[0098] In some embodiments, in Formula III and Formula IIIA, CPT is a compound represented by Formula I or Formula IA below, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, and the corresponding specific compound:

[0099] In Formula I, and in the context of the present invention, if not otherwise specified, R1, R2, R3, R4 are independently hydrogen, halogen, hydroxyl, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3, R4 together with the carbon atom to which they are connected constitute a C3-6 cyclic alkyl. When R1, R2, R3, R4 are independently C1-6 alkoxy, the C1-6 alkoxy includes straight or branched C1-6 alkoxy, preferably straight or branched C1-3 alkoxy, more preferably methoxy. When R1, R2, R3, R4 are independently substituted amino, the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl. When R1, R2, R3, and R4 are independently C1-7 alkyl or substituted C1-7 alkyl, the C1-7 alkyl or substituted C1-7 alkyl includes linear or branched C1-7 alkyl or substituted C1-7 alkyl, and the substituted C1-7 alkyl is a C1-7 alkyl substituted by one or more substituents selected from cyclopropyl and cyclobutyl; alternatively, the linear or branched C1-7 alkyl or substituted C1-7 alkyl is preferably a C1-3 alkyl or substituted C1-3 alkyl, such as methyl, halogenated methyl (preferably trifluoromethyl).

[0100] In formula I, and in the context of the present invention, unless otherwise stated, G is hydrogen, halogen, methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

[0101] In Formula I, and in the context of the present invention, unless otherwise specified, Y is oxygen, sulfur, sulfone, sulfoxide, methylene, or substituted methylene. A substituted methylene group may have one or both hydrogen atoms replaced, and the substituent may be benzyl or alkyl. When the substituent is an alkyl group, the alkyl group, R3 and / or R4, and the carbon atoms to which they are attached may form a C3-6 membered cyclic or spirocyclic structure. When Y is a substituted methylene group, the substituent is preferably an alkyl group, more preferably a linear or branched C1-4 alkyl group. Preferably, Y is oxygen, sulfur, sulfone, or sulfoxide; alternatively, preferably, Y is oxygen, sulfur, or methylene.

[0102] In formula I, and unless otherwise stated in the context of the present invention, X is oxygen or sulfur.

[0103] In formula I, and in the context of the present invention unless otherwise stated, n=0 or 1.

[0104] In some embodiments of Formula I, when R1, R2, R3, and R4 are simultaneously hydrogen, X is oxygen, and n=0, when Y is methylene, G cannot be hydrogen or fluorine; and when Y is oxygen or sulfur, G cannot be hydrogen.

[0105] Preferably, R1, R2, R3, and R4 are independently hydrogen, halogen (e.g., fluorine), C1-7 alkyl, or substituted C1-7 alkyl, or any two of R1, R2, R3, and R4, together with the carbon atoms to which they are attached, form a C3-6 cyclic alkyl (e.g., a C3-5 cyclic alkyl). Furthermore, R1 and R2 may be the same; and / or R3 and R4 may be the same.

[0106] Preferably, Y is a methylene group substituted by an alkyl group, and the alkyl group, R3 and / or R4, and the carbon atoms to which they are connected can form a C3-6 membered cyclic or spirocyclic structure.

[0107] Preferably, X may be oxygen.

[0108] Preferably, X is oxygen, G is hydrogen, halogen (such as fluorine or chlorine), methyl or methoxy, and Y and R1, R2, R3, R4 are as defined above.

[0109] Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, and R4 are as defined above.

[0110] Preferably, X is oxygen, G is fluorine, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, and R4 are as defined above.

[0111] Preferably, X is oxygen, G is chlorine, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, and R4 are as defined above.

[0112] Preferably, X is oxygen, G is methyl, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, and R4 are as defined above.

[0113] Preferably, X is oxygen, G is methoxy, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, and R4 are as defined above.

[0114] Preferably, X is oxygen, G is hydrogen, Y is oxygen, sulfone or sulfoxide, and R1, R2, R3, and R4 are as defined above.

[0115] Preferably, X is oxygen, G is hydrogen, Y is sulfone or sulfoxide, and R1, R2, R3, and R4 are as defined above.

[0116] Preferably, the compound represented by formula I is further represented by formula IA:

[0117] In formula IA, the groups R1, R2, R3, and R4 are the same as those defined above in formula I, and R1, R2, R3, and R4 may be hydrogen at the same time or at different times.

[0118] In some embodiments, the camptothecin compound is a compound represented by Formula II or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0119] In Formula II, and unless otherwise specified in the context of the present invention, R5 is C1-5 alkyl or C1-5 alkyl substituted with one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted with one or more substituents, phenyl, or substituted phenyl. When R5 is C1-5 alkyl or substituted C1-5 alkyl, the C1-5 alkyl includes straight-chain or branched C1-5 alkyl. Further, R5 is C1-4 straight-chain alkyl. When R5 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl, and C3-6 cyclic alkyl; and wherein the substituted amino is amino substituted with one or more substituents selected from methyl and ethyl. When R5 is substituted phenyl, the substituent is selected from alkyl (e.g., C1-6 alkyl, preferably C1-3) or halogen.

[0120] In formula II, and unless otherwise stated in the context of the present invention, G is hydrogen, halogen (eg fluorine), methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

[0121] In formula II, X is oxygen or sulfur.

[0122] In formula II, n=0 or 1.

[0123] Preferably, in formula II, when X is oxygen, G is hydrogen, and n=0, R5 cannot be n-butyl.

[0124] Preferably, the compound represented by formula II is further represented by formula IIA:

[0125] In Formula IIA, the group R5 is the same as the group R5 in Formula II above. In some embodiments, R5 cannot be n-butyl. In some embodiments, R5 can be n-butyl.

[0126] In some embodiments, the camptothecin compound (CPT) has the following structure:

[0127] In Formula III and Formula IIIA, the compound represented by Formula I or Formula IA is connected to the carboxyl group of A through an amide bond via its amino group (see Formula I or Formula IA, respectively), that is, the amino group of the compound represented by Formula I or Formula IA forms an amide bond with the carboxyl group of A in Formula III or IIIA.

[0128] For example, in Formula III and Formula IIIA, the compound represented by CPT Formula II or Formula IIA or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, and the corresponding specific compound.

[0129] In Formula III and Formula IIIA, the compound represented by Formula II or Formula IIA is connected to the carboxyl group of A through an amide bond via its amino group (see Formula II or Formula IIA, respectively), that is, the amino group of the compound represented by Formula II or Formula IIA forms an amide bond with the carboxyl group of A in Formula III or IIIA.

[0130] Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14-P, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or pharmaceutically acceptable salts, stereoisomers, solvates, or prodrugs thereof presented above can be used as "CPT" in formula III and linked to the carboxyl group of A in formula III or IIIA via an amide bond via its amino group.

[0131] For example, in Formula III and Formula IIIA, CPT can be an exteacan derivative shown in Formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0132] In formula IV, and unless otherwise stated in the context of the present invention, R8 is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, or halogen.

[0133] When R8 is a substituted C1-5 alkyl group or a substituted C3-6 cyclic alkyl group, the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino group is an amino group substituted by one or more substituents selected from methyl and ethyl.

[0134] In formula III and formula IIIA, the compound shown in formula IV is connected to the carboxyl group of A through a self-releasing structure via its hydroxyl group connected to the same carbon as R8 (see formula IV), and the self-releasing structure is, for example The solid line indicates the site of attachment to the carboxyl group of A in Formula III or Formula IIIA, and the wavy line indicates the site of attachment to the hydroxyl group in Formula IV.

[0135] In particular, for the compound represented by formula IIIA, when R6 and R7 are Ar'S, M is preferably a phenylene or substituted phenylene, and SP1 is a C1-21 (preferably C1-16, more preferably C1-11) straight-chain heteroalkylene, wherein the straight-chain heteroalkylene contains 1-11 (preferably 1-6) heteroatoms selected from N, O or S.

[0136] In some embodiments, in Formula III' and Formula IIIA', D' is a compound represented by the following Formula I' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound:

[0137] where R a1 、R a2 、R a3 、R a4 、R a5 and R a8 Each independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;

[0138] R a6 and R a7 Each independently selected from C 1-8 alkoxy, such as methoxy, ethoxy or propoxy;

[0139] R a9 Selected from C 1-8 Alkyl and COOH; preferably C 1-4 Alkyl, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;

[0140] R a10 Selected from OH and H.

[0141] In some embodiments, the compound of formula I' is further a compound of formula IA':

[0142] where R a1 -R a10 As defined above;

[0143] In some embodiments, the compound of Formula I' is selected from:

[0144] In formula III' and formula IIIA', the compound represented by formula I' is connected to the carboxyl group of A via an amide bond via its amino group, that is, the left secondary amino group of the compound represented by formula I' forms an amide bond with the carboxyl group of A in formula III' or IIIA'.

[0145] In some embodiments, the compound represented by Formula III and Formula IIIA is further represented by Formula V.

[0146] In formula V, R6 and R7 are independently Ar'S, Ar' is phenyl or phenyl substituted by one or more substituents, in which the substituents are selected from alkyl (e.g., C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group. Preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl

[0147] In formula V, and unless otherwise stated in the context of the present invention, Xh and Yh are independently hydrogen, halogen, haloalkyl (e.g. haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl) or alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy).

[0148] In formula V, and unless otherwise specified in the context of the present invention, m is any integer from 1 to 10, preferably from 1 to 5, more preferably from 3 to 5.

[0149] In formula V, A represents 2-4 amino acids, as defined above.

[0150] In Formula V, the definition of CPT and its linkage in Formula V are the same as those of CPT in Formula III and Formula IIIA, as defined above.

[0151] In some embodiments, the compound of formula IIIA' is further a compound of formula V',

[0152] where R 6 、R 7 , Xh, Yh, m and A are as defined above (especially in formula V), and D' is as defined above.

[0153] In some embodiments, the compound of formula V' is further a compound of formula V-A',

[0154] wherein A and D' are as defined above.

[0155] In some embodiments, the compound of formula V is further represented by formula VA:

[0156] In formula VA, the groups A, G, Y, R1, R2, R3, R4, X, and n are the same as those defined above in formula III or formula IIIA.

[0157] Preferably, in formula VA, G is hydrogen, fluorine or chlorine.

[0158] Preferably, in formula VA, Y is methylene, sulfur or oxygen.

[0159] In formula VA, and in the context of the present invention, unless otherwise indicated, "-A-NH-" indicates that the amino group indicated forms an amide bond with the carboxyl group in A.

[0160] In some embodiments, the compound represented by Formula VA is further represented by Formula VA-1:

[0161] Alternatively, the compound represented by formula V is further represented by formula VB:

[0162] In formula VB, the groups A, G, R5, X, and n are the same as those defined above in formula III or formula IIIA.

[0163] In formula VB, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group shown forms an amide bond with the carboxyl group in A.

[0164] Preferably, the compound represented by formula VB is further represented by formula VB-1:

[0165] Alternatively, the compound represented by formula V is further represented by formula VC:

[0166] In formula VC, the groups A and R8 are the same as those defined above for the groups A and R8 in formula III or IIIV.

[0167] In formula VC, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group shown forms an amide bond with the carboxyl group in A.

[0168] In formula VC, and in the context of the present invention unless otherwise specified, The self-releasing structure is the same as that when the CPT in the above formula III or formula IIIV is formula IV.

[0169] According to a specific embodiment of the present invention, the compound represented by formula V is further represented by formula VI:

[0170] In formula VI, A represents 2 to 4 amino acids, as defined above.

[0171] In Formula VI, CPT is a camptothecin compound. The definition of CPT and its connection relationship in Formula VI are the same as those of CPT in Formula III and Formula IIIA, as defined above.

[0172] Preferably, the compound represented by Formula VI provided by the present invention is further represented by Formula VI-A:

[0173] In formula VI-A, the groups A, G, Y, R1, R2, R3, R4, X, and n are the same as those defined above in formula III or formula IIIA.

[0174] Preferably, in Formula VI-A, G is hydrogen, fluorine or chlorine.

[0175] Preferably, in Formula VI-A, Y is methylene, sulfur or oxygen.

[0176] In formula VI-A, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group indicated forms an amide bond with the carboxyl group in A.

[0177] Preferably, the compound represented by formula VI-A is further represented by formula VI-A-1:

[0178] Alternatively, the compound represented by Formula VI is further represented by Formula VI-B:

[0179] In formula VI-B, the groups A, G, R5, X, and n are the same as those defined above in formula III or formula IIIA.

[0180] In Formula VI-B, and unless otherwise specified in the context of the present invention, "-A-NH-" indicates that the amino group in the CPT shown forms an amide bond with the carboxyl group in A.

[0181] More preferably, the compound represented by formula VI-B is further represented by formula VI-B-1:

[0182] Alternatively, the compound represented by Formula VI is further represented by Formula VI-C:

[0183] In formula VI-C, the definitions of group A and R8 are the same as those of group A and R8 in formula III or IIIA above.

[0184] In Formula VI-C, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group indicated forms an amide bond with the carboxyl group in A.

[0185] When the self-releasing structure in VC is When , Formula VC can be further structured as shown in Formula VI-C.

[0186] In some embodiments, the drug-containing linker is selected from the following structures:

[0187] It should be understood that the present invention also relates to the specific methods disclosed in the embodiments of the present invention. In addition, the technical solutions obtained by replacing or combining certain technical features in the specific embodiments of the present invention with the technical features in the above-mentioned embodiments are also considered in the present invention.

[0188] Some small molecule drugs, drug-containing linkers and / or intermediates thereof, and / or antibody-drug conjugates in the present invention are described in Chinese patent application 202211627164.8, and the entire content of this patent application is introduced into this application as part of the content of this application.

[0189] The steps of the above preparation method are interrelated and interact with each other to produce the technical effect of improving the purity of the DAR2-ADC conjugate, and this technical effect is produced by the steps as a whole.

[0190] Second aspect

[0191] The present invention provides an antibody drug conjugate having the general formula The structure shown in FIG5 , wherein mAb represents an antibody or antibody fragment, such as an antibody as defined above, and groups M, SP1, SP2, A, L2, and D are as defined above in the first aspect. y is 1 to 10, preferably 1 to 8 (e.g., 1 to 5), more preferably 1 to 3, and most preferably 2.

[0192] Among them, E L is selected from the following groups, wherein, Indicates that it is linked to the cysteine ​​in the mAb, and the other The bond of is connected to M:

[0193] and / or where n is as defined above for variable E.

[0194] In the general structure, the mAb may be an IgG antibody or a fragment thereof, preferably an IgG1 subtype antibody or a fragment thereof.

[0195] In some embodiments, the antibody drug conjugate is prepared by the method of the present invention described in the first aspect.

[0196] In some embodiments, the antibody drug conjugate has an average DAR of about 2.

[0197] In some embodiments, the antibody drug conjugate (DAR2-ADC) has a purity greater than 90%, such as greater than 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% purity, as measured, for example, by HIC-HPLC.

[0198] In some embodiments, the antibody drug conjugate has the structure shown in the following formula VII, VII', VIII, or VIII':

[0199] and / or

[0200] wherein y is 1 to 10, preferably 1 to 8 (eg, 1 to 5), and more preferably 1, 2, or 3.

[0201] Wherein, the groups A, CPT, and D' are the same as defined in the first aspect above.

[0202] The third aspect

[0203] In a third aspect, the present invention provides the small molecule drugs, drug-containing linkers and / or intermediates thereof described in the preparation method of the first aspect of the present invention (including but not limited to Formula AI, I, I', IA, IA', II, II-A, III, III', III-A, III-A', V, VA, VA-1, VB, VB-1, VC, VI, VI-A, VI-A-1, VI-B, VI-B-1, VI-C), preferably including but not limited to the compounds disclosed in the examples.

[0204] The fourth aspect

[0205] The present invention provides a pharmaceutical composition comprising the small molecule drug, drug-containing linker, intermediate and / or antibody-drug conjugate described in the first to third aspects of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

[0206] In some embodiments, the pharmaceutical composition is a liquid preparation or a lyophilized preparation.

[0207] In some embodiments, the pharmaceutical composition comprises a buffer, a stabilizer, and a surfactant.

[0208] In a fifth aspect, the present invention provides use of a compound according to the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, antibody-drug conjugate or pharmaceutical composition thereof in the preparation of a medicament for treating tumors.

[0209] Preferably, the tumor is cancer. Preferably, the tumor is expressed in a tumor-associated antigen such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue Factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, or a combination thereof. C), mesothelin (Mesothelin), SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and Claudin 18.2 positive or high expression.

[0210] Preferably, the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.

[0211] In a sixth aspect, the present invention provides a method for treating tumors using a compound according to the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, antibody-drug conjugate or pharmaceutical composition thereof, the method comprising administering the compound or antibody-drug conjugate to a subject in need thereof.

[0212] Preferably, the tumor is cancer. Preferably, the tumor is expressed in a tumor-associated antigen such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue Factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, or a combination thereof. C), mesothelin (Mesothelin), SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and Claudin 18.2 positive or high expression.

[0213] Preferably, the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.

[0214] Preferably, the subject is a mammal, preferably a primate, more preferably a human.

[0215] The methods and / or products of the present invention have the following advantages and / or benefits:

[0216] (1) The method of the present invention can provide highly homogeneous antibody conjugate molecules, the main peak of which accounts for more than 99% of DAR2;

[0217] (2) The preparation method of the present invention has fewer steps and is simple to operate, which is conducive to industrial scale-up production. At the same time, the prepared product has fewer impurities and higher purity, which significantly improves drug safety and reduces production costs; and / or

[0218] (3) The antitumor drug prepared by the coupling method of the present invention has excellent antitumor effect and safety, including high stability and less side effects, expanding the therapeutic window, improving the therapeutic effect, and reducing toxic reactions.

[0219] definition:

[0220] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0221] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0222] The term "antibody drug conjugate" or "ADC" refers to a substance obtained by linking a bioactive compound fragment (small molecule drug) to an antibody or its antigen-binding fragment. The bioactive compound fragment is connected to the targeting portion via a linker. In some cases, the linker can be broken in a specific environment (e.g., a low pH environment in the cell) or under a specific action (e.g., the action of a lysosomal protease), thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the bioactive compound fragment is directly linked to the antibody or its antigen-binding fragment via a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.

[0223] The term "linker" refers to a fragment that connects a biologically active compound fragment (small molecule drug) to an antibody portion. In this regard, the linker, before being attached to the antibody or antigen-binding fragment thereof (i.e., a linker precursor), has a functional group that can form a bond with a functional group of the antibody or antigen-binding fragment thereof.

[0224] The term "small molecule drug" refers to a low molecular weight compound that can regulate biological processes. "Small molecule" is defined as a molecule with a molecular weight of less than 10kD, typically less than 2kD, and preferably less than 10kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules. Small molecule drugs include, but are not limited to, cytotoxic agents, such as camptothecins, calicheamicin, maytansinoids, dolastatin, auristatin compounds, and trichothecene. Preferably, the maytansinoid derivative is selected from DM1, DM3 and DM4; the auristatin compound is selected from the auristatin compounds mentioned in the embodiments of the present invention, including but not limited to MMAE and MMAF; the camptothecin compound is selected from camptothecin, 10-hydroxycamptothecin, exitecan, SN-38 and topotecan, as well as the camptothecin compounds mentioned in the embodiments of the present invention.

[0225] In this disclosure, when a small molecule drug is part of an ADC or a drug-containing linker, it refers to a structural fragment derived from the small molecule drug, such as a structural fragment formed by the bond between the hydroxyl or amino group of the small molecule drug and the linker, which can form a portion (fragment or group) of a biologically active drug (such as a small molecule cytotoxic drug, including a group after the loss of an atom or atomic group) or its derivative (such as a precursor) after the linker is cleaved / degraded / enzymatically cleaved between tumor tissues or within tumor cells. For the avoidance of doubt, "small molecule drugs" do not only refer to "drugs" that have been approved by pharmaceutical regulatory authorities, but also include any compound with potential therapeutic biological activity in clinical practice, or in R&D and academic research.

[0226] The term "drug-containing linker" refers to a compound formed by linking a bioactive compound fragment (small molecule drug) to a linker, and is sometimes referred to herein as a "drug-linker conjugate."

[0227] Metal ion chelating agents are compounds that, through their strong binding with metal ions, encapsulate metal ions into the interior of the chelating agent, turning them into stable compounds with larger molecular weight, thereby preventing the metal ions from acting. Metal ion chelating agents include but are not limited to ethylenediaminetetraacetic acid,

[0228] The term "pharmaceutical composition" refers to a composition that is in form permitting the active ingredient contained therein to be effective for biological activity, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0229] The terms "treat," ...

[0230] The term "prevent" includes the inhibition of the development or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevent" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0231] The term "alkyl" refers to a straight-chain, branched, fully saturated hydrocarbon group, preferably C1-C 10 , more preferably C1-C8, C1-C6, or C1-C4 alkyl. Examples of alkyl are methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl or n-decyl, and the like.

[0232] The term "alkylene" refers to a divalent group formed by removing a hydrogen atom from an alkyl group as defined above.

[0233] The term "cycloalkyl" refers to a cyclic alkyl group, preferably a C3-C 15 Cycloalkyl, for example C3-C8 cycloalkyl, C3-C6 cycloalkyl. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl or cycloheptyl, etc.

[0234] The term "cycloalkylene" refers to a divalent group formed by removing a hydrogen atom from a cycloalkyl group as defined above.

[0235] The term "heteroalkylene" refers to an alkyl group as defined above, which contains one or more heteroatoms selected from N, O or S in the chain. It should be understood that heteroalkylene does not include a group formed by two or more N, O or S heteroatoms directly linked to each other. Preferably, heteroalkylene is a C1-21 (e.g., C1-10, C1-6) straight chain heteroalkylene group containing 1-11 (preferably 1-6, 1-3) heteroatoms selected from N, O or S.

[0236] The term "heterocyclyl" refers to a partially unsaturated or fully unsaturated 3-10 membered cyclic group, preferably a 3-10 membered cyclic group, more preferably a 3-6 membered cyclic group, and even more preferably a 5-6 membered cyclic group, which may be optionally substituted and contains one or more (e.g., 1, 2, 3, or 4) heteroatoms selected from N, O, S, SO, SO2, or P. The heterocyclyl group contains 1-9 carbon atoms, preferably 2-8 carbon atoms, and more preferably 3-5 carbon atoms. Examples of heterocyclyl groups include 1,3-dioxolanyl, 1,3-dioxanyl, 1,4-dioxanyl, imidazolinyl, indolinyl, morpholinyl, pyridone, 2-pyrrolidone, piperazinyl, homopiperazinyl, piperidinyl, pyrazinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrahydrothienyl, oxanyl, oxathiolanyl, thianyl, and the like.

[0237] "Chemical bond" refers to the bond connecting the groups on both sides. For example, for the group -A-L2-D, when L2 is a chemical bond, the group can be written as -AD.

[0238] The term "halogen" refers to F, Cl, Br, I.

[0239] The term "reducing agent" refers to an agent capable of selectively reducing disulfide bonds. The reducing agent is selected from, but not limited to, tris(2-carboxyethyl)phosphine (TCEP) (including tris(2-carboxyethyl)phosphine hydrochloride), dithiothreitol (DTT), mercaptoethylamine, mercaptoethanol, and combinations thereof.

[0240] The term "antibody molar equivalent" refers to the molar equivalent relative to the antibody.

[0241] It should be understood that for the divalent groups in the general structure of the present application, the listed groups are connected to the groups on both sides in the direction shown, unless the structure obtained by such connection is unreasonable or deviates from the present invention, or is inconsistent with the context. For example, when L2 is When L2 is connected to A, the amino group on the left side of L2 is connected to A, and the carbonyl group on the right side of L2 is connected to the small molecule drug (D).

[0242] The term "drug to antibody ratio" or "DAR" refers to the ratio of the number of small molecule drug moieties (D) coupled to an antibody moiety (mAb) described herein to the number of antibody moieties. The DAR of an ADC can range from 1 to 10, but higher loadings are possible depending on the number of attachment sites on the antibody. The term DAR can be used to refer to the amount of drug loaded onto a single antibody. The DAR can also be calculated as the average DAR of a population of molecules in a product, i.e., the overall ratio (molar ratio) of the small molecule drug moiety (D) coupled to the mAb moiety described herein to the Ab moiety in the product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), which DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the antibody drug conjugate of the invention is 1.0-10.0, e.g., 4.0-10.0, 5.0-9.0, 6.0-8.0, 1.0-8.0, 2.0-6.0, e.g., 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.10, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6. 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0, and ranges having two of these values ​​as endpoints.

[0243] The term "DAR2-ADC" refers to an ADC with a DAR of 2, that is, an ADC in which an antibody is linked to two small molecule drugs.

[0244] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

[0245] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0246] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, combinations of the recited elements, integers, or steps are also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence. DETAILED DESCRIPTION

[0247] The present disclosure is further illustrated below by describing specific embodiments, but this is not intended to limit the present disclosure. Those skilled in the art may make various modifications or improvements based on the teachings of this disclosure without departing from the basic concept and scope of this disclosure. All instruments or equipment used without indicating their source are commercially available. All reagents used without indicating their source are commercially available or synthesized by conventional methods.

[0248] The abbreviations herein have the following meanings. Abbreviations without given meanings have the meanings commonly understood in the art:

[0249] Abbreviated name

[0250] KHMDS Potassium Hexamethyldisilazide

[0251] NFSI N-Fluorobis(benzenesulfonyl)imide

[0252] DIPEA N,N-Diisopropylethylamine

[0253] PE petroleum ether

[0254] EA Ethyl acetate

[0255] eq equivalent

[0256] MeOH methanol

[0257] DMF dimethylformamide

[0258] PB Disodium hydrogen phosphate and sodium dihydrogen phosphate buffer solution

[0259] IPA Isopropyl alcohol

[0260] MTS Abcam, Cat. No. ab223881

[0261] Example 1 Synthesis of small molecules

[0262] Example 1.1 Synthesis of Compound MWC-1

[0263] According to the method of WO2020200880A1, MWC-1 was prepared using B-1 as an intermediate as a yellow solid. LC-MS (ESI): [M+1]+=404

[0264] Example 1.2 Synthesis of Compound 3

[0265] Step 1

[0266] In a 500 ml three-necked flask, under nitrogen protection, compound B (2.45 g, 7.7 mmol, 1 eq) was dissolved in tetrahydrofuran (200 ml), and the reaction solution was cooled to -70 to -60 ° C in a dry ice-acetone bath. KHMDS (1 M, 31 ml, 31.0 mmol, 4 eq) was slowly added dropwise. After the addition, the mixture was stirred at -70 to -60 ° C for 10 minutes; a tetrahydrofuran solution of NFSI (7.35 g, 23 mmol, 3 eq) was added dropwise. ) was dissolved in tetrahydrofuran (70 ml). After addition, the mixture was stirred at -70 to -60°C for 10 minutes, then naturally warmed to 20 to 30°C and stirred for 3 hours. Saturated aqueous ammonium chloride solution (80 ml) was added to quench the reaction. The reaction solution was extracted with ethyl acetate (150 ml * 3). The organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product. The crude product was purified by column chromatography using petroleum ether:ethyl acetate = 1:0-1:3 as eluent to obtain compound 3a as a yellow solid, 0.85 g, in a yield of 31.2%. LC-MS (ESI): [M+1]+ = 355.8

[0267] 1 H NMR (400MHz, CDCl3) δ11.49(s,1H),8.64(d,J=9.2Hz,1H),7.74(d,J=8.5Hz,1H) ,6.06(s,1H),3.01(t,J=6.4Hz,2H),2.55–2.39(m,2H),2.19(s,3H),1.44(s,9H)

[0268] Step 2

[0269] In a 100ml single-necked flask, compound 3a (0.85g, 2.4mmol) was mixed with 6N hydrochloric acid (30ml) and heated under reflux for 4 hours. Heating was stopped, and the reaction solution was cooled to room temperature. The pH was adjusted to 7-8 with sodium bicarbonate and extracted with dichloromethane (30ml*3). The organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain the crude product. Purification by column chromatography with petroleum ether:ethyl acetate = 1:0 to 1:3 as eluent yielded compound 3b as a brown solid (0.25g, 49.1% yield). LC-MS (ESI): [M+1]+ = 213.9

[0270] Step 3

[0271] In a 50 ml single-necked reaction flask, under nitrogen protection, compound 3b (120 mg, 0.59 mmol, 1 eq), CDE tricyclic compound (180 mg, 0.68 mmol, 1.2 eq) and PPTS (35 mg, 0.14 mmol, 0.25 eq) were mixed in 15 ml of acetic acid and heated under reflux for 3 to 4 hours. Most of the acetic acid was removed by distillation under reduced pressure, and the residue was crudely separated by column chromatography with an eluent of dichloromethane:methanol = 15:1 to obtain 80 mg of a crude product, which was then purified by preparative liquid phase separation to obtain camptothecin compound 3 as a khaki solid. LC-MS (ESI): [M+1]+ = 440.8

[0272] 1 H NMR (400MHz, Acetone) δ7.89(d,J=9.1Hz,1H),7.48(d,J=9.1Hz,1H),7.37(s,1H),7.31(d,J=8.9Hz,0H), 5.59–5.26(m,5H),3.11–3.05(m,4H),2.73–2.59(m,3H),1.98(dt,J=14.0,7.0Hz,1H),1.05–0.98(m,3H)

[0273] 1 H NMR (400MHz, DMSO) δ7.87(d,J=9.1Hz,0H),7.41(d,J=9.1Hz,0H),7.21(s,0H),6.50(s,0H),6.15(s,1H),5.36(d ,J=46.6Hz,1H),2.93(t,J=6.5Hz,1H),2.66–2.54(m,1H),1.97–1.68(m,3H),1.24(s,1H),0.88(t,J=7.3Hz,3H)

[0274] Synthesis of compound B

[0275] Compound B-1 was synthesized according to the method of Jornal of Medical Chemstry, 1998, 41(13), 2308-2318. Compound B-1 was a brown solid. LC-MS (ESI): [M+1]+=177

[0276] 1H NMR(400MHz,DMSO-d6)δ6.76(d,J=8.7Hz,1H),6.67(bs,2H),6.42(d,J=8.7Hz, 1H),4.20(bs,2H),2.56(t,J=6.2Hz,2H),2.48–2.41(m,2H),1.96–1.84(m,2H)

[0277] In a 250 ml three-necked flask, compound B-1 (2.18 g, 124 mmol, 1 eq) was dissolved in tetrahydrofuran (100 ml), and Boc anhydride (8.2 g, 376 mmol, 3 eq) was added. The mixture was stirred at 40-45 °C for 5 hours. The reaction solution was directly concentrated to dryness and purified by column chromatography with petroleum ether:ethyl acetate = 1:0-1:3 as eluent to obtain compound B-2 as a yellow solid (3.0 g, yield 90%).

[0278] 1 H NMR (400MHz, CDCl3) δ7.25 (s, 1H), 6.41 (t, J = 10.2Hz, 3H), 5.90 (s, 1H), 2.7 3(t,J=6.2Hz,2H),2.61–2.47(m,2H),2.02–1.86(m,3H),1.49–1.36(m,9H)

[0279] In a 250 ml three-necked flask, compound B-2 (3.0 g, 10.8 mmol, 1 eq) and DIPEA (3.5 g, 27.1 mmol, 1.5 eq) were mixed in dichloromethane (125 ml). The reaction solution was cooled to -5 to 0 ° C with an ice-salt bath, and acetyl chloride (1.3 g, 16.5 mmol, 2 eq) was added dropwise. After the addition was completed, the ice-salt bath was removed, the temperature was naturally raised, and the reaction was stirred at room temperature for 4 hours; the reaction solution was concentrated to dryness, and the residue was purified by column chromatography with petroleum ether: ethyl acetate = 1:0 to 1:3 as the eluent to obtain compound B as a light yellow solid, 3.48 g, with a yield of 100%.

[0280] 1 H NMR (400MHz, CDCl3) δ12.01(s,1H),8.55(d,J=9.1Hz,1H),7.57(t,J=36.2Hz,1H),6.09(s, 1H),2.80(t,J=6.2Hz,2H),2.65–2.58(m,2H),2.15(s,3H),2.07–1.98(m,2H),1.44(s,9H)

[0281] Example 2 Synthesis of Novel Drug-Containing Linkers

[0282] Example 2.1 Synthesis of drug-containing linker BL20E (C-3)

[0283] The BL20E compound was prepared by condensing the BL linker compound with VC-PABC-MMAE according to the method disclosed in WO2018 / 095422A1. The synthetic route is as follows:

[0284] The BL20E compound was a yellow solid. LC-MS (ESI): [M+1]+ was 1963 (C-3).

[0285] Example 2.2 Synthesis of drug-containing linker MWD-L1

[0286] The MWD-L1 compound is synthesized from the BL linker compound and GGFG-Dxd. The synthetic route is as follows:

[0287] The BL linker compound (857 mg, 1 mmol), GGFG-Dxd (synthesized according to the method described in patent publication (US20190151328A1)) (840 mg, 1 mmol, 1 eq), DIPEA (323 mg, 2.5 mmol, 2.5 eq), and HATU (570 mg, 1.5 mmol, 1.5 eq) were dissolved in 30 ml of DCM and stirred for 2 h. The reaction solution was cooled to 5-10°C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The liquid was separated, and the aqueous phase was extracted with DCM (30 ml x 2). The organic phases were combined. The organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by column chromatography, eluent: DCM:MeOH = 50:1-10:1, afforded the drug-containing linker MWD-L1 as a yellow solid, 500 mg, yield 29.8%, LC-MS (ESI): M+1 = 1681, (M+1) / 2 = 840.9.

[0288] Example 2.3 Synthesis of drug-containing linker MWC-L2

[0289] Step 1

[0290] Boc-Val-Ala-OH (288 mg, 1 mmol), compound A1 (synthesized according to the method in Journal of Medicinal Chemistry, 1998, vol. 41, #13, pp. 2308-2318) (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), and HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 mL of DCM and stirred for 2 h. The reaction solution was dried and purified by column chromatography using PE:EA = 10:1 to 5:1 as the eluent to obtain intermediate 2-1 quantitatively as a gray-green solid (450 mg).

[0291] Step 2

[0292] Intermediate 2-1 (450 mg, 1 mmol), the CDE tricyclic compound (263 mg, 1 mmol, 1 eq), and pyridinium p-toluenesulfonate (PPTS) (251 mg, 1 mmol, 1 eq) were suspended in 30 ml of toluene and heated under reflux for 2 hours. Heating was stopped, and the reaction mixture was allowed to cool. The solid precipitate was collected as the crude product of Intermediate 2-2, a brown solid (750 mg). LC-MS (ESI): M+1 = 574. The product was used directly in the next step without purification.

[0293] Step 3

[0294] The BL linker compound (857 mg, 1 mmol), HoSu (138 mg, 1.2 mmol, 1.2 eq), and DCC (310 mg, 1.5 mmol, 1.5 eq) were dissolved in 30 ml of DCM and stirred at room temperature for 3 h. The reaction solution was then filtered, and the filtrate was the DCM solution of A-Osu. The filtrate was added to a mixed solution of the crude intermediate 2-2, DIPEA (323 mg, 2.5 mmol, 2.5 eq), and DCM (30 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The layers were separated, the aqueous phase was extracted with DCM (30 ml x 2), and the organic phases were combined. The organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by column chromatography, eluent: DCM:MeOH = 50:1 to 10:1, to obtain linker MWC-L2 as an orange-red solid, 325 mg, yield 23.0%, LC-MS (ESI): M+1 = 1414, (M+1) / 2 = 707

[0295] Example 2.4 Synthesis of drug-containing linker MWC-L3

[0296] Step 1

[0297] Boc-Gly-OH (175 mg, 1 mmol), compound A1 (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), and HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 mL of DCM and stirred for 2 h. The reaction solution was then dried and purified by column chromatography using an eluent of PE:EA (10:1 to 5:1) to quantitatively obtain 335 mg of intermediate 3-1 as a light green solid.

[0298] Step 2

[0299] Intermediate 3-1 (335 mg, 1 mmol), the CDE tricyclic compound (263 mg, 1 mmol, 1 eq), and PPTS (251 mg, 1 mmol, 1 eq) were suspended in 30 ml of toluene and heated under reflux for 2 hours. Heating was stopped, and the reaction solution was allowed to cool. The solid precipitate was collected as the crude product of Intermediate 3-2, a brown solid (560 mg). LC-MS (ESI): M+1 = 461. The product was used in the next step without purification.

[0300] Step 3

[0301] Fmoc-Gly-Gly-Phe-OH (501 mg, 1 mmol), crude intermediate 3-2 (560 mg), DIPEA (322 mg, 2.5 mmol, 2.5 eq), and HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 20 ml of DMF and stirred for 3 h. 1N hydrochloric acid (30 ml) was added and stirred for 0.5 h. Extraction was performed with DCM (30 ml x 2), and the organic phases were combined. The organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by column chromatography (eluent: DCM:MeOH = 50:1 to 10:1) afforded intermediate 3-3 as a brown solid (350 mg). LC-MS (ESI): M+1 = 945

[0302] Step 4

[0303] Intermediate 3-3 (350 mg, 0.37 mmol) was dissolved in methanol (20 ml), and diethylamine (2 ml) was added. The mixture was stirred for 3 h. The reaction solution was dried to give the crude intermediate 3-4 as a brown viscous product. LC-MS (ESI): M+1=722. The crude product was directly used for the next step without purification.

[0304] Step 5

[0305] The BL linker compound (318 mg, 0.37 mmol), HoSu (51 mg, 0.44 mmol, 1.2 eq), and DCC (114 mg, 0.56 mmol, 1.5 eq) were dissolved in 30 ml of DCM and stirred at room temperature for 3 h. The reaction solution was then filtered. The filtrate was added to a mixed solution of the crude intermediate 3-4, DIPEA (120 mg, 0.93 mmol, 2.5 eq), and DCM (30 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The layers were separated, the aqueous phase was extracted with DCM (30 ml x 2), and the organic phases were combined. The organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by column chromatography, eluent: DCM:MeOH = 50:1 to 10:1, to obtain linker MWC-L3 as an orange-red solid (120 mg, yield 20.8%), LC-MS (ESI): M+1 = 1562, (M+1) / 2 = 781

[0306] Example 2.5 Synthesis of drug-containing linker MWF-L6

[0307] According to the synthesis method of MWC-L2, MWF-L6 was prepared by replacing compound A1 with compound A7. The product was an orange-red solid. LC-MS (ESI): M+1=1450.

[0308] Compound A7 was synthesized as follows:

[0309] In a 250 ml three-necked flask, compound A1 (2.18 g, 124 mmol, 1 eq) was dissolved in tetrahydrofuran (100 ml), and Boc anhydride (8.2 g, 376 mmol, 3 eq) was added. The mixture was stirred at 40-45°C for 5 hours. The reaction solution was directly concentrated to dryness and purified by column chromatography with petroleum ether:ethyl acetate = 1:0-1:3 as eluent to obtain compound A7-1 as a yellow solid (3.0 g, 90% yield).

[0310] 1 H NMR (400MHz, CDCl3) δ7.25 (s, 1H), 6.41 (t, J = 10.2Hz, 3H), 5.90 (s, 1H), 2.7 3(t,J=6.2Hz,2H),2.61–2.47(m,2H),2.02–1.86(m,3H),1.49–1.36(m,9H)

[0311] In a 250 ml three-necked flask, compound A7-1 (3.0 g, 10.8 mmol, 1 eq) and DIPEA (3.5 g, 27.1 mmol, 1.5 eq) were mixed in dichloromethane (125 ml). The reaction solution was cooled to -5 to 0°C with an ice-salt bath. Acetyl chloride (1.3 g, 16.5 mmol, 2 eq) was added dropwise. After the addition was complete, the ice-salt bath was removed, the temperature was naturally raised, and the reaction was stirred at room temperature for 4 hours. The reaction solution was concentrated to dryness, and the residue was purified by column chromatography with petroleum ether:ethyl acetate = 1:0 to 1:3 as the eluent to obtain compound A7-2 as a light yellow solid, 3.48 g, in a yield of 100%.

[0312] 1 H NMR (400MHz, CDCl3) δ12.01(s,1H),8.55(d,J=9.1Hz,1H),7.57(t,J=36.2Hz,1H),6.09(s, 1H),2.80(t,J=6.2Hz,2H),2.65–2.58(m,2H),2.15(s,3H),2.07–1.98(m,2H),1.44(s,9H)

[0313] In a 500 ml three-necked flask, under nitrogen protection, compound A7-2 (2.45 g, 7.7 mmol, 1 eq) was dissolved in tetrahydrofuran (200 ml), and the reaction solution was cooled to -70 to -60 ° C in a dry ice-acetone bath. KHMDS (1 M, 31 ml, 31.0 mmol, 4 eq) was slowly added dropwise. After the addition, the mixture was stirred at -70 to -60 ° C for 10 minutes; a tetrahydrofuran solution of NFSI (7.35 g, 23 mmol, 3 eq) was added dropwise. ) was dissolved in tetrahydrofuran (70 ml). After addition, the mixture was stirred at -70 to -60°C for 10 minutes, then naturally warmed to 20 to 30°C and stirred for 3 hours. Saturated aqueous ammonium chloride solution (80 ml) was added to quench the reaction. The reaction solution was extracted with ethyl acetate (150 ml * 3). The organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product. The crude product was purified by column chromatography using petroleum ether:ethyl acetate = 1:0-1:3 as eluent to obtain compound A7-3 as a yellow solid, 0.85 g, in a yield of 31.2%. LC-MS (ESI): [M+1]+ = 355.8

[0314] 1 H NMR (400MHz, CDCl3) δ11.49(s,1H),8.64(d,J=9.2Hz,1H),7.74(d,J=8.5Hz,1H) ,6.06(s,1H),3.01(t,J=6.4Hz,2H),2.55–2.39(m,2H),2.19(s,3H),1.44(s,9H)

[0315] In a 100ml single-necked flask, compound A7-3 (0.85g, 2.4mmol) was mixed with 6N hydrochloric acid (30ml) and heated under reflux for 4 hours. Heating was stopped, and after the reaction solution cooled to room temperature, the pH was adjusted to 7-8 with sodium bicarbonate. The mixture was extracted with dichloromethane (30ml*3). The organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product. The crude product was purified by column chromatography using petroleum ether:ethyl acetate = 1:0 to 1:3 as eluent to obtain compound A7 as a brown solid (0.25g, yield 49.1%). LC-MS (ESI): [M+1]+ = 213.9

[0316] Example 2.6 Synthesis of drug-containing linker DT-1

[0317] DT-1 was synthesized according to the method of CN114106088A as a yellow solid. LC-MS (ESI): [M+1]+=1474.4

[0318] Example 2.7 Synthesis of drug-containing linker DT-2

[0319] According to the synthesis method of BL20E compound, DT-2-A compound was used instead of BL linker compound to synthesize DT-2, which was a light yellow solid. LC-MS (ESI): [M+1]+=1339.7, wherein DT-2-A compound was synthesized according to the literature Chem. Sci., 2022, 13, 8781–8790.

[0320] Example 2.8 Synthesis of drug-containing linker DT-3

[0321] DT-3 was synthesized according to the method of WO2022253033A as a light yellow solid, LC-MS (ESI): [M+1]+=1631.5.

[0322] Example 2.9 Synthesis of drug-containing linker DT-4

[0323] DT-4 was synthesized according to the method of CN103933575B as a light yellow solid. LC-MS (ESI): [M+1]+=1414.7

[0324] Example 2.10 Synthesis of drug-containing linker DT-5

[0325] DT-5 was synthesized according to the method of CN109232464A as a yellow solid. LC-MS (ESI): [M+1]+=1591.8

[0326] Example 2.11 Synthesis of drug-containing linker DT-6

[0327] DT-6 was synthesized according to the method of KR2019014324A as a yellow solid. LC-MS (ESI): [M+1]+=1619.6

[0328] Example 2.12 Synthesis of drug-containing linker DT-7

[0329] DT-7 was synthesized according to the method of JP2018138538A as a yellow solid. LC-MS (ESI): [M+1]+=1733.7

[0330] MC-VC-PAB-MMAE used in the examples was purchased from MCE.

[0331] Example 3 Coupling process study

[0332] Example 3.1 Antibody reduction conditions and metal ion screening

[0333] Exchange the trastuzumab antibody solution to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer (pH 6.5-7.4) and dilute to 5-15mg / mL. Sequentially add aqueous solutions of various metal ions and aqueous tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution, and react overnight at 0-15°C. The reaction equivalents are shown in Table 1-1. Then, add a 3-fold molar equivalent of BL20E (C-3) in DMA to the reaction solution. Incubate at 0-15°C for 2 hours, then exchange the solution to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer (pH 6.5-7.4). Adjust the reaction solution concentration to approximately 5-15mg / mL, add 4-fold equivalent of ethylenediaminetetraacetic acid (EDTA), and react at room temperature for 30 minutes before exchange to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer (pH 7.4-8.0). Adjust the reaction mixture to a concentration of approximately 5-15 mg / mL, add 8 times the antibody equivalent of dehydroascorbic acid (DHAA), and incubate at 25-35°C for 2 hours. Replace the reaction mixture with 50 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer (pH 7.4). This yields the antibody-drug conjugate product. Samples were collected and analyzed to determine the antibody-drug conjugate ratio (DAR). The results are shown in Table 1-1.

[0334] Table 1-1. Screening results of different reducing agent equivalents and different metal ions

[0335] It can be seen that the use of the above metal ions or metal complexes and reactant equivalents in the preparation method of the present application can achieve a HIC peak value of 61-74% for DAR2-ADC.

[0336] Example 3.2 Screening of Antibody Coupling Conditions

[0337] The trastuzumab antibody solution was exchanged into 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15mg / mL. A ZnCl2 solution (2x the molar equivalent of the antibody) and a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution (4.0-4.5x the molar equivalent of the antibody) were sequentially added. The reaction was allowed to react overnight at 0-15°C, after which the solution was exchanged into 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solutions of varying pH values. Subsequently, a BL20E (C-3) solution in DMA (3x the molar equivalent of the antibody) was added to the reaction solution. The coupling reaction temperature and pH of the coupling solution are shown in Table 1-2. After completion of the coupling reaction, the solution was exchanged into 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 6.5-7.4. The reaction solution was adjusted to a concentration of approximately 5-15 mg / mL, and ethylenediaminetetraacetic acid (EDTA) was added at 4 times the antibody equivalent. The reaction mixture was allowed to react at room temperature for 30 minutes, and then the solution was exchanged to a 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at a pH of 7.4-8.0. The reaction solution was adjusted to a concentration of approximately 5-15 mg / mL, and dehydroascorbic acid (DHAA) was added at 8 times the antibody molar equivalent. The reaction mixture was allowed to react at 25-35°C for 2 hours, and then the solution was exchanged to a 50 mM sodium dihydrogen phosphate-disodium dihydrogen phosphate buffer solution at a pH of 7.4. The antibody-drug conjugate product was obtained. Samples were taken for analysis, and the antibody coupling ratio (DAR) and purity were determined. The test results are shown in Tables 1-2.

[0338] Table 1-2. Screening results of different coupling reaction temperatures and different coupling solution pH

[0339] It can be seen that the HIC peak value of DAR2-ADC can be achieved to be 61-78% by adopting the above coupling reaction temperature and coupling solution pH in the preparation method of the present application.

[0340] Example 3.3 Screening of antibody purification conditions

[0341] Exchange trastuzumab antibody solution to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and dilute to 5-15mg / mL. Sequentially add 2 times the antibody molar equivalent of aqueous ZnCl2 solution and 4.0-4.5 times the antibody molar equivalent of aqueous tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution. Incubate at 0-15°C overnight. Then, add 3 times the antibody molar equivalent of BL20E (C-3) in DMA to the reaction solution. Incubate at 0-15°C for 2 hours before exchanging the solution to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4. Adjust the reaction solution concentration to approximately 10mg / mL, add 4 times the antibody molar equivalent of ethylenediaminetetraacetic acid (EDTA), incubate at room temperature for 30 minutes, and then exchange the solution to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4-8.0. Adjust the concentration of the reaction solution to approximately 5-15 mg / mL, add 8 times the antibody equivalent of dehydroascorbic acid (DHAA), and react at 25-35°C for 2 h. After the reaction, exchange the solution with 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.5), dilute to approximately 5-20 mg / mL, and add ammonium sulfate solution to adjust the conductivity. Purify the antibody-drug conjugate product by hydrophobic interaction chromatography (HIC). Purification conditions are shown in Table 1-3. Samples of the purified product were analyzed by HIC. The HIC test results are shown in Table 1-3.

[0342] Table 1-3. Screening results of different purification conditions

[0343] It can be seen that the use of the above fillers, salt types and salt concentrations (as phase A) in the preparation method of the present application can achieve an HIC peak value of 98-100% for DAR2-ADC.

[0344] Example 3.4 Conjugation experiments of different drug-containing linkers and different antibodies

[0345] Exchange the antibody solution to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and dilute to 5-15mg / mL. Sequentially add 2 times the antibody molar equivalent of aqueous ZnCl2 and 4.0-4.5 times the antibody molar equivalent of aqueous tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution and react overnight at 0-15°C. Then, add 2.5-5.5 times the antibody molar equivalent of a drug-linker solution in DMA to the reaction solution. React at 0-15°C for 2 hours, then exchange the solution to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4. Adjust the reaction solution concentration to approximately 5-15mg / mL, add 4 times the antibody molar equivalent of ethylenediaminetetraacetic acid (EDTA), react at room temperature for 30 minutes, and then exchange the solution to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4-8.0. The reaction solution was adjusted to a concentration of approximately 5-15 mg / mL, and 8 times the molar equivalent of the antibody in dehydroascorbic acid (DHAA) was added. The reaction was allowed to react at 25-35°C for 2 h. After the reaction, the solution was exchanged to 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer at pH 7.5 and diluted to approximately 5-20 mg / mL. Ammonium sulfate was added to adjust the conductivity. A solution of 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate at pH 7.5 plus 0.45-0.75 M ammonium sulfate was used as phase A, and a solution of 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate at pH 7.5 was used as phase B. Purification was performed using Butyl 650M filler. The resulting purified solution was concentrated and exchanged to 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer at pH 7.4. After purification, samples were collected and analyzed to determine the antibody coupling ratio (DAR) and purity of the resulting antibody-drug conjugate product. The test results are shown in Tables 1-4.

[0346] Table 1-4. Experimental results of different antibodies coupled with different small molecules

[0347] Among them, antibody h23-12 was prepared according to the method described in patent WO2021068949A1, and antibody hH2L1 was prepared according to the method described in patent CN113527486A.

[0348] Example 3.5 Comparative Example

[0349] Trastuzumab-MC-VC-PAB-MMAE, an antibody-drug conjugate, was prepared according to the method disclosed in patent WO2020 / 164561A1. A brief description is as follows:

[0350] (1) ZnCl2 (0.04 mM) and TCEP (0.08 mM) were added to the trastuzumab antibody solution and reacted at 4°C overnight;

[0351] (2) Add 0.12 mM DMA solution of MC-VC-PAB-MMAE to the reaction solution of (1) above and react at 4°C for 2 h; the molar equivalent ratio of antibody to drug-containing linker is 1:3.

[0352] (3) adding cysteine ​​(0.08 mM) to the reaction solution of (2) above to remove excess MC-VC-PAB-MMAE;

[0353] (4) Add EDTA (0.08 mM) to the reaction solution from (3) above, followed by DHAA (0.16 mM);

[0354] (5) The reaction mixture was purified using a desalting column (Model: 40K, 0.5 mL, REF: 87766, Lot: SJ251704, Thermo).

[0355] Table 1-5. Comparison results of HIC analysis

[0356] Example 4 Physicochemical Characterization of Antibody Drug Conjugates

[0357] Example 4.1 Physicochemical Characterization of Antibody Drug Conjugates

[0358] a. Determination of DAR by hydrophobic interaction chromatography (HIC-HPLC)

[0359] Mobile phase A: 2.0 M (NH4)2SO4 + 50 mM PB, pH 7.0

[0360] Mobile phase B: 50 mM PB + 15% IPA, pH 7.0

[0361] Flow rate: 0.6 mL / min

[0362] Injection concentration: 3-5 mg / mL

[0363] Injection volume: 10 μL

[0364] Analytical column: TOSOH Butyl NPR, 4.6*100mm, 2.5μm, PN0042168, CN081D00130D

[0365] Column temperature: 25°C

[0366] Detection wavelength: 280nm

[0367] gradient:

[0368] Sample injection analysis: For samples with a concentration exceeding 5 mg / mL, dilute the sample with 50% phase A solution and filter through a 0.2 μm filter before injection analysis. For samples with a concentration of 5 mg / mL or less, filter directly before injection analysis.

[0369] DAR = ∑ (weighted peak area) / 100, that is, DAR = (D0 peak area ratio × 0 + D1 peak area ratio × 1 + D2 peak area ratio × 2 + D3 peak area ratio × 3 + D4 peak area ratio × 4 + D5 peak area ratio × 5 + D6 peak area ratio × 6 + D7 peak area ratio × 7 + D8 peak area ratio × 8) / 100.

[0370] b. Purity determination by non-reducing capillary electrophoresis NR-CE-SDS

[0371] The determination was based on the monoclonal antibody size variant determination method in General Chapter 3127 of the Chinese Pharmacopoeia Volume IV.

[0372] Example 5 Bioactivity Analysis of Antibody Drug Conjugates

[0373] 5.1 Study on the killing effect of antibody-drug conjugates in vitro

[0374] Exemplarily, the following method was used to conduct the experiment.

[0375] This experiment used human gastric cancer cells NCI-N87 (purchased from ATCC). The cell density of NCI-N87 cells was adjusted to 15×10 4 Afterwards, 100 μl / well was added to the cell culture plate and cultured overnight. The next day, the ADC samples were diluted with the above complete medium to 50 ug / ml, and then diluted 4 times in a gradient, with a total of 9 gradients plus a zero point. All samples were set up with 3 replicates. Set up a negative control (cells + culture medium) and a blank control (no cells, pure culture medium); the diluted ADC samples were added to the cell culture plate cultured overnight in sequence, 100 μl / well. Then placed in a cell culture incubator and incubated for 120 hours. Remove the cell culture plate, add 40 μl / well of MTS (abcam, product number ab223881), and react in a 37°C incubator for 2-4 hours; remove the cell plate and read the OD value at 490 nm.

[0376] Table 1-6 Comparative study results of the activity of different antibody drug conjugates

Claims

1. A method for preparing an antibody-drug conjugate (ADC), the method comprising the following steps: (a) adding a metal salt or a metal complex and a reducing agent to a buffer solution containing an antibody, followed by incubation; (b) adding a drug-containing linker to the reaction solution in (a) for coupling; and (c) Using hydrophobic interaction chromatography, a hydrophobic gel-type filler and a salt solution to purify the conjugate product to obtain a high-purity DAR2-ADC conjugate.

2. The method according to claim 1, wherein the metal in the metal salt or metal complex in step (a) can be selected from Zn, Cd and Hg, etc. Preferably, the metal salt or metal complex in step (a) is selected from one or more of the following: hydrochlorides or sulfates of Zn, Cd and Hg, Zn(NH2CH3)2 2+ ,Zn(NH2CH2CH3)2 2+ ,Zn(NH2CH2CH2CH3)2 2+ ,Zn(NH2CH(CH3)2)2 2+ ,Zn(NH2C(CH3)3)2 2+ ,Zn(NH2CH2C(CH3)3)2 2+ ,Zn(NH(CH3)2)2 2+ ,Zn(NH(CH2CH3)2)2 2+ ,Zn(NH(CH(CH3)2)2)2 2+ ,Zn(NH(C(CH3)3)2)2 2+ ,Zn(NH(CH(CH2CH3)2)2)2 2+ ,Zn(NH(CH2C(CH3)3)2)2 2+ ,Zn(NH(CH2C(CH2CH3)3)2)2 2+ ,Zn(NH(CH2CH2C(CH3)3)2)2 2+ ,Zn(NH2CH2CH2OH)2 2+ ,Zn(NH(CH2CH2OH)2)2 2+ ,Zn(N(CH2CH2OH)3)2 2+ ,Zn(NH2CH2COOH)2 2+ ,Zn(NH2CH2CONH2)2 2+ ,Zn(NH2CH2COOCH3)2 2+ ,Zn(NH2CH2COOCH2CH3)2 2+ ,Zn(NH2CH2COOC(CH3)3)2 2+ ,Zn(NH2CH2COOCH(CH3)2)2 2+ ,Zn(NH2CH2CH2COOH)2 2+ ,Zn(NH(CH2COOH)2)2 2+ ,Zn(N(CH2CH2COOH)3)2 2+ ,Zn(NH2CH3)4 2+ ,Zn(NH2CH2CH3)4 2+ ,Zn(NH2CH2CH2CH3)4 2+ ,Zn(NH2CH(CH3)2)4 2+ ,Zn(NH2C(CH3)3)4 2+ ,Zn(NH2CH2C(CH3)3)4 2+ ,Zn(NH(CH3)2)4 2+ ,Zn(NH(CH2CH3)2)4 2+ ,Zn(NH(CH(CH3)2)2)4 2+ ,Zn(NH(C(CH3)3)2)4 2+ ,Zn(NH(CH(CH2CH3)2)2)4 2+ ,Zn(NH(CH2C(CH3)3)2)4 2+ ,Zn(NH(CH2C(CH2CH3)3)2)4 2+ ,Zn(NH(CH2CH2C(CH3)3)2)4 2+ ,Zn(NH2CH2CH2OH)4 2+ , More preferably, the metal salt or metal complex in step (a) is selected from ZnCl2, CdCl2, HgCl2, One or more of .

3. The method according to claim 1 or 2, wherein The reducing agent in step (a) is selected from tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), mercaptoethylamine and mercaptoethanol, preferably TCEP.

4. The method of any one of claims 1 to 3, wherein the concentration of the antibody in step (a) is 0.5-150 mg / mL, such as 4-30 mg / mL, The amount of the reducing agent used in step (a) is 1-6 times the antibody molar equivalent, for example 3.5-5.0 times the antibody molar equivalent, The amount of the metal salt or metal complex in step (a) is 1-5 times the molar equivalent of the antibody, for example 1-3 times the molar equivalent of the antibody. The buffer solution in step (a) has a pH of 5-8, such as a pH of 6.5-7.5, preferably a pH of 6.9, and / or The buffer solution in step (a) is selected from phosphate buffer, acetate buffer, citrate buffer, succinate buffer, preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, preferably, the buffer concentration in the buffer solution is 5-100mM, such as 10-30 or mM.

5. The method according to any one of claims 1 to 4, wherein the amount of the drug-containing linker in step (b) is 1 to 8 times the molar equivalent of the antibody, such as 3 to 5 times the molar equivalent of the antibody, Preferably, the reaction solution in step (b) has a pH of 5-8, such as a pH of 6.5-7.

5.

6. The method according to any one of claims 1 to 5, wherein the hydrophobic gel filler in step (c) is selected from Butyl-650M, Butyl-4FF, Butyl-650S, Butyl-ImpRes and Phenyl Sepharose HP, and / or The salt type in step (c) is selected from sulfate and chloride, preferably ammonium sulfate and sodium chloride; preferably, the salt concentration is 0.1-5 mol / L, such as 0.6-1.2 mol / L.

7. The method according to any one of claims 1 to 6, wherein after step (b), step (b') is included: adding a metal ion chelating agent such as ethylenediaminetetraacetic acid (EDTA) to react, Preferably, the amount of the metal ion chelator used is 1-8 times the molar equivalent of the antibody, such as 3-5 times the molar equivalent of the antibody.

8. The method according to any one of claims 1 to 7, wherein The method comprises the step (b") after the step (b'): adding dehydroascorbic acid (DHAA) to react, Preferably, the amount of DHAA used is 1-16 times the antibody molar equivalent, such as 6-12 times the antibody molar equivalent.

9. The method according to any one of claims 1 to 8, wherein The DAR2-ADC obtained by the method has a purity greater than 90%, as measured, for example, by HIC-HPLC.

10. The method of any one of claims 1 to 9, wherein the antibody is selected from the group consisting of a humanized antibody, a murine antibody, a human antibody, a chimeric antibody, a single-chain antibody, and a bispecific antibody, Preferably, the antibody is an antibody that binds to the following antigens: HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C C), Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2; Preferably, the antibody comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs) of adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12 or hH2L1. CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3), or VH and / or VL of the antibody, or heavy chain and / or light chain of the antibody; Preferably, the antibody is selected from trastuzumab, pertuzumab, h23-12 and hH2L1.

11. The method according to any one of claims 1 to 10, wherein the drug-containing linker in step (b) is a compound represented by formula AI or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, in, E is selected from the following groups: Among them, with The bond of represents the attachment site to M: n is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R6 and R7 are independently halogen or Ar'S-, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituents are selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group. Preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-formylmorpholine substituted phenyl M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in the substituted phenylene group, the substituent group is selected from an alkyl group (e.g., a C1-C6 alkyl group, preferably a C1-C4 alkyl group), a haloalkyl group (e.g., a halo C1-C6 alkyl group, preferably a halo C1-C4 alkyl group, such as a trifluoromethyl group), an alkoxy group (e.g., a C1-C6 alkoxy group, preferably a C1-C4 alkoxy group, preferably a methoxy group), a halogen, an ester group, an amide group, and a cyano group; preferably, M is a phenylene group substituted by a halogen; SP1 is selected from C1-8 alkylene, C1-8 alkylene phenylene-, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11) straight chain heteroalkylene, wherein the C1-21 straight chain heteroalkylene contains 1-11 (preferably 1-6) heteroatoms selected from N, O or S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxyl, oxo, amino, sulfonic acid and cyano; SP2 is selected from -NH(CH2CH2O)aCH2CH2CO-, -NH(CH2CH2O)aCH2CO-, -S(CH2)aCO- or a chemical bond, wherein a is an integer of 1-20, preferably an integer of 1-10, more preferably an integer of 1-6; A represents 2 to 4 amino acids, wherein when A represents 2 amino acids, it can be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO or NH-Val-Cit-CO; when A represents 3 amino acids, it can be NH-Glu-Val-Ala-CO. la-CO, NH-Glu-Val-Cit-CO or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO; when A represents 4 amino acids, it can be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO, preferably, A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO or NH-Ala-Ala-Ala-CO; Preferably, A is selected from the group consisting of NH-Val-Cit-CO, NH-Val-Ala-CO and NH-Gly-Gly-Phe-Gly-CO; L2 is selected from and chemical bonds; D is a small molecule drug.

12. The method of claim 11, wherein M is selected from 13. The method of claim 11 or 12, wherein Selected from wherein q is an integer selected from 1-10; p is an integer selected from 1-20; Preferably, Selected from 14. The method of any one of claims 11 to 13, wherein D is selected from the group consisting of camptothecins, calicheamicin, maytansinoids, dolastatins, auristatins and trichothecenes; Preferably, the maytansinoid derivative is selected from DM1, DM3 and DM4; the auristatin compound is selected from MMAE and MMAF; the camptothecin compound is selected from camptothecin, 10-hydroxycamptothecin, exitecan, SN-38, topotecan and camptothecin derivatives.

15. The method of any one of claims 11 to 14, wherein: The drug-containing linker is a compound represented by Formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein E, M, SP1, SP2 and A are as defined in any one of claims 11 to 14, and CPT is a camptothecin compound; or is a compound represented by formula III' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein E, M, SP1, SP2 and A are as defined in any one of claims 11 to 14, and D' is an auristatin compound.

16. The method of claim 15, wherein the compound of formula III is further a compound of formula IIIA: in, R6 and R7 are independently halogen or Ar'S-, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclyl -CO-, halogen, ester group, amide group and cyano group, preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl wherein M, SP1, SP2, A and CPT are as defined in claim 15; and The compound of formula III' is further a compound of formula IIIA': wherein R6, R7, M, SP1, SP2, A and D′ are as defined in claim 15.

17. The method of claim 15 or 16, wherein CPT is a compound represented by the following formula I or formula IA or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound: Wherein R1, R2, R3, and R4 are independently hydrogen, halogen, hydroxyl, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3, and R4 together with the carbon atoms to which they are connected constitute a C3-6 cyclic alkyl. When R1, R2, R3, and R4 are independently C1-6 alkoxy, the C1-6 alkoxy includes a straight-chain or branched C1-6 alkoxy, preferably a straight-chain or branched C1-3 alkoxy, more preferably a methoxy. When R1, R2, R3, and R4 are independently substituted amino, the substituted amino is selected from An amino group substituted with one or more substituents selected from methyl and ethyl, when R1, R2, R3, and R4 are independently C1-7 alkyl or substituted C1-7 alkyl, the C1-7 alkyl or substituted C1-7 alkyl includes a straight chain or branched C1-7 alkyl or substituted C1-7 alkyl, and the substituted C1-7 alkyl is a C1-7 alkyl substituted with one or more substituents selected from cyclopropyl and cyclobutyl; or, the straight chain or branched C1-7 alkyl or substituted C1-7 alkyl is preferably a C1-3 alkyl or substituted C1-3 alkyl, such as methyl, halomethyl (preferably trifluoromethyl); G is hydrogen, halogen, methyl or methoxy, preferably, G is hydrogen, fluorine or chlorine; Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene. The substituted methylene may be a methylene in which one hydrogen is substituted or two hydrogens are substituted simultaneously. The substituent may be a benzyl or an alkyl. When the substituent is an alkyl, the alkyl and R3 and / or R4 and the carbon atoms to which they are connected may form a C3-6-membered cyclic or spirocyclic structure. When Y is a substituted methylene, the substituent of the substituted methylene is preferably an alkyl, more preferably a linear or branched C1-4 alkyl. Preferably, Y is oxygen, sulfur, sulfone or sulfoxide. Alternatively, preferably, Y is oxygen, sulfur or methylene. X is oxygen or sulfur; and n = 0 or 1; Preferably, the compound represented by formula I is further represented by formula IA: In formula IA, the groups R1, R2, R3, and R4 have the same definitions as the groups R1, R2, R3, and R4 in formula I.

18. The method of claim 15 or 16, wherein the camptothecin compound is a compound of formula II or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof: wherein R5 is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, phenyl or substituted phenyl; when R5 is C1-5 alkyl or substituted C1-5 alkyl, the C1-5 alkyl includes straight chain or branched C1-5 alkyl; further, R5 is C1-4 straight chain alkyl; when R5 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein, Substituted amino is amino substituted by one or more substituents selected from methyl and ethyl. When R5 is substituted phenyl, the substituent is selected from alkyl (e.g. C1-6 alkyl, preferably C1-3) or halogen; G is hydrogen, halogen (such as fluorine), methyl or methoxy, preferably, G is hydrogen, fluorine or chlorine; X is oxygen or sulfur; and n = 0 or 1; Preferably, the compound represented by formula II is further represented by formula IIA: In formula IIA, the group R5 has the same meaning as the group R5 in formula II.

19. The method of claim 15 or 16, wherein: The camptothecin compound (CPT) has the following structure:

20. The method according to any one of claims 14 to 19, wherein the amino group of the camptothecin compound and the carboxyl group of A form an amide bond.

21. The method of any one of claims 15-20, wherein CPT is an exitecan derivative represented by formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof: wherein R8 is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, or halogen; When R8 is a substituted C1-5 alkyl or a substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein, The substituted amino group is an amino group substituted with one or more substituents selected from the group consisting of methyl and ethyl; Preferably, the compound represented by formula IV is connected to the carboxyl group of A via a self-releasing structure via its hydroxyl group connected to the same carbon as R8 (see formula IV), and the self-releasing structure is, for example The solid line indicates the site of attachment to the carboxyl group of A in Formula III or Formula IIIA, and the wavy line indicates the site of attachment to the hydroxyl group in Formula IV.

22. The method of any one of claims 15 to 21, wherein In Formula III' and Formula IIIA', D' is a compound shown in the following Formula I' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound: Where R a1 , R a2 , R a3 , R a4 , R a5 and R a8 Each independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; R a6 and R a7 Each independently selected from C 1-8 Alkoxy, such as methoxy, ethoxy or propoxy; R a9 Selected from C 1-8 Alkyl and COOH; preferably C 1-4 Alkyl, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; R a10 Selected from OH and H; Preferably, the compound of formula I' is further a compound of formula IA': Where R a1 -R a10 As defined in formula I'; More preferably, the compound of formula I' is selected from: Preferably, in formula III' and formula IIIA', the left secondary amino group of the compound represented by formula I' forms an amide bond with the carboxyl group of A in formula III' or IIIA'.

23. The method of any one of claims 15-22, wherein The compounds represented by formula III and formula IIIA are further represented by formula V, In formula V, R6 and R7 are independently Ar'S, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group, preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl Xh and Yh are independently hydrogen, halogen, haloalkyl (e.g. haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl) or alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy); m is an integer from 1 to 10; A and CPT are as defined in any one of claims 15 to 22; And the compound of formula IIIA' is further a compound of formula V', Where R 6 , R 7 , Xh, Yh, m, A and D' are as defined in any one of claims 16 to 22; Preferably, the compound of formula V' is further a compound of formula V-A', wherein A and D' are as defined in any one of claims 16-22.

24. The method of claim 23, wherein: The compound of formula V is further a compound of formula VA: wherein the variables are as defined in claim 23; Preferably, the compound represented by formula VA is further represented by formula VA-1: wherein the variables are as defined in claim 23; Alternatively, the compound represented by formula V is further represented by formula VB: wherein the variables are as defined in claim 23; Preferably, the compound represented by formula VB is further represented by formula VB-1: wherein the variables are as defined in claim 23; Alternatively, the compound represented by formula V is further represented by formula VC: The variables are as defined in claim 23.

25. The method according to claim 23, wherein the compound of formula V is further a compound of formula VI: wherein the variables are as defined in claim 23; Preferably, the compound represented by formula VI is further represented by formula VI-A: wherein the variables are as defined in claim 23; Preferably, the compound represented by formula VI-A is further represented by formula VI-A-1: wherein the variables are as defined in claim 23; Alternatively, the compound represented by formula VI is further represented by formula VI-B: wherein the variables are as defined in claim 23; More preferably, the compound represented by formula VI-B is further represented by formula VI-B-1: wherein the variables are as defined in claim 23; Alternatively, the compound represented by formula VI is further represented by formula VI-C: Wherein the variables are as defined in claim 23 In formula VI-C, the definitions of group A and R8 are the same as those of group A and R8 in formula III or IIIA above.

26. The method according to any one of claims 1 to 25, wherein the drug-containing linker is selected from the following compounds or pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs thereof:

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