Method for preparing antibody drug conjugate

Abstract

The invention relates to a method for preparing an antibody drug conjugate, which is characterized in that highly homogeneous antibody conjugate molecules are obtained through a specific chemical fixed-point coupling method, and the DAR2 main peak proportion of the antibody conjugate molecules is more than 99%. The preparation method disclosed by the invention is few in steps, simple to operate and beneficial to industrial large-scale production, meanwhile, the prepared product is few in impurity and high in purity, the medication safety is obviously improved, and the production cost is also reduced.

Description

Technical Field

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

[0002] Background Overview

[0003] Antibody-drug conjugates (ADCs) are a new form of drug that connects small molecule drugs to monoclonal antibodies. They use the targeting function of monoclonal antibodies to transport small molecule drugs to target sites to exert their efficacy, reduce toxic side effects, and increase the therapeutic window. In recent years, antibody-drug conjugates have been one of the hot directions in precision tumor treatment, bringing hope to tumor treatment. Antibody-drug conjugates (ADCs) are composed of antibody drugs targeting specific antigens and small molecule cytotoxic drugs coupled through linkers. They have both the powerful killing effect of traditional small molecule chemotherapy and the tumor targeting of antibody drugs. So far, more than a dozen ADC drugs have been approved for marketing worldwide.

[0004] Antibody-drug conjugates consist of several parts, such as antibodies, linkers, and payloads. Among them, antibodies and drug-linkers can be connected in different coupling modes. The coupling methods are mainly divided into non-fixed site coupling and fixed site coupling. In the early days, non-fixed site coupling methods were used, mainly lysine coupling and cysteine ​​coupling, which directly coupled drugs to amino acid residues on antibodies using chemical methods, without involving antibody transformation or modification. The number of toxin molecules and coupling sites coupled to them cannot be determined, and the uniformity is poor. The currently commonly used fixed-site coupling method is to perform specific coupling through genetic engineering sites or special connectors to achieve more uniform coupling, and can achieve the connection of cytotoxins at specific sites. Antibody-drug conjugates produced by fixed-site coupling can reduce the fluctuations in efficacy, pharmacokinetics, and quality control caused by different coupling sites and coupling numbers. At present, common fixed-site coupling methods include THIOMAB technology, non-natural amino acid coupling technology, glutamine enzymatic coupling technology, Sortase transpeptidase coupling technology, and Thiobridge technology. Among them, the modification of antibodies by antibody engineering or enzymatic coupling may have a certain impact on the structural stability of the antibody, and at the same time have certain requirements for CMC. In addition, the Thiobridge technology using chemical coupling also has certain defects, such as the replacement of DBM (dibromomaleimides)-type linkers and other thiol-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 ADC drugs, which represents the average number of small molecule drugs coupled to antibodies. The degree of drug coupling will affect the stability and aggregation tendency of ADC drugs. Therefore, the DAR value is a key quality attribute for ADC drug analysis and an important quality control link in the ADC drug development process. Fixed-site coupling technology can achieve fixed-site and quantitative coupling of antibodies and small molecule toxins. The ADC obtained by this technology has a suitable drug-to-antibody ratio (DAR), high uniformity, good stability, high batch-to-batch reproducibility, better activity and pharmacokinetic properties, and is also more suitable for large-scale production of ADC. The current development trend of ADC drugs is to use a site-specific coupling method to couple highly active small molecule drugs to antibodies to form a site-specific DAR2 ADC, such as the approved targeted CD19-ADC (loncastuximabtesirine, Zynlonta) that couples PBD small molecules to antibodies through glycosylation site-specific coupling technology to achieve site-specific DAR2, as well as HER2-targeted ADC (ARX788) and FRα-targeted ADC (MORAb-202), which both use non-natural amino acid mutations to achieve site-specific DAR2. However, these methods either require the use of enzymes or the modification of antibody sequences, and the manufacturing process is 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 (tri(2-carboxyethyl)phosphine) solution and mix well, and leave at room temperature for 2 hours. Add 5-10 times the amount of the antibody to the above solution system, mix well, and let stand at room temperature for 20 hours. After completion, 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. According to sample measurement, the DAR of the ADC conjugate prepared by the above non-site-specific coupling method in this patent is 4, and there is no record of a DAR of 2.

[0007] WO2022078524A2 discloses a site-specific coupling method for antibody conjugates. After reducing and incubating the antibody with a variety of zinc complexes and TCEP, 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 or UF / DF or ion exchange chromatography. The results of the DAR analysis of the conjugates using HIC-HPLC 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 by 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 comprises the following steps:

[0009] (a) Incubate a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)) and an antibody to be conjugated in a buffer system (e.g., Hepes, histidine buffer, PBS, MES, etc.) in the presence of an effective amount of transition metal ions (e.g., Zn2+, etc.) to selectively reduce intrachain disulfide bonds in the antibody; (b) react an excess of a payload carrying a reactive group (e.g., a maleimide-linked drug) with the reduced thiol group obtained in step (a); and (c) add an effective amount of an oxidant (e.g., dehydroascorbic acid (DHAA)) to reoxidize the unreacted thiol group, and then recover the obtained 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 smaller than the weight ratio of DAR4.

[0010] For ADC, the DAR value is the key quality attribute of ADC. Although high DAR can bring more small molecule drugs to the target site and may have a better therapeutic effect, the design of high DAR will sacrifice the stability and uniformity of ADC drugs, which may cause the aggregation of antibodies, thereby affecting the efficacy of ADC drugs and causing greater toxic side effects. The preparation of ADCs with high uniformity and / or purity of DAR2 can avoid the adverse effects of high DAR and can be beneficial to the stability and efficacy of ADC drugs. However, as mentioned above, the prior art can achieve fixed-point DAR2 through glycosylation fixed-point coupling technology. However, these methods either require the use of enzymes or the modification of antibody sequences, and the manufacturing process is complicated. The chemical fixed-point coupling method currently reported for the preparation of ADC, although the process is relatively simple, can only achieve ADC products with a main DAR of 4.

[0011] Therefore, there is an urgent need in the art for a simpler and more effective method for preparing site-directed DAR2-ADC. Summary of the invention

[0012] The present invention provides a new preparation method for antibody-drug conjugates, which obtains highly homogeneous antibody conjugate molecules through a specific chemical site-specific coupling method, and its DAR2 main peak accounts for more than 99%. The preparation method of the present invention has fewer steps and simple operation, which is conducive to industrial scale-up production. At the same time, the prepared product has fewer impurities and higher purity, which significantly improves the safety of medication and reduces production costs. The anti-tumor drug prepared by the coupling method of the present invention has excellent anti-tumor effects and safety (including higher stability and less side effects).

[0013] First aspect

[0014] The present invention provides a method for preparing an antibody-drug conjugate (ADC), the method comprising the following steps:

[0015] (a) adding a metal salt or a metal complex and a reducing agent to a buffer solution containing an antibody, followed by incubation;

[0016] (b) adding a drug-containing linker to the reaction solution in (a) for coupling; and

[0017] (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.

[0018] In some embodiments, the metal in the metal salt or metal complex described in step (a) can be selected from Zn, Cd, Hg, and the like.

[0019] 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

[0020]

[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(NH2CH2CH20H)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(NH2CH2C00C(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)42+ ,

[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(NH2CH2CH20H)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, such as 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, such as 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 antibody molar equivalent, such as 2-6, 3-5, 3.5-5.0 or 4.0-4.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 times the antibody molar equivalent.

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

[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 disodium hydrogen 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,

[0043] 3-10 hours, 4-8 hours or 5-24 hours, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 35 hours.

[0044] In some embodiments, the amount of the drug-containing linker in step (b) is 1-8 times the antibody molar equivalent, such as 2-6, 2.5-5.5, 3-5 or 3.5-4.5 times the antibody molar equivalent, 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 antibody molar equivalent.

[0045] In some embodiments, the coupling reaction temperature in step (b) is 0-30°C, such as 0-15°C or 3-15°C.

[0046] 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.

[0047] In some embodiments, the drug-containing linker is provided as a DMA solution.

[0048] 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,

[0049] 1.5-5 hours, 1-3 hours or 1-4 hours, for example 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 hours.

[0050] 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.

[0051] In some embodiments, the type of salt in step (c) is selected from sulfates, chlorides, etc., such as alkali metal (such as lithium, sodium, potassium) salts of sulfuric acid or hydrochloric acid, alkaline earth metal (such as calcium, magnesium) salts, salts formed by amines or ammonia and sulfuric acid or hydrochloric acid, preferably ammonium sulfate and 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.

[0052] In some embodiments, the salt solution described in step (c) is used as mobile phase A for hydrophobic interaction chromatography.

[0053] 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.

[0054] In some embodiments, mobile phase A and / or mobile phase B may further contain other solvents such as isopropanol.

[0055] In some embodiments, step (b) is followed by step (b'): adding a metal ion chelating agent such as ethylenediaminetetraacetic acid (EDTA) to react.

[0056] In some embodiments, step (b') is performed at room temperature, for example, for 5 min-5 hours, 10 min-45 min, or 30 min.

[0057] In some embodiments, the amount of metal ion chelator is 1-8 times the antibody molar equivalent, such as 2-6, 2.5-5.5,

[0058] 3-5 or 3.5-4.5 times antibody molar equivalents, 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 antibody molar equivalents.

[0059] In some embodiments, step (b") is followed by step (b'): adding dehydroascorbic acid (DHAA) to react.

[0060] In some embodiments, step (b") is performed at 25-35°C, for example 0.2-24 hours, for example 0.5-12 hours, 1-10 hours, 1.5-5 hours, 1-3 hours or 1-4 hours, for example 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 hours.

[0061] In some embodiments, the amount of DHAA used is 1-16 times the antibody molar equivalent, such as 2-10, 2-6, 6-16 or 6-12 times the antibody molar equivalent, such as 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14 or 16 times the antibody molar equivalent.

[0062] In some embodiments, the DAR2-ADC obtained by the method 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.

[0063] 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.

[0064] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

[0065] 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.

[0066] In some embodiments, the antibody is an anti-HER2 antibody.

[0067] 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.

[0068] In some embodiments, the antibody is selected from adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12, and hH2L1.

[0069] 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.

[0070] In some embodiments, the antibody is selected from trastuzumab, pertuzumab, h23-12, and hH2L1.

[0071] 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,

[0072] Wherein L1 represents a linker unit for connecting to an antibody,

[0073] A represents one or more (e.g. 1, 2, 3 or 4) amino acids,

[0074] L2 represents a linker unit for connecting to a small molecule drug, and

[0075] D is a small molecule drug.

[0076] 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,

[0077]

[0078] In the structural formula AI, and in the context of the present invention, if not otherwise specified, E is selected from the following groups, wherein The bond of represents the attachment site to M:

[0079]

[0080] n is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0081] 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

[0082] 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 group, an ester group, an amide group and a cyano group; preferably, M is a halogen-substituted phenylene group.

[0083] 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;

[0084] 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;

[0085] 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. 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;

[0086] Preferably, A is selected from the group consisting of NH-Val-Cit-CO, NH-Val-Ala-CO and NH-Gly-Gly-Phe-Gly-CO;

[0087] It should be understood that the NH on the left side of A is the amino group in the left amino acid in A, and the CO on the right side of A is the carbonyl group in the right amino acid in A.

[0088] L2 is selected from In some embodiments, M is selected from

[0089] In some embodiments, Selected from

[0090]

[0091] wherein q is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0092] 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;

[0093] In some embodiments, Selected from

[0094]

[0095] In some embodiments, D is selected from the group consisting of camptothecins, calicheamicins, maytansinoids, dolastatins, auristatins, and trichothecenes.

[0096] In some embodiments, 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, exatecan, SN-38, topotecan and camptothecin derivatives.

[0097] In some embodiments, the drug-containing linker is a compound of Formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

[0098]

[0099] wherein E, M, SP1, SP2 and A are as defined above, and CPT is a camptothecin compound.

[0100] In some embodiments, the drug-containing linker is a compound represented by Formula III' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,

[0101]

[0102] wherein E, M, SP1, SP2 and A are as defined above, and D' is an auristatin compound.

[0103] In some embodiments, the compound of formula III is further a compound of formula IIIA:

[0104]

[0105] 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

[0106] 4-Methylcarbamoylphenyl or 4-morpholinoformylphenyl wherein M, SP1, SP2, A and CPT are as defined above.

[0107] In some embodiments, the compound of formula III' is further a compound of formula IIIA':

[0108]

[0109] wherein R6, R7, M, SP1, SP2, A and D' are as defined above.

[0110] In some embodiments, in Formula III and Formula IIIA, CPT is a compound as shown in Formula I or Formula IA below, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound:

[0111]

[0112] In formula I, and in the context of the present invention, unless 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 atoms to which they are attached constitute a C3-6 cyclic alkyl. When R1, R2, R3, R4 are independently C1-6 alkoxy, the C1-6 alkoxy includes a straight or branched C1-6 alkoxy, preferably a straight or branched C1-3 alkoxy, more preferably a 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; or, 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).

[0113] In formula I, and unless otherwise stated in the context of the present invention, G is hydrogen, halogen, methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

[0114] In formula I, and in the context of the present invention, unless otherwise specified, Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene. The substituted methylene may be a methylene hydrogen substituted, or two hydrogens substituted simultaneously, and 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 attached 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; or, preferably, Y is oxygen, sulfur or methylene.

[0115] In formula I, and unless otherwise stated in the context of the present invention, X is oxygen or sulfur.

[0116] In formula I, and unless otherwise stated in the context of the present invention, n=0 or 1.

[0117] 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.

[0118] 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 constitute a C3-6 cyclic alkyl (e.g., a C3-5 cyclic alkyl). Further, R1 and R2 may be the same; and / or, R3 and R4 may be the same.

[0119] 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.

[0120] Preferably, X may be oxygen.

[0121] Preferably, X is oxygen, G is hydrogen, halogen (eg fluorine or chlorine), methyl or methoxy, and Y and R1, R2, R3, R4 are as defined above.

[0122] Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, R4 are as defined above.

[0123] Preferably, X is oxygen, G is fluorine, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, R4 are as defined above.

[0124] Preferably, X is oxygen, G is chlorine, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, R4 are as defined above.

[0125] Preferably, X is oxygen, G is methyl, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, R4 are as defined above.

[0126] Preferably, X is oxygen, G is methoxy, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, R4 are as defined above.

[0127] Preferably, X is oxygen, G is hydrogen, Y is oxygen, sulfone or sulfoxide, and R1, R2, R3, R4 are as defined above.

[0128] Preferably, X is oxygen, G is hydrogen, Y is sulfone or sulfoxide, and R1, R2, R3, R4 are as defined above.

[0129] Preferably, the compound represented by formula I is further represented by formula IA:

[0130]

[0131] In formula IA, the groups R1, R2, R3, and R4 are the same as the definitions of the groups R1, R2, R3, and R4 in formula I above, and R1, R2, R3, and R4 may be hydrogen at the same time or at different times.

[0132] In some embodiments, the camptothecin compound is a compound represented by Formula II or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

[0133]

[0134] In formula II, and in the context of the present invention, unless otherwise specified, 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 a straight or branched C1-5 alkyl. Further, R5 is a C1-4 straight chain alkyl. When R5 is a substituted C1-5 alkyl or a substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, mesyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl. When R5 is a substituted phenyl, the substituent is selected from alkyl (e.g., C1-6 alkyl, preferably C1-3) or halogen.

[0135] 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.

[0136] In formula II, X is oxygen or sulfur.

[0137] In formula II, n=0 or 1.

[0138] Preferably, in formula II, when X is oxygen, G is hydrogen, and n=0, R5 cannot be n-butyl.

[0139] Preferably, the compound represented by formula II is further represented by formula IIA:

[0140]

[0141] In Formula IIA, the group R5 is the same as the definition of the group R5 in Formula II above. In some embodiments, R5 may not be n-butyl. In some embodiments, R5 may be n-butyl.

[0142] In some embodiments, the camptothecin compound (CPT) has the following structure:

[0143]

[0144]

[0145]

[0146]

[0147] 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.

[0148] For example, in Formula III and Formula IIIA, the compound shown in CPT Formula II or Formula IIA or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, and the corresponding specific compound.

[0149] 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.

[0150] 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 connected to the carboxyl group of A in formula III or IIIA through an amide bond via its amino group.

[0151] 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:

[0152]

[0153] In formula IV, and unless otherwise specified 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.

[0154] 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 is an amino substituted by one or more substituents selected from methyl and ethyl.

[0155] 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.

[0156] In particular, for the compound represented by formula IIIA, when R6 and R7 are Ar'S, M is preferably a phenylene or a 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.

[0157] In some embodiments, in Formula III' and Formula IIIA', D' is a compound as shown in the following Formula I' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound:

[0158]

[0159] 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;

[0160] R a6 and R a7 Each independently selected from C 1-8 Alkoxy, such as methoxy, ethoxy or propoxy;

[0161] 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;

[0162] R a10 Selected from OH and H.

[0163] In some embodiments, the compound of formula I' is further a compound of formula IA':

[0164]

[0165] Where R a1 -R a10 As defined above;

[0166] In some embodiments, the compound of Formula I' is selected from:

[0167]

[0168]

[0169] In III' and 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'.

[0170] In some embodiments, the compound represented by Formula III and Formula IIIA is further represented by Formula V.

[0171]

[0172] 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

[0173] In formula V, and unless otherwise specified 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).

[0174] 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.

[0175] In formula V, A represents 2 to 4 amino acids, as defined above.

[0176] In Formula V, the definition of CPT and its linkage in Formula V are the same as CPT in Formula III and Formula IIIA, as defined above.

[0177] In some embodiments, the compound of formula IIIA' is further a compound of formula V',

[0178]

[0179] Where R 6 , R 7 , Xh, Yh, m and A are as defined above (especially formula V), and D' is as defined above.

[0180] In some embodiments, the compound of formula V' is further a compound of formula V-A',

[0181]

[0182] wherein A and D' are as defined above.

[0183] In some embodiments, the compound of formula V is further a compound of formula VA:

[0184]

[0185] In formula VA, the groups A, G, Y, R1, R2, R3, R4, X, and n are the same as the definitions of the groups A, G, Y, R1, R2, R3, R4, X, and n in formula III or formula IIIA above.

[0186] Preferably, in formula VA, G is hydrogen, fluorine or chlorine.

[0187] Preferably, in formula VA, Y is methylene, sulfur or oxygen.

[0188] In formula VA, and unless otherwise specified in the context of the present invention, "-A-NH-" indicates that the amino group indicated forms an amide bond with the carboxyl group in A.

[0189] In some embodiments, the compound represented by Formula VA is further represented by Formula VA-1:

[0190]

[0191] Alternatively, the compound represented by formula V is further represented by formula VB:

[0192]

[0193] In Formula VB, the groups A, G, R5, X, and n are the same as those defined above in Formula III or Formula IIIA.

[0194] In Formula VB, 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.

[0195] Preferably, the compound represented by formula VB is further represented by formula VB-1:

[0196]

[0197] Alternatively, the compound represented by formula V is further represented by formula VC:

[0198]

[0199] In formula VC, the groups A and R8 are the same as defined above in formula III or formula IIIV.

[0200] In formula VC, 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.

[0201] In formula VC, and in the context of the present invention unless otherwise specified, The self-releasing structure is the same as when the CPT in the above formula III or formula IIIV is formula IV.

[0202] According to a specific embodiment of the present invention, the compound represented by formula V is further represented by formula VI:

[0203]

[0204] In formula VI, A represents 2 to 4 amino acids, as defined above.

[0205] 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.

[0206] Preferably, the compound represented by Formula VI provided by the present invention is further represented by Formula VI-A:

[0207]

[0208] In formula VI-A, the groups A, G, Y, R1, R2, R3, R4, X, and n have the same definitions as the groups A, G, Y, R1, R2, R3, R4, X, and n in formula III or IIIA above.

[0209] Preferably, in Formula VI-A, G is hydrogen, fluorine or chlorine.

[0210] Preferably, in Formula VI-A, Y is methylene, sulfur or oxygen.

[0211] In Formula VI-A, and in the context of the present invention, unless otherwise specified, "-A-NH-" means that the amino group indicated forms an amide bond with the carboxyl group in A.

[0212] Preferably, the compound represented by formula VI-A is further represented by formula VI-A-1:

[0213]

[0214] Alternatively, the compound represented by formula VI is further represented by formula VI-B:

[0215]

[0216] 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.

[0217] 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.

[0218] More preferably, the compound represented by formula VI-B is further represented by formula VI-B-1:

[0219]

[0220] Alternatively, the compound represented by formula VI is further represented by formula VI-C:

[0221]

[0222] 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.

[0223] 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.

[0224] When the self-releasing structure in VC is When, formula VC can be further structured as shown in formula VI-C.

[0225] In some embodiments, the drug-containing linker is selected from the following structures:

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234] 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.

[0235] Some small molecule drugs, drug-containing linkers and / or intermediates thereof, and / or antibody-drug conjugates in the present invention are recorded in Chinese patent application 202211627164.8, and the entire content of the patent application is introduced into this application as part of the content of this application.

[0236] The steps of the above preparation method are interrelated and interact with each other to produce a technical effect of improving the purity of the DAR2-ADC conjugate, and this technical effect is produced by the steps as a whole.

[0237] Second aspect

[0238] The present invention provides an antibody drug conjugate having the general formula The structure shown, wherein mAb represents an antibody or antibody fragment, such as the antibody defined above, and groups M, SP1, SP2, A, L2 and D are as defined above in the first aspect. y is 1-10, preferably 1-8 (e.g. 1-5), more preferably 1-3, and most preferably 2.

[0239] 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 key indicates connection with M:

[0240] and / or where n is as defined above in variable E.

[0241] In the general structure, mAb may be an IgG type antibody or a fragment thereof, preferably an IgG1 subtype antibody or a fragment thereof.

[0242] In some embodiments, the antibody drug conjugate is prepared by the method of the present invention as described in the first aspect.

[0243] In some embodiments, the antibody drug conjugate has an average DAR of about 2.

[0244] 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%, such as measured by HIC-HPLC.

[0245] In some embodiments, the antibody drug conjugate has the structure shown in the following general formula VII, VII', VIII, VIII':

[0246]

[0247]

[0248] and / or

[0249] Here, y is 1-10, preferably 1-8 (eg, 1-5), and more preferably 1, 2 or 3.

[0250] Wherein, the groups A, CPT and D' are the same as defined in the first aspect above.

[0251] The third aspect

[0252] In the third aspect, the present invention provides the small molecule drugs, drug-containing linkers and / or intermediates thereof (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) described in the preparation method of the first aspect of the present invention, preferably including but not limited to the compounds disclosed in the embodiments.

[0253] The fourth aspect

[0254] 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.

[0255] In some embodiments, the pharmaceutical composition is a liquid preparation or a lyophilized preparation.

[0256] In some embodiments, the pharmaceutical composition comprises a buffer, a stabilizer, and a surfactant.

[0257] Fifth Aspect

[0258] The present invention provides use of the compound or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, antibody-drug conjugate or pharmaceutical composition according to the present invention in preparing a drug for treating tumors.

[0259] Preferably, the tumor is cancer. Preferably, the tumor is associated with tumor-associated antigens 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, 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, and Claudin 18.2 positive or high expression.

[0260] 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.

[0261] The sixth aspect

[0262] 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 thereof, an antibody-drug conjugate or a pharmaceutical composition thereof, the method comprising administering the compound or antibody-drug conjugate to a subject in need thereof.

[0263] Preferably, the tumor is cancer. Preferably, the tumor is associated with tumor-associated antigens 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, 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, and Claudin 18.2 positive or high expression.

[0264] 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.

[0265] Preferably, the subject is a mammal, preferably a primate, more preferably a human.

[0266] The method and / or product of the present invention has the following advantages and / or benefits:

[0267] (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;

[0268] (2) The preparation method of the present invention has few steps and is simple to operate, which is conducive to industrial scale-up production. At the same time, the prepared product has few impurities and high purity, which significantly improves the safety of drug use and reduces production costs; and / or

[0269] (3) The anti-tumor drug prepared by the coupling method of the present invention has excellent anti-tumor effect and safety, including high stability and less side effects, expanding the treatment window, improving the treatment effect, and reducing toxic reactions.

[0270] definition:

[0271] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, scheme and reagent described herein, because these can change. It should also be understood that the terms used in this article are only for describing specific embodiments, and are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs.

[0272] 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.

[0273] The term "antibody drug conjugate" or "ADC" refers to a substance obtained by connecting 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 through 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 connected to the antibody or its antigen-binding fragment through 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.

[0274] 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 has a functional group that can form a bond with a functional group of the antibody or its antigen-binding fragment before being attached to the antibody or its antigen-binding fragment (i.e., a linker precursor).

[0275] 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 less than 10kD, usually 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 immune responses than macromolecules. Small molecule drugs include but are not limited to cytotoxic agents, such as camptothecin compounds, calicheamicin (calicheamicin), maytansinoids (maytansinoids), dolastatin (dolastatin), auristatin compounds and trichothecene (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 and the camptothecin compounds mentioned in the embodiments of the present invention.

[0276] In the present disclosure, when a small molecule drug is part of an ADC or a drug-containing linker, it refers to a structural fragment from a small molecule drug, such as a structural fragment formed by bonding a hydroxyl group or an amino group of a small molecule drug to a linker, which can form a part (fragment or group) of a biologically active drug (such as a small molecule cytotoxic drug, the drug includes a group after losing an atom or atom group) or a derivative (such as a precursor thereof) after the linker is cleaved / degraded / enzymatically cleaved between tumor tissues or in tumor cells. In order to avoid ambiguity, "small molecule drugs" do not only refer to "drugs" that have been approved by the pharmaceutical regulatory authorities, but also include any compounds with potential therapeutic biological activity in clinical practice, or in research and development and academic research.

[0277] The term "drug-containing linker" refers to a compound formed by linking a bioactive compound fragment (small molecule drug) to a linker, which is sometimes referred to herein as a "drug-linker conjugate".

[0278] Metal ion chelators are compounds that, through their strong binding with metal ions, encapsulate metal ions into the interior of the chelator, turning them into stable compounds with larger molecular weight, thereby preventing the metal ions from taking effect. Metal ion chelators include but are not limited to ethylenediaminetetraacetic acid,

[0279] The term "pharmaceutical composition" refers to a composition that is in a form that permits 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.

[0280] The terms "treat," ...

[0281] The term "prevention" includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

[0282] 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, etc.

[0283] The term "alkylene" refers to a divalent group formed by removing a hydrogen atom from an alkyl group as defined above.

[0284] 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 and the like.

[0285] The term "cycloalkylene" refers to a divalent group formed by removing a hydrogen atom from a cycloalkyl group as defined above.

[0286] 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 connected to each other. Preferably, heteroalkylene is a C1-21 (e.g., C1-10, C1-6) straight chain heteroalkylene, which contains 1-11 (preferably 1-6, 1-3) heteroatoms selected from N, O or S.

[0287] 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 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.

[0288] "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.

[0289] The term "halogen" refers to F, Cl, Br, I.

[0290] 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.

[0291] The term "antibody molar equivalent" refers to the molar equivalent relative to the antibody.

[0292] It should be understood that for the divalent groups in the general structure of the present application, the listed groups are connected to the two side groups in the direction shown, unless the structure obtained by such connection is unreasonable or deviates from the present invention, or contradicts the context. For example, when L2 is When , 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).

[0293] The term "drug to antibody ratio" or "DAR" refers to the ratio of the number of small molecule drug moieties (D) coupled to the antibody moiety (mAb) described herein to the antibody moiety. The DAR of an ADC can be in the range of 1 to 10, but higher loadings are possible depending on the number of attachment sites on the antibody. The term DAR can be used when referring to the number of drugs 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 a product 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, such as 4.0-10.0, 5.0-9.0, 6.0-8.0, 1.0-8.0, 2.0-6.0, such as 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, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6. 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9,

[0294] The term “DAR2-ADC” refers to an ADC with a DAR of 2, i.e., an ADC in which an antibody is linked to two small molecule drugs.

[0295] 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.

[0296] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0297] As used herein, the term "comprising" or "including" means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the combination of the elements, integers or steps described. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence. DETAILED DESCRIPTION

[0298] The disclosure is further described below by describing specific embodiments, but this is not a limitation of the disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of the disclosure without departing from the basic idea and scope of the disclosure. The instruments or equipment used without indicating the source are all products that can be obtained commercially. The reagents used without indicating the source are products that can be obtained commercially or synthesized by conventional methods.

[0299] The abbreviations herein have the following meanings, and abbreviations without meanings provided have the meanings commonly understood in the art:

[0300] Abbreviated name

[0301] KHMDS Potassium Hexamethyldisilazide

[0302] NFSI N-Fluoro-bis(benzenesulfonyl)imide

[0303] DIPEA N,N-Diisopropylethylamine

[0304] PE Petroleum Ether

[0305] EA Ethyl acetate

[0306] eq equivalent

[0307] MeOH Methanol

[0308] DMF Dimethylformamide

[0309] PB Disodium hydrogen phosphate and sodium dihydrogen phosphate buffer solution

[0310] IPA Isopropyl Alcohol

[0311] MTS Abcam, Catalog Number ab223881

[0312] Example 1 Synthesis of small molecules

[0313] Example 1.1 Synthesis of Compound MWC-1

[0314]

[0315] According to the patent method of WO2020200880A1, MWC-1 was prepared using B-1 as an intermediate. It was a yellow solid. LC-MS (ESI): [M+1]+=404

[0316] Example 1.2 Synthesis of compound 3

[0317]

[0318] Step 1

[0319] 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 with a dry ice-acetone bath, and 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 (NFSI (7.35 g, 23 mmol, 3 eq) was added dropwise. ) was dissolved in tetrahydrofuran (70 ml), and after the addition, the mixture was stirred at -70 to -60 °C for 10 minutes, and the temperature was naturally raised to 20 to 30 °C and stirred for 3 hours; a saturated aqueous solution of ammonium chloride (80 ml) was added to quench the reaction, and 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, which was purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 1:0-1:3 to obtain compound 3a as a yellow solid, 0.85 g, with a yield of 31.2%. LC-MS (ESI): [M+1]+ = 355.8

[0320] 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)

[0321] Step 2

[0322] 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. The heating was stopped, and after the reaction solution was cooled to room temperature, the pH was adjusted to 7-8 with sodium bicarbonate, extracted with dichloromethane (30ml*3), the organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product, which was purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 1:0-1:3 to obtain compound 3b, 0.25g of brown solid, with a yield of 49.1%. LC-MS (ESI): [M+1]+ = 213.9

[0323] Step 3

[0324] In a 50 ml single-mouth reaction bottle, 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 distilled off 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 separated and purified by preparative liquid phase separation to obtain camptothecin compound 3 as a khaki solid. LC-MS (ESI): [M+1]+=440.8

[0325] 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)

[0326] 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)

[0327] Synthesis of compound B

[0328]

[0329] Compound B-1 was synthesized according to the method of Jornal of Medcinal Chemstry, 1998, 41(13), 2308-2318. Compound B-1 was a brown solid. LC-MS (ESI): [M+1]+=177

[0330] 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)

[0331] In a 250ml three-necked flask, compound B-1 (2.18g, 124mmol, 1eq) was dissolved in tetrahydrofuran (100ml), and Boc anhydride (8.2g, 376mmol, 3eq) 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 the eluent being petroleum ether: ethyl acetate = 1:0-1:3 to obtain compound B-2 as a yellow solid (3.0g). The yield was 90%.

[0332] 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)

[0333] In a 250ml three-necked flask, compound B-2 (3.0g, 10.8mmol, 1eq) and DIPEA (3.5g, 27.1mmol, 1.5eq) were mixed in dichloromethane (125ml), and the reaction solution was cooled to -5~0°C with an ice-salt bath, and acetyl chloride (1.3g, 16.5mmol, 2eq) 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 the eluent being petroleum ether: ethyl acetate = 1:0~1:3 to obtain compound B as a light yellow solid, 3.48g, with a yield of 100%.

[0334] 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)

[0335] Example 2 Synthesis of novel drug-containing linkers

[0336] Example 2.1Synthesis of drug-containing linker BL20E (C-3)

[0337]

[0338] The BL20E compound is obtained by condensing the BL linker compound with VC-PABC-MMAE according to the method disclosed in WO2018 / 095422A1. The synthesis route is as follows:

[0339]

[0340] The BL20E compound was a yellow solid. LC-MS (ESI): [M+1]+ was 1963 (C-3).

[0341] Example 2.2 Synthesis of drug-containing linker MWD-L1

[0342]

[0343] The MWD-L1 compound is synthesized from the BL linker compound and GGFG-Dxd. The synthesis route is as follows:

[0344]

[0345] BL linker compound (857 mg, 1 mmol), GGFG-Dxd [synthesized according to the method described in the patent disclosure document (US20190151328A1)] (840 mg, 1 mmol, 1 eq), DIPEA (323 mg, 2.5 mmol, 2.5 eq), HATU (570 mg, 1.5 mmol, 1.5 eq), dissolved in 30 ml DCM, 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, the aqueous phase was extracted with DCM (30 ml * 2), and the organic phase was combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and dried. Purification by column chromatography, eluent: DCM:MeOH=50:1-10:1, to obtain 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.

[0346] Example 2.3 Synthesis of drug-containing linker MWC-L2

[0347]

[0348] Step 1

[0349] Boc-Val-Ala-OH (288 mg, 1 mmol), A1 compound [synthesized according to the method of Journal of Medicinal Chemistry, 1998, vol. 41, #13, p. 2308-2318] (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 ml DCM and stirred for 2 h. The reaction solution was dried and purified by column chromatography, eluent: PE: EA = 10: 1 to 5: 1, and intermediate 2-1 was quantitatively obtained as a gray-green solid (450 mg).

[0350] Step 2

[0351] Intermediate 2-1 (450 mg, 1 mmol), CDE tricyclic compound (263 mg, 1 mmol, 1 eq), p-toluenesulfonic acid pyridinium salt (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 after the reaction solution was cooled, the solid precipitate was collected as the crude intermediate 2-2, 750 mg of brown solid. LC-MS (ESI): M+1=574. The product was directly used for the next step without purification.

[0352] Step 3

[0353] BL linker compound (857mg, 1mmol), HoSu (138mg, 1.2mmol, 1.2eq), DCC (310mg, 1.5mmol, 1.5eq) were dissolved in 30ml DCM, stirred at room temperature for 3h, and the reaction solution was filtered. The filtrate was the DCM solution of A-Osu. The filtrate was added to the mixed solution of the crude intermediate 2-2, DIPEA (323mg, 2.5mmol, 2.5eq) and DCM (30ml), and the reaction was stirred for 3h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h. The liquid was separated, the aqueous phase was extracted with DCM (30ml*2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Column chromatography purification, eluent: DCM: MeOH = 50: 1 to 10: 1, the linker MWC-L2 was obtained as an orange-red solid, 325 mg, yield 23.0%, LC-MS (ESI): M+1 = 1414, (M+1) / 2 = 707

[0354] Example 2.4 Synthesis of drug-containing linker MWC-L3

[0355]

[0356] Step 1

[0357] Boc-Gly-OH (175 mg, 1 mmol), A1 compound (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 ml DCM and stirred for 2 h. The reaction solution was spin-dried and purified by column chromatography, eluent: PE: EA = 10: 1 to 5: 1, and intermediate 3-1 was quantitatively obtained as a light green solid 335 mg.

[0358] Step 2

[0359] Intermediate 3-1 (335 mg, 1 mmol), CDE tricyclic compound (263 mg, 1 mmol, 1 eq), 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 after the reaction solution was cooled, the solid precipitate was collected as the crude intermediate 3-2, 560 mg of brown solid. LC-MS (ESI): M+1=461. The product was directly used for the next step without purification.

[0360] Step 3

[0361] Fmoc-Gly-Gly-Phe-OH (501 mg, 1 mmol), crude intermediate 3-2 (560 mg), DIPEA (322 mg, 2.5 mmol, 2.5 eq), HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 20 ml DMF and stirred for 3 h. 1N hydrochloric acid (30 ml) was added and stirred for 0.5 h. Extracted with DCM (30 ml*2) and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and dried. Purified by column chromatography, eluent: DCM: MeOH = 50: 1 ~ 10: 1, intermediate 3-3 was obtained as a brown solid, 350 mg, LC-MS (ESI): M+1 = 945

[0362] Step 4

[0363] Intermediate 3-3 (350 mg, 0.37 mmol) was dissolved in methanol (20 ml), diethylamine (2 ml) was added, and the mixture was stirred for 3 h. The reaction solution was spin-dried to obtain a crude intermediate 3-4 as a brown viscous substance, LC-M (ESI): M+1=722, and the crude product was directly used for the next step without purification.

[0364] Step 5

[0365] BL linker compound (318 mg, 0.37 mmol), HoSu (51 mg, 0.44 mmol, 1.2 eq), DCC (114 mg, 0.56 mmol, 1.5 eq) were dissolved in 30 ml DCM, stirred at room temperature for 3 h, and the reaction solution was 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 liquid was separated, the aqueous phase was extracted with DCM (30 ml * 2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Column chromatography purification, eluent: DCM: MeOH = 50: 1 to 10: 1, to obtain linker MWC-L3, an orange-red solid 120 mg, yield 20.8%, LC-MS (ESI): M+1 = 1562, (M+1) / 2 = 781

[0366] Example 2.5 Synthesis of drug-containing linker MWF-L6

[0367]

[0368] 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.

[0369] Compound A7 was synthesized as follows:

[0370]

[0371] 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 the eluent to obtain compound A7-1 as a yellow solid (3.0 g). The yield was 90%.

[0372] 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)

[0373] In a 250ml three-necked flask, A7-1 compound (3.0g, 10.8mmol, 1eq) and DIPEA (3.5g, 27.1mmol, 1.5eq) were mixed in dichloromethane (125ml), and the reaction solution was cooled to -5~0°C with an ice-salt bath, and acetyl chloride (1.3g, 16.5mmol, 2eq) 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 = 1:0~1:3 as the eluent to obtain A7-2 compound as a light yellow solid, 3.48g, with a yield of 100%.

[0374] 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)

[0375] 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 with a dry ice-acetone bath, and 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), and after the addition, the mixture was stirred at -70 to -60 °C for 10 minutes, and the temperature was naturally raised to 20 to 30 °C and stirred for 3 hours; a saturated aqueous solution of ammonium chloride (80 ml) was added to quench the reaction, and 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, which was purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 1:0-1:3 to obtain compound A7-3 as a yellow solid, 0.85 g, with a yield of 31.2%. LC-MS (ESI): [M+1]+ = 355.8

[0376] 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)

[0377] 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. The heating was stopped, and after the reaction solution was cooled to room temperature, the pH was adjusted to 7-8 with sodium bicarbonate, extracted with dichloromethane (30ml*3), the organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product, which was purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 1:0-1:3 to obtain compound A7, a brown solid of 0.25g, with a yield of 49.1%. LC-MS (ESI): [M+1]+ = 213.9

[0378] Example 2.6 Synthesis of drug-containing linker DT-1

[0379]

[0380] DT-1 was synthesized according to the method of CN114106088A as a yellow solid, LC-MS (ESI): [M+1]+=1474.4

[0381] Example 2.7 Synthesis of drug-containing linker DT-2

[0382]

[0383] According to the synthesis method of BL20E compound, DT-2-A compound was used to replace the BL linker compound to synthesize DT-2, which was a light yellow solid. LC-MS (ESI): [M+1]+=1339.7

[0384] The DT-2-A compound was synthesized according to the literature Chem.Sci., 2022, 13, 8781–8790.

[0385] Example 2.8 Synthesis of drug-containing linker DT-3

[0386]

[0387] DT-3 was synthesized according to the method of WO2022253033A as a light yellow solid, LC-MS (ESI): [M+1]+=1631.5.

[0388] Example 2.9 Synthesis of drug-containing linker DT-4

[0389]

[0390] DT-4 was synthesized according to the method of CN103933575B as a light yellow solid. LC-MS (ESI): [M+1]+=1414.7

[0391] Example 2.10 Synthesis of drug-containing linker DT-5

[0392]

[0393] DT-5 was synthesized according to the method of CN109232464A as a yellow solid, LC-MS (ESI): [M+1]+=1591.8

[0394] Example 2.11 Synthesis of drug-containing linker DT-6

[0395]

[0396] DT-6 was synthesized according to the method of KR2019014324A as a yellow solid, LC-MS (ESI): [M+1]+=1619.6

[0397] Example 2.12 Synthesis of drug-containing linker DT-7

[0398]

[0399] DT-7 was synthesized according to the method of JP2018138538A as a yellow solid, LC-MS (ESI): [M+1]+=1733.7. The MC-VC-PAB-MMAE used in the example was purchased from MCE.

[0400] Example 3 Coupling process study

[0401] Example 3.1 Antibody reduction conditions and metal ion screening

[0402] The trastuzumab antibody solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15mg / mL. Different metal ion aqueous solutions and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solutions were added in sequence and reacted at 0-15°C overnight. The reaction equivalents are shown in Table 1-1. Then, 3 times the antibody molar equivalent of BL20E (C-3) DMA solution was added to the above reaction solution, reacted at 0-15°C for 2h, and then the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 6.5-7.4. The concentration of the above reaction solution was adjusted to about 5-15mg / mL, 4 times the antibody equivalent of ethylenediaminetetraacetic acid (EDTA) was added, and the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 7.4-8.0 after reacting at room temperature for 30min. The concentration of the reaction solution was adjusted to about 5-15 mg / mL, 8 times the antibody equivalent of dehydroascorbic acid (DHAA) was added, and the solution was changed to 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 after reacting at 25-35°C for 2 hours. The antibody drug conjugate product was obtained. Samples were taken for analysis, and the antibody coupling ratio (DAR) was determined. The test results are shown in Table 1-1.

[0403] Table 1-1. Screening results of different reducing agent equivalents and different metal ions

[0404]

[0405]

[0406] 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.

[0407] Example 3.2 Screening of antibody coupling conditions

[0408] The trastuzumab antibody solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15mg / mL. A ZnCl2 aqueous solution of 2 times the antibody molar equivalent and a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution of 4.0-4.5 times the antibody molar equivalent were added in sequence, and the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution of different pH values ​​after overnight reaction at 0-15°C. Then, a DMA solution of BL20E (C-3) of 3 times the antibody molar equivalent was added to the above reaction solution. The coupling reaction temperature and the pH of the coupling solution are shown in Table 1-2. After the above coupling reaction was completed, the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 6.5-7.4. The concentration of the above reaction solution was adjusted to about 5-15 mg / mL, 4 times the antibody equivalent of ethylenediaminetetraacetic acid (EDTA) was added, and the solution was changed to 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 7.4-8.0 after reacting at room temperature for 30 minutes. The concentration of the above reaction solution was adjusted to about 5-15 mg / mL, 8 times the antibody molar equivalent of dehydroascorbic acid (DHAA) was added, and the solution was changed to 50 mM sodium dihydrogen phosphate-disodium dihydrogen phosphate buffer with a pH of 7.4 after reacting at 25-35°C for 2 hours. 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 Table 1-2.

[0409] Table 1-2. Screening results of different coupling reaction temperatures and different coupling solution pH

[0410] Coupling reaction temperature (℃) Coupling solution pH HIC (main peak D2%) NR-CE-SDS (purity %) 4 6.5 75 78.8 4 6.9 78 79.4 10 6.5 61 69.5 10 6.9 65 67.4

[0411] It can be seen that the HIC peak value of DAR2-ADC can be achieved to be 61-78% by using the above coupling reaction temperature and coupling solution pH in the preparation method of the present application.

[0412] Example 3.3 Screening of antibody purification conditions

[0413] The trastuzumab antibody solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15mg / mL. A ZnCl2 aqueous solution of 2 times the antibody molar equivalent and a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution of 4.0-4.5 times the antibody molar equivalent were added in sequence, and reacted at 0-15°C overnight. Then, a DMA solution of BL20E (C-3) of 3 times the antibody molar equivalent was added to the above reaction solution, and the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 6.5-7.4 after reacting at 0-15°C for 2h. The concentration of the above reaction solution was adjusted to about 10mg / mL, and ethylenediaminetetraacetic acid (EDTA) of 4 times the antibody equivalent was added. After reacting at room temperature for 30min, the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 7.4-8.0. The concentration of the above reaction solution was adjusted to about 5-15 mg / mL, 8 times the antibody equivalent of dehydroascorbic acid (DHAA) was added, and the reaction was carried out at 25-35°C for 2 hours. After the reaction, the solution was changed to 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 7.5, diluted to about 5-20 mg / mL, and ammonium sulfate solution was added to adjust the conductivity. The antibody drug conjugate product was purified by hydrophobic interaction chromatography (HIC). The purification conditions are shown in Table 1-3. The purified product was sampled for HIC analysis, and the HIC test results are shown in Table 1-3.

[0414] Table 1-3. Screening results of different purification conditions

[0415] Filler Type Salt Type Salt concentration (mol / L) Yield (%) HIC-HPLC (D2%) Butyl-650M Ammonium sulfate 0.60 30 100 Butyl-650M Ammonium sulfate 0.65 50 99.1 Butyl-650M Ammonium sulfate 0.75 43 99.4 Butyl-650M Sodium chloride 2 48 99.1 Butyl-4FF Ammonium sulfate 0.75 42 98.8 Butyl-650S Ammonium sulfate 0.60 35 100 Butyl-650S Ammonium sulfate 0.65 43 99.6 Butyl-ImpRes Ammonium sulfate 0.60 34 100 Butyl-ImpRes Ammonium sulfate 0.65 40 99.8 Butyl-ImpRes Sodium chloride 1.50 NA, sample flowthrough NA Butyl-ImpRes Sodium chloride 2.00 37 100 Phenyl Sepharose HP Ammonium sulfate 0.6 32 100 Phenyl Sepharose HP Sodium chloride 1.50 NA, sample flowthrough NA Phenyl Sepharose HP Sodium chloride 2.00 NA, sample flowthrough NA

[0416] 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 a HIC peak value of 98-100% for DAR2-ADC.

[0417] Example 3.4 Conjugation experiments of different drug-containing linkers and different antibodies

[0418] The antibody solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15mg / mL. A ZnCl2 aqueous solution of 2 times the antibody molar equivalent and a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution of 4.0-4.5 times the antibody molar equivalent were added in sequence, and reacted at 0-15°C overnight. Then, a DMA solution containing a drug linker of 2.5-5.5 times the antibody molar equivalent was added to the above reaction solution, and the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 6.5-7.4 after reacting at 0-15°C for 2h. The concentration of the above reaction solution was adjusted to about 5-15mg / mL, ethylenediaminetetraacetic acid (EDTA) of 4 times the antibody molar equivalent was added, and the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 7.4-8.0 after reacting at room temperature for 30min. The concentration of the above reaction solution was adjusted to about 5-15 mg / mL, 8 times the antibody molar equivalent of dehydroascorbic acid (DHAA) was added, and the reaction was carried out at 25-35°C for 2 hours. After the reaction was completed, the solution was changed to a 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 7.5, diluted to about 5-20 mg / mL, and an ammonium sulfate solution was added to adjust the conductivity. A 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate + 0.45-0.75M ammonium sulfate solution at pH 7.5 was used as phase A, and a 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate solution at pH 7.5 was used as phase B. Purification was performed using Butyl 650M filler, and the purified solution was concentrated and changed to a 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 7.4. After purification, samples were taken for analysis, and the antibody coupling ratio (DAR) and purity in the obtained antibody-drug conjugate product were determined. The test results are shown in Tables 1-4.

[0419] Table 1-4. Experimental results of different antibodies coupled to different small molecules

[0420]

[0421] 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.

[0422] Example 3.5 Comparative Example

[0423] According to the preparation method disclosed in patent WO2020 / 164561A1, the antibody drug conjugate Trastuzumab-MC-VC-PAB-MMAE was prepared. The brief description is as follows:

[0424] (1) Add ZnCl2 (0.04 mM) and TCEP (0.08 mM) to the Trastuzumab antibody solution and react overnight at 4°C;

[0425] (2) Add DMA solution (0.12 mM) 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.

[0426] (3) adding cysteine ​​(0.08 mM) to the reaction solution of (2) above to remove excess MC-VC-PAB-MMAE; (4) adding EDTA (0.08 mM) to the reaction solution of (3) above, followed by adding DHAA (0.16 mM);

[0427] (5) The reaction mixture was purified using a desalting column (Model: 40K, 0.5 mL, REF: 87766, Lot: SJ251704, Thermo).

[0428] Table 1-5. HIC analysis comparison results

[0429]

[0430] Example 4 Physicochemical Characterization of Antibody Drug Conjugates

[0431] Example 4.1 Physicochemical Characterization of Antibody Drug Conjugates

[0432] a. Determination of DAR by hydrophobic interaction chromatography (HIC-HPLC)

[0433] Mobile phase A: 2.0M (NH4)2SO4 + 50mM PB, pH 7.0

[0434] Mobile phase B: 50 mM PB + 15% IPA, pH 7.0

[0435] Flow rate: 0.6mL / min

[0436] Injection concentration: 3-5 mg / mL

[0437] Injection volume: 10 μL

[0438] Analytical column: TOSOH Butyl NPR, 4.6*100mm, 2.5μm, PN0042168, CN081D00130D

[0439] Column temperature: 25°C

[0440] Detection wavelength: 280nm

[0441] gradient:

[0442] time ％A ％B initial 100 0 15 0 100 20 0 100 20.1 100 0 30 100 0

[0443] Sample injection analysis: For samples with a concentration exceeding 5 mg / mL, dilute the sample with 50% phase A solution, filter through a 0.2 μm filter, and then perform sample injection analysis; for samples with a concentration of 5 mg / mL or less, filter directly before sample injection analysis.

[0444] 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.

[0445] b. Purity determination by non-reducing capillary electrophoresis NR-CE-SDS

[0446] The method was determined according to the monoclonal antibody molecular size variant determination method in General Chapter 3127 of the Chinese Pharmacopoeia Volume IV.

[0447] Example 5 Bioactivity Analysis of Antibody Drug Conjugates

[0448] 5.1 Study on the killing effect of antibody-drug conjugates in vitro

[0449] Exemplarily, the following method was used to conduct the experiment.

[0450] This experiment used human gastric cancer cell line NCI-N87 (purchased from ATCC). The cell density of NCI-N87 cells was adjusted to 15×10 using complete culture medium (45 ml RPMI 1640 culture medium, 5 ml FBS, mixed before use). 4 After that, 100 μl / well was added to the cell culture plate and cultured overnight. On the second day, the ADC samples were diluted with the above complete medium to 50ug / ml, and then diluted 4 times in a gradient, with a total of 9 gradients plus zero point, and 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); add the above diluted ADC samples to the cell culture plate cultured overnight in sequence, 100 μl / well. Then place it in a cell culture incubator and incubate for 120 hours. Take out the cell culture plate, add MTS (abcam, product number ab223881) at 40 μl / well, and react in a 37°C incubator for 2-4 hours; take out the cell plate and read the OD value at 490nm.

[0451] Table 1-6 Comparative study results of the activity of different antibody-drug conjugates

[0452]

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: Zn, Cd and Hg hydrochloride or sulfate, 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 antibody molar equivalent, for example 1-3 times the antibody molar equivalent, 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, i.e. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12 or hH2L1, or comprises VH and / or VL of the antibody, or comprises the 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 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-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 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 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 R6 , 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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