Preparation method of antibody-drug conjugate

Abstract

The invention provides a preparation method of an antibody-drug conjugate. Specifically, provided is a method for preparing an antibody-drug conjugate (ADC) having improved homogeneity. Compared with a conventional method, the method has the advantages that the process is simple, the steps of reaction quenching and oxidation are not needed, and the coupling reaction is not limited by conditions such as temperature.

Description

Method for preparing antibody-drug conjugates

[0001] This disclosure claims priority to Chinese patent application No. 202210553520.X, filed on May 20, 2022, and this disclosure cites the full text of the above-mentioned Chinese patent application. Technical Field

[0002] The present disclosure relates to a method for specific reduction of antibodies and a method for preparing an antibody-drug conjugate (ADC), as well as an antibody-drug conjugate (ADC) with improved homogeneity. Background Art

[0003] Antibody-drug conjugates (ADCs) are created by linking an antibody or antigen-binding fragment to a biologically active toxin via a stable chemical linker. They combine the potent lethality of small molecule drugs with the specificity of antibodies, achieving the specificity of antibodies for binding to surface antigens on both normal and tumor cells and the high efficacy of cytotoxicity, while avoiding the drawbacks of antibodies, such as low efficacy and the excessive side effects of cytotoxicity. ADCs can precisely bind to tumor cells while minimizing the effects on normal cells, thereby reducing adverse reactions during treatment (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329–332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

[0004] As of 2021, ten ADC drugs have been launched on the market, namely Germtuzumab Ozogamicin (Mylotarg), Brentuximab Vedotin (Adcetris), Trastuzumab emtansine (Kadc yla, T-DM1), Inotuzumab Ozogamicin (Besponsa), Polatuzumab Vedotinpilig (Poliv y), Enfortumab Vedotin-ejfv (Padcev), fam-Trastuzumab Deruxtecan-nxki (Enhertu), Sacituzumab govitecan (Trodelvy), belantamab mafodotin (Blenrep), and Ioncastuxi mab tesirine-lpyl (ZYNLONTA).

[0005] ADCs often link antibodies and toxins through two chemical strategies. One is conjugation based on lysine residues on the antibody, as exemplified by the marketed drugs Mylotarg, Kadcyla, and Besponsa. These drugs all utilize a linker's succinimide group to bind to lysine side-chain amino groups, attaching the toxin to the monoclonal antibody. Since an antibody molecule contains 80–90 lysine residues, conjugation can occur at nearly 40 different lysine residues, resulting in multiple and non-fixed conjugation sites. The other is conjugation based on cysteine ​​residues on the antibody. Compared to the stable intrachain disulfide bonds on the antibody, interchain disulfide bonds are easily partially reduced to free sulfhydryl groups, which can then bind to linker groups (such as maleimide groups). Both IgG1 and IgG4 antibodies contain four pairs of interchain disulfide bonds. With appropriate reducing agents and conjugation, ADCs with drug-to-antibody ratios (DARs) of 0–8 can be obtained, as demonstrated by seven drugs, including Adcetris, Polivy, and Padcev. Therefore, compared with lysine coupling, cysteine ​​coupling contains fewer coupling sites and is the preferred connection strategy for random coupling due to its simple coupling process.

[0006] However, current cysteine ​​technology still has shortcomings. For example, the ADC components of ADC samples with a mean DAR of 4 are normally distributed, making the ADC drug non-uniform. In addition, ADC samples with a mean DAR of 4 also contain a certain proportion of ADC components connected to 6 and 8 toxins, making the ADC drug more easily cleared from the body (JY, et al., 2021, Bioconjugate Chem, 18; 32(8): 1525-1534).

[0007] With the development of protein engineering technology, in order to obtain uniform and highly stable ADC drugs, various technologies have been used to try to introduce cysteine ​​into the antibody structure, thereby introducing a sulfhydryl group and achieving site-specific conjugation between the antibody and the drug molecule. For example, a literature indicates that inserting a cysteine ​​after amino acid 239 of the antibody heavy chain, introducing a free sulfhydryl group and then conjugating it with a toxin can produce a stable and uniform ADC (Nazzareno Dimasi, Mol. Pharmaceutics 2017, 14, 5, 1501–1516). However, the introduction of additional cysteine ​​by this method may lead to disulfide bond mismatch, thereby affecting the stability of the ADC drug.

[0008] In addition, some scholars have chosen to perform cysteine ​​mutations at specific sites on antibodies. The mutated antibodies contain free sulfhydryl groups and can also meet the requirements of ADC uniformity. However, this method is a modification of the antibody structure. In order to find mutation sites that do not affect the antibody structure and function, a large amount of site screening is required, which is costly (Shiraishi Y, et al., Bioconjug Chem, 2015, 26(6):1032-1040; Shinmi D, et al Bioconjugate Chem, 2016, 27(5):1324-1331).

[0009] WO2020164561A1 relates to a method for preparing ADC, which uses TCEP as a reducing agent and utilizes zinc ions to form chemical coordination bonds with the sulfhydryl groups in the hinge region of the antibody, thereby increasing the probability of conjugation of the reduced sulfhydryl groups in the Fab region to toxins. This method significantly increases the proportion of DAR 4 components. However, this method, because excessive use of reducing agents in the reduction stage leads to a large number of openings of interchain disulfide bonds, must perform toxin quenching and reoxidation steps after the coupling is completed, which is complicated to operate and easily introduces process impurities. Based on this, the present disclosure adopts process optimization means to improve the uniformity of ADCs based on cysteine ​​coupling.

[0010] The present disclosure provides a method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, and produces an ADC with improved homogeneity. The preparation method is simple, and there is no need to quench the reaction to terminate the coupling reaction after the coupling reaction, and there is no need for a reoxidation step to reoxidize the unreacted sulfhydryl groups of the antibody. At the same time, the coupling reaction is not limited by temperature and can be carried out at -10°C to 40°C. The coupling substrate has no effect on the selectivity of the coupling reaction. It can limit and increase the proportion of DAR 4 product (D4) in the ADC, with optimized safety and efficacy.

[0011] Summary of the Invention

[0012] The present disclosure provides a method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0013] (a) reacting a reducing agent and an antibody in the presence of a transition metal ion in a buffer system to selectively reduce interchain disulfide bonds within the antibody to sulfhydryl groups;

[0014] (b) reacting the antibody having a thiol group obtained in step (a) with a drug linker intermediate or a pharmaceutically acceptable salt thereof.

[0015] In some embodiments, the method further comprises or does not comprise step (c): step (b) is performed without a quenching step and / or a reoxidation step to obtain an antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments, the present disclosure provides a method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0017] (a) reacting a reducing agent and an antibody in a buffer system in the presence of an effective amount of a transition metal ion to selectively reduce interchain disulfide bonds within the antibody to sulfhydryl groups;

[0018] (b) reacting the antibody having a thiol group obtained in step (a) with a drug linker intermediate or a pharmaceutically acceptable salt thereof;

[0019] (c) Obtaining an antibody-drug conjugate or a pharmaceutically acceptable salt thereof by performing step (b) without the quenching step and / or the reoxidation step.

[0020] In some embodiments, the present disclosure provides a method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0021] (a) reacting a reducing agent with an antibody in the presence of a transition metal ion to selectively reduce interchain disulfide bonds within the antibody to sulfhydryl groups;

[0022] (b) reacting the antibody having a thiol group obtained in step (a) with a drug linker intermediate or a pharmaceutically acceptable salt thereof;

[0023] (c) Obtaining an antibody-drug conjugate or a pharmaceutically acceptable salt thereof by performing step (b) without the quenching step and / or the reoxidation step.

[0024] The quenching step refers to the step of inactivating the reactivity of the unreacted drug-linker intermediate to terminate the conjugation reaction. For example, the conjugation reaction is terminated by inactivating the reactivity of the unreacted drug-linker intermediate with a sulfhydryl-containing reagent (e.g., N-acetamidocysteine, cysteine).

[0025] The reoxidation step refers to adding an effective amount of an oxidizing agent (eg, DHAA) to reoxidize the unreacted sulfhydryl groups on the antibody after reduction.

[0026] In some embodiments, the transition metal ion in step (a) is selected from Zn 2+ 、Cd 2+ 、Hg 2+ or a combination thereof; some embodiments transition metal ions are selected from Zn 2+ .

[0027] In some embodiments, appropriate transition metal salts are added in step (a) as long as they are soluble in the reaction solution so that free transition metal ions can be released into the reaction solution.

[0028] In some embodiments, suitable zinc salts include, but are not limited to, ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, zinc formate, and zinc tetrafluoroborate.

[0029] In some embodiments, a suitable soluble and free Cd-releasing agent is added in step (a). 2+ or Hg 2+ Ionic transition metal salts, including but not limited to: CdCl2, Cd(NO3)2, CdSO4, Cd(CH3COO)2, CdI2, CdBr2, cadmium formate and cadmium tetrafluoroborate; HgCl2, Hg(NO3)2, HgSO4, Hg(CH3COO)2, HgBr2, mercury (II) formate, and mercury (II) tetrafluoroborate, etc.

[0030] In some embodiments, the reducing agent in step (a) is a reducing agent containing a diphenylphosphine group, or a salt thereof; in some embodiments, the reducing agent is selected from diphenylphosphinoacetic acid (DPA),

[0031] 2-[2-(Diphenylphosphino)ethyl]pyridine,

[0032] 3-(Diphenylphosphino)benzenesulfonic acid,

[0033] 4-(Diphenylphosphino)benzoic acid,

[0034] 2-(Diphenylphosphino)ethylamine,

[0035] 3-(diphenylphosphino)propylamine,

[0036] 3-(Diphenylphosphino)propionic acid,

[0037] 2-(diisopropylphosphino)ethylamine (2-(diisopropylphosphino)ethylamine),

[0038] 2-(diphenylphosphino)benzoic acid,

[0039] (2-hydroxyphenyl)diphenylphosphine, or a salt thereof; in some embodiments, the reducing agent is selected from diphenylphosphinoacetic acid, or a salt thereof.

[0040] In some embodiments, the final concentration of the antibody is selected from about 0.01 mM to about 0.50 mM, for example, about 0.01 mM, about 0.02 mM, about 0.03 mM, about 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.09 mM, about 0.10 mM, about 0.11 mM, about 0.12 mM, about 0.13 mM, about 0.14 mM, about 0.15 mM, about 0.20 mM, about 0.25 mM, about 0.30 mM, about 0.35 mM, about 0.40 mM, about 0.45 mM, about 0.50 mM, or any value or range between any two values; in some embodiments, the final concentration of the antibody is selected from about 0.10 mM to about 0.20 mM. In some embodiments, the final concentration of the antibody is selected from about 0.12 mM to about 0.14 mM.

[0041] In some embodiments, the final concentration of the transition metal ion is selected from about 0.01 mM to about 0.50 mM, such as about 0.01 mM, about 0.02 mM, about 0.03 mM, about 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.09 mM, about 0.10 mM, about 0.15 mM, about 0.20 mM, about 0.21 mM, about 0.22 mM, about 0.23 mM, about 0.24 mM. , about 0.25 mM, about 0.26 mM, about 0.27 mM, about 0.28 mM, about 0.29 mM, about 0.30 mM, about 0.31 mM, about 0.32 mM, about 0.33 mM, about 0.34 mM, about 0.35 mM, about 0.40 mM, about 0.45 mM, about 0.50 mM, or any value or range between any two values; in some embodiments, the final concentration of the transition metal ion is selected from about 0.27 mM to about 0.28 mM.

[0042] In some embodiments, the final concentration of the reducing agent is selected from about 0.20 mM to about 1.00 mM, for example, about 0.20 mM, about 0.25 mM, about 0.30 mM, about 0.35 mM, about 0.40 mM, about 0.45 mM, about 0.50 mM, about 0.55 mM, about 0.60 mM, about 0.65 mM, about 0.70 mM, about 0.75 mM, about 0.80 mM, about 0.85 mM, about 0.90 mM, about 0.95 mM, about 1.00 mM, or any value or range between any two values; in some embodiments, the final concentration of the reducing agent is selected from about 0.35 mM to about 0.50 mM, about 0.38 mM to about 0.45 mM.

[0043] In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the reducing agent is about 3:1 to about 1:10, for example, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, or any value or range therebetween. In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the reducing agent is about 1:2.5 to about 1:3.5.

[0044] In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the transition metal ion is 5:1 to about 1:5, for example, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, or any value or range therebetween. In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the transition metal ion is about 1:2.

[0045] In some embodiments, the buffer system used in step (a) is selected from: Hepes buffer, histidine buffer, PBS buffer, MES buffer, citrate buffer, tris buffer, gluconate buffer, adipic acid buffer, lactate buffer, acetate buffer or succinate buffer; in some embodiments, the buffer system is selected from histidine buffer.

[0046] In some embodiments, the buffer system used in step (a) depends on a transition metal ion.

[0047] In some embodiments, the buffer system may or may not contain a metal chelator. In some embodiments, the buffer system does not contain a metal chelator.

[0048] In some embodiments, the pH of the buffer system used in step (a) is selected from about 4 to about 10, for example, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or any value or range between any two values; in some embodiments, selected from about 5.5 to about 8, about 6 to about 7.5; in some embodiments, selected from 6.0-7.0; in some embodiments, selected from 6.5.

[0049] In some embodiments, step (a) is carried out at about -10°C to about 40°C, for example, about -5°C, about -3°C, about -2°C, about -1°C, about 0°C, about 2°C, about 3°C, about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 37°C, or any value or range therebetween; in some embodiments, at about -5°C to about 37°C; in some embodiments, at about 0°C to about 37°C; in some embodiments, at about 0°C to about 4°C; in some embodiments, at about 0°C to about 37°C; in some embodiments, at about 10°C to about 25°C; in some embodiments, at about -5°C to about 5°C; in some embodiments, at 0°C to 30°C; in some embodiments, at 0°C to 25°C.

[0050] In some embodiments, the reaction time of step (a) is selected from 1 h to 24 h, such as 2 h, 4 h, 8 h, 10 h, 12 h, 16 h; in some embodiments, the reaction time of step (a) is 16 h; in some embodiments, the reaction time of step (a) is 2 h.

[0051] In some embodiments, the reaction conditions of step (a) are selected from standing at 0-4°C overnight, standing at 0-4°C for 16 hours, or standing at 25°C for 2 hours.

[0052] In some embodiments, the reaction conditions of step (a) depend on the specific antibody to be conjugated. The determination of the incubation period and temperature based on the specific antibody is within the ability of those of ordinary skill in the art. For example, the antibody to be conjugated is typically incubated overnight at 4°C with a reducing agent in the presence of a transition metal ion.

[0053] In some embodiments, the method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof described in the present disclosure further comprises the step of adding a metal chelator between step (a) and step (b).

[0054] In some embodiments, the metal chelator is removed by subsequent dialysis, ultrafiltration, or gel filtration.

[0055] In some embodiments, the metal chelator is used to chelate transition metal ions; in some embodiments, the metal chelator is selected from ethylenediaminetetraacetic acid (hereinafter also referred to as "EDTA"); in some embodiments, the metal chelator is selected from ethylenediaminetetraacetic acid, disodium salt of ethylenediaminetetraacetic acid, dicalcium salt of ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid or a mixture thereof.

[0056] In some embodiments, the final concentration equivalent ratio (mM / mM) of the transition metal ion to the metal chelator is 1:1 to about 1:5, for example, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, or any value or range therebetween. In some embodiments, the final concentration equivalent ratio (mM / mM) of the transition metal ion to the metal chelator is about 1:2.

[0057] The drug linker intermediate or its pharmaceutically acceptable salt disclosed herein is not particularly limited, as long as it is a compound capable of reacting with the interchain sulfhydryl group of an antibody, such as a drug linker intermediate having an N-substituted maleimide group. In some embodiments, the drug linker intermediate or its pharmaceutically acceptable salt contains a reactive group capable of reacting with a sulfhydryl group; in some embodiments, the drug linker intermediate or its pharmaceutically acceptable salt contains a maleimide group (such as an N-substituted maleimide group), a bromine group, an iodine group, a fluorine group, an olefin group, an alkenyl group, or a sulfonyl group.

[0058] In some specific embodiments, the drug-linker intermediate is selected from mc-vc-pab-MMAE;

[0059] In some specific embodiments, the drug linker intermediate is selected from the compound represented by formula (III-A') or (III-B') or a pharmaceutically acceptable salt thereof:

[0060] in,

[0061] Ring A is The ring A is optionally substituted with one or more substituents Q1;

[0062] Ring B is The ring B is optionally substituted with one or more substituents Q1;

[0063] X1 is -(CR 5a R 5b )m- or a 6- to 10-membered aryl or 5- to 10-membered heteroaryl group optionally substituted by one or more substituents Q1, wherein the heteroaryl group contains at least one nitrogen atom;

[0064] R 5a and R 5bare independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k and C1-C6 alkoxy, or R 5a and R 5b Together they form oxo or thio;

[0065] Ring C is selected from The ring C is optionally substituted with one or more substituents Q1;

[0066] Ring D is The ring D is optionally substituted with one or more substituents Q1;

[0067] X2 is selected from -(CR 6a R 6b )n-、-O-、-S-、-NR 6c -、-CH2S-、-CH2O-、-NHCR 6d R 6e - and 6 to 10 membered aryl or 5 to 10 membered heteroaryl, optionally substituted by one or more substituents Q1;

[0068] R 6a and R 6b are independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k and C1-C6 alkoxy;

[0069] R 6c 、R 6d and R 6e Each independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl and C1-C6 alkoxy;

[0070] R2 is each independently selected from -CH2OH, -CH2SH, -CH2Cl, -SCH2Cl, -SCH2F, -SCH2CF3, -OH, -OCH2CN, -OCH2Cl, -OCH2F, -OCH3, -OCH2CH3, -SCH2CN,

[0071] R 2a is hydrogen or C1-C6 alkyl;

[0072] R 2b is a C1-C6 alkyl group or a C1-C6 alkoxy group;

[0073] R 2c Selected from hydrogen, C1-C6 alkyl, -CH2OH and C1-C6 alkoxy;

[0074] R 2d and R 2e Each is independently hydrogen or C1-C6 alkyl;

[0075] R3 are each independently hydrogen or halogen;

[0076] m and n are each independently an integer from 1 to 6;

[0077] The substituent groups Q1 are each independently selected from halogen, hydroxy, mercapto, deuterium, oxo, thioxo, cyano, amino, carboxyl, C1-C6 alkyl and C1-C6 alkoxy;

[0078] R i and R j Each independently selected from a hydrogen atom, a hydroxyl group, a C1-C6 alkyl group and a C1-C6 alkoxy group;

[0079] R k independently selected from hydrogen atoms, C1-C6 alkyl groups, C1-C6 haloalkyl groups, C1-C6 alkoxy groups, hydroxyl groups and -NR i R j ;

[0080] R 1a Each independently selected from hydrogen, C1-C6 alkyl and C1-C6 alkoxy;

[0081] R 1b Each independently selected from hydrogen, PG-, H-L1-, PG-L1-,

[0082] p is each independently 1, 2, 3, 4, 5 or 6;

[0083] L1 is an amino acid unit, preferably -glycine-glutamic acid- or

[0084] X is a halogen;

[0085] PG is an amino protecting group; and

[0086] The condition is that when R 5a When R is hydrogen or alkyl, 5b Not hydrogen or alkyl.

[0087] In some specific embodiments, the drug-linker intermediate is selected from:

[0088] or a pharmaceutically acceptable salt thereof.

[0089] In some specific embodiments, the drug-linker intermediate is selected from: or a pharmaceutically acceptable salt thereof; in some more preferred embodiments, X is halogen, in some more preferred embodiments, X is chlorine or bromine, in some more preferred embodiments, X is bromine.

[0090] In some embodiments, the drug in the drug-linker intermediate is bound to the antibody via a linker. The linker used in the present disclosure is not particularly limited, as long as the linker contains at least two reactive groups, one of which can covalently bind to the drug molecule and the other can covalently couple to the antibody. In some embodiments, the linker includes a cleavable linker and a non-cleavable linker. In some embodiments, the cleavable linker is selected from a peptide linker that is cleaved by an intracellular protease (such as a lysozyme, in some embodiments, a somatic protease or an endosomal protease).

[0091] In some embodiments, the linker comprises an amino acid unit L1, which preferably comprises 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, homolysine, N-methyl-valine, (q is an integer of 1-6) peptide residues composed of amino acids, exemplary amino acid units include, but are not limited to, valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys), phenylalanine-homolysine (Phe-Homolys), N-methyl-valine-citrulline (Me-Val-Cit), alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala), glycine-lysine (Gly-Lys), glycine-valine-citrulline (Glv-Val-Cit) and glycine-glycine-glycine (Gly-Gly-Gly) and

[0092] In certain embodiments, the linker comprises a stretching unit, which is a chemical structural fragment covalently linked to the antibody through a carbon atom at one end and to an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptide chemical moiety at the other end. Exemplary stretching units include, but are not limited to:

[0093] In certain embodiments, the stretching unit is selected from:

[0094] wherein p is each independently 1, 2, 3, 4, 5 or 6.

[0095] In certain embodiments, the linker is selected from:

[0096] In certain embodiments, the linker is selected from:

[0097] In some embodiments, the linker is selected from:

[0098] Depending on the desired drug and the selected linker, one skilled in the art can select an appropriate method to couple them together. For example, some conventional coupling methods, such as amine coupling, can be used to form the desired drug-linker intermediate, which still contains a reactive group for conjugation to the antibody via covalent attachment, such as a drug-maleimide intermediate (i.e., a maleimide-linked drug).

[0099] In some embodiments, the amount of the drug linker intermediate or its pharmaceutically acceptable salt in step (b) is in excess. In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the drug linker intermediate or its pharmaceutically acceptable salt (based on the drug linker intermediate) is about 1:1 to about 1:20, for example, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, or any value or range between any two values. In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the drug linker intermediate or its pharmaceutically acceptable salt (based on the drug linker intermediate) is about 1:6.

[0100] In some embodiments, the drug-linker intermediate is dissolved in a solution and added to the antibody system having a thiol group obtained in step (a) so that they can react with each other.

[0101] Examples of solvents that can be used to dissolve the drug-linker intermediates in the present disclosure include organic solvents such as 50% acetone in water, 80% ethanol in water, 80% methanol in water, 80% isopropanol in water, 80% dimethyl sulfoxide in water, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA) and N-methyl-2-pyrrolidone (NMP); in some embodiments, the solvent for dissolving the drug-linker intermediate is selected from 100% DMSO.

[0102] In some embodiments, step (b) is carried out at about -10°C to about 40°C, for example, about -5°C, about -3°C, about -2°C, about -1°C, about 0°C, about 2°C, about 3°C, about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 37°C, or any value or range therebetween; in some embodiments, at about -5°C to about 37°C; in some embodiments, at about 0°C to about 4°C; in some embodiments, at about 0°C to about 37°C; in some embodiments, at about 10°C to about 25°C; in some embodiments, at about -5°C to about 5°C.

[0103] In some embodiments, the reaction time of step (b) is selected from 1 h to 24 h, such as 2 h, 4 h, 8 h, 12 h, 16 h; in some embodiments, the reaction time of step (b) is 1 h to 2 h.

[0104] In some embodiments, the reaction conditions of step (b) are selected from 0°C or room temperature for 1-2 hours.

[0105] In some embodiments, step (c) further comprises a step of purifying the reaction solution, wherein the purification is selected from the step of using a desalting column or an ultrafiltration centrifuge tube to remove impurities such as free toxins and organic solvents.

[0106] Those skilled in the art can select an appropriate purification method to recover the obtained antibody-drug conjugate or a pharmaceutically acceptable salt thereof. Many ADC purification methods are well known in the art. For example, the obtained antibody-drug conjugate or a pharmaceutically acceptable salt thereof can be purified by using a desalting column, size exclusion chromatography, ultrafiltration, dialysis, UF-DF, etc.

[0107] In some embodiments, based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains D4 in an amount greater than about 50 wt%, such as greater than about 50 wt%, greater than about 55 wt%, greater than about 60 wt%, greater than about 61 wt%, greater than about 62 wt%, greater than about 63 wt%, greater than about 64 wt%, greater than about 65 wt%, greater than about 66 wt%, greater than about 67 wt%, greater than about 68 wt%, greater than about 69 wt%, greater than about 70 wt%, greater than about 71 wt%, greater than about 72 wt%, greater than about 73 wt%, greater than about 74 wt%, greater than about 75 wt%, greater than about 76 wt%, greater than about 77 wt%, greater than about 78 wt%, greater than about 79 wt%, greater than about 80 wt%, greater than about 81 wt%, greater than about 82 wt%, greater than about 83 wt%, greater than about 84 wt%, greater than about 85 wt%, greater than about 86 wt%, greater than about 87 wt%, greater than about 88 wt%, greater than about 89 wt%, greater than about 90 wt%, greater than about 91 wt%, greater than about 92 wt%, greater than about 93 wt%, greater than about 94 wt%, greater than about 95 wt%, greater than about 96 wt%, greater than about 97 wt%, greater than about 98 wt%, greater than about 99 wt%, greater than about 100 wt%, greater than about 101 wt%, greater than about 102 wt%, greater than about 103 wt%, greater than about 104 wt%, greater than about 105 wt%, greater than about 106 %, higher than about 69 wt %, higher than about 70 wt %, higher than about 71 wt %, higher than about 72 wt %, higher than about 73 wt %, higher than about 74 wt %, higher than about 75 wt %; in some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains D4 in an amount selected from about 55 wt % to about 75 wt %, about 60 wt % to about 70 wt %, about 55 wt % to about 65 wt %.

[0108] In some embodiments, based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains a higher content of D4 than that obtained by using TCEP.

[0109] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure comprises D4a, wherein D4a is selected from an antibody-drug conjugate or its pharmaceutically acceptable salt having and only having 4 drug linkers, and the 4 drug linkers are bound to the heavy-light chain intersulfhydryl group. Based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure comprises D4a in an amount greater than about 50 wt%, for example, greater than about 50 wt%, greater than about 55 wt%, greater than about 60 wt%, greater than about 61 wt%, greater than about 62 wt%, greater than about 63 wt%, greater than about 64 wt%, greater than about 65 wt%, greater than about 66 wt%, greater than about 67 wt%, greater than about 68 wt%, greater than about 69 wt%, greater than about 70 wt%, greater than about 80 wt%, greater than about 81 wt%, greater than about 82 wt%, greater than about 83 wt%, greater than about 84 wt%, greater than about 85 wt%, greater than about 86 wt%, greater than about 87 wt%, greater than about 88 wt%, greater than about 89 wt%, greater than about 90 wt%, greater than about 91 wt%, greater than about 92 wt%, greater than about 93 wt%, greater than about 94 wt%, greater than about 95 wt%, greater than about 96 wt%, greater than about 97 wt%, greater than about 98 wt%, greater than about 99 wt%, greater than about 100 wt%, greater than about 101 wt%, greater than about 102 wt%, greater than about 103 wt%, greater than about 104 wt%, greater than about 105 wt%, greater than about 10 Higher than about 71 wt%, higher than about 72 wt%, higher than about 73 wt%, higher than about 74 wt%, higher than about 75 wt%; in some embodiments, based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains D4a in an amount selected from about 55 wt% to about 75 wt%, about 60 wt% to about 70 wt%, about 55 wt% to about 65 wt%.

[0110] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure comprises D4b, wherein D4b is selected from an antibody-drug conjugate or its pharmaceutically acceptable salt having and only having 4 drug linkers, and wherein the 4 drug linkers are bound to the heavy-heavy interchain sulfhydryl group. Based on the total weight of D0, D2, D4, D6 and D8, the content of D4b contained in the antibody-drug conjugate or its pharmaceutically acceptable salt is less than about 40 wt%, such as less than about 40 wt%, less than about 30 wt%, less than about 20 wt%, less than about 15 wt%, less than about 10 wt%, or less than about 5 wt%. In some embodiments, based on the total weight of D0, D2, D4, D6 and D8, the content of D4b contained in the antibody-drug conjugate or its pharmaceutically acceptable salt is selected from about 5 wt% to about 30 wt%, about 5 wt% to about 20 wt%, or about 5 wt% to about 10 wt%.

[0111] In some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure comprises less than about 20 wt% of DO + D8, such as less than about 19 wt%, less than about 18 wt%, less than about 17 wt%, less than about 16 wt%, less than about 15 wt%, less than about 14 wt%, less than about 13 wt%, less than about 12 wt%, less than about 11 wt%, less than about 10 wt%, less than about 9 wt%, less than about 8 wt%, less than about 7 wt%, less than about 6 wt%, less than about 5 wt%, less than about 4 wt%, less than about 3 wt%. In some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure comprises less than about 3 wt% to about 20 wt%, about 5 wt% to about 15 wt%, about 3 wt% to about 10 wt%.

[0112] In some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure comprises less than about 20 wt% of D6, such as less than about 19 wt%, less than about 18 wt%, less than about 17 wt%, less than about 16 wt%, less than about 15 wt%, less than about 14 wt%, less than about 13 wt%, less than about 12 wt%, less than about 11 wt%, less than about 10 wt%, less than about 9 wt%, less than about 8 wt%, less than about 7 wt%, less than about 6 wt%, or less than about 5 wt%. In some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure comprises D6 in an amount selected from about 5 wt% to about 20 wt%, about 5 wt% to about 15 wt%, or about 5 wt% to about 10 wt%.

[0113] In some embodiments, the total weight of D0, D2, D4, D6 and D8 refers to the total weight of D0, D2, D4, D6 and D8 obtained without purification after the coupling reaction.

[0114] In some embodiments, the total weight of D0, D2, D4, D6, and D8 refers to the total weight of D0, D2, D4, D6, and D8 obtained after purification to remove only small molecule process impurities (e.g., free toxins and organic solvents, etc.), wherein the purification step does not affect the different drug-loaded components of the prepared ADC.

[0115] In some embodiments, based on the sum of peak areas, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure comprises a peak area percentage of D4 greater than about 50%, such as greater than about 50%, greater than about 55%, greater than about 60%, greater than about 61%, greater than about 62%, greater than about 63%, greater than about 64%, greater than about 65%, greater than about 66%, greater than about 67%, greater than about 68%, greater than about 69%, greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, or greater than about 75%. In some embodiments, based on the sum of peak areas, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure comprises a peak area percentage of D4 selected from about 55% to about 75%, about 60% to about 70%, or about 55% to about 65%.

[0116] In some embodiments, based on the sum of peak areas, the peak area percentage of D4 contained in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the disclosed method is greater than the content of D4 obtained using TCEP.

[0117] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure comprises D4a, wherein D4a is selected from an antibody-drug conjugate or a pharmaceutically acceptable salt thereof having only four drug linkers, and the four drug linkers are bound to the sulfhydryl groups between the heavy and light chains. Based on the sum of peak areas, the peak area percentage of D4a contained in the antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is greater than about 50%, for example, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 61%, greater than about 62%, greater than about 63%, greater than about 64%, greater than about 65%, greater than about 66%, greater than about 67%, greater than about 68%, greater than about 69%, greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, greater than about 75%, or any value or range between any two values; in some embodiments, based on the sum of peak areas, the peak area percentage of D4a contained in the antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is selected from about 55% to about 75%, about 60% to about 70%, or about 55% to about 65%.

[0118] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure comprises D4b, wherein D4b is selected from an antibody-drug conjugate or its pharmaceutically acceptable salt having only four drug linkers, and wherein the four drug linkers are bound to a heavy-heavy interchain sulfhydryl group. Based on the total peak area, the peak area percentage of D4b contained in the antibody-drug conjugate or its pharmaceutically acceptable salt is less than about 40%, for example, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%; in some embodiments, based on the total peak area, the peak area percentage of D4b contained in the antibody-drug conjugate or its pharmaceutically acceptable salt is selected from about 5% to about 30%, about 5% to about 20%, or about 5% to about 10%.

[0119] In some embodiments, based on the sum of peak areas, the peak area percentage of D0+D8 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure is less than about 20%, such as less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%. In some embodiments, based on the sum of peak areas, the peak area percentage of D0+D8 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure is selected from about 3% to about 20%, about 5% to about 15%, about 3% to about 10%.

[0120] In some embodiments, based on the total peak area, the peak area percentage of D6 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure is less than about 20%, such as less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%. In some embodiments, based on the total peak area, the peak area percentage of D6 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure is selected from about 5% to about 20%, about 5% to about 15%, about 5% to about 10%.

[0121] Based on the total peak area, the peak area percentage refers to: the antibody-drug conjugate (ADC) prepared by the present disclosure or a pharmaceutically acceptable salt thereof is tested by the HIC-HPLC method, and the sum of the areas of each chromatographic peak after deducting the blank solution is calculated by comparing the chromatogram with the blank solution. The chromatogram is integrated to obtain the total peak area, and the peak area of ​​each component (e.g., D0, D2, D4, D6 or D8) is calculated as a percentage of the total peak area using the area normalization method.

[0122] In some embodiments, the HIC-HPLC method is as described in Example 1.

[0123] In some embodiments, the HIC-HPLC method is as described in Example 2.

[0124] Based on the sum of the peak areas of D0, D2, D4, D6 and D8, the peak area percentage of D4 contained in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is greater than about 50%, for example, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 61%, greater than about 62%, greater than about 63%, greater than about 64%, greater than about 65%, greater than about 66%, greater than about 67%, greater than about 68%, greater than about 69%, greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, or greater than about 75%. In some embodiments, based on the sum of the peak areas of D0, D2, D4, D6 and D8, the peak area percentage of D4 contained in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is selected from about 55% to about 75%, about 60% to about 70%, or about 55% to about 65%.

[0125] In some embodiments, based on the sum of the peak areas of D0, D2, D4, D6, and D8, the peak area percentage of D4 contained in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is greater than the content of D4 obtained using TCEP.

[0126] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure comprises D4a, wherein D4a is selected from an antibody-drug conjugate or a pharmaceutically acceptable salt thereof having only four drug linkers, and the four drug linkers are bound to the sulfhydryl groups between the heavy and light chains. Based on the sum of the peak areas of D0, D2, D4, D6 and D8, the peak area percentage of D4a contained in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is greater than about 50%, for example, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 61%, greater than about 62%, greater than about 63%, greater than about 64%, greater than about 65%, greater than about 66%, greater than about 67%, greater than about 68%, greater than about 69%, greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, or greater than about 75%. In some embodiments, based on the sum of the peak areas of D0, D2, D4, D6 and D8, the peak area percentage of D4a contained in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is selected from about 55% to about 75%, about 60% to about 70%, or about 55% to about 65%.

[0127] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure comprises D4b, wherein D4b is selected from an antibody-drug conjugate or its pharmaceutically acceptable salt having only four drug linkers, and wherein the four drug linkers are bound to a heavy-heavy interchain sulfhydryl group. Based on the sum of the peak areas of D0, D2, D4, D6, and D8, the peak area percentage of D4b contained in the antibody-drug conjugate or its pharmaceutically acceptable salt is less than about 40%, for example, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. In some embodiments, based on the total weight of D0, D2, D4, D6, and D8, the peak area percentage of D4b contained in the antibody-drug conjugate or its pharmaceutically acceptable salt is selected from about 5% to about 30%, about 5% to about 20%, or about 5% to about 10%.

[0128] In some embodiments, based on the sum of the peak areas of D0, D2, D4, D6, and D8, the peak area percentage of D0+D8 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure is less than about 20%, such as less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%. In some embodiments, based on the sum of the peak areas of D0, D2, D4, D6, and D8, the peak area percentage of D0+D8 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure is selected from about 3% to about 20%, about 5% to about 15%, about 3% to about 10%.

[0129] In some embodiments, based on the sum of the peak areas of D0, D2, D4, D6, and D8, the peak area percentage of D6 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure is less than about 20%, such as less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, or less than about 5%. In some embodiments, based on the sum of the peak areas of D0, D2, D4, D6, and D8, the peak area percentage of D6 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure is selected from about 5% to about 20%, about 5% to about 15%, or about 5% to about 10%.

[0130] The peak area percentage based on the sum of the peak areas of D0, D2, D4, D6, and D8 refers to: the antibody-drug conjugate (ADC) prepared by the present disclosure or a pharmaceutically acceptable salt thereof is detected by HIC-HPLC, and the sum of the areas of D0, D2, D4, D6, and D8 after deducting the blank solution is calculated by comparing the chromatogram with the blank solution. The chromatogram is integrated to obtain the sum of the peak areas based on D0, D2, D4, D6, and D8. The peak area of ​​each component (e.g., D0, D2, D4, D6, or D8) is calculated as a percentage of the sum of the peak areas based on D0, D2, D4, D6, and D8 using the area normalization method.

[0131] In some embodiments, the HIC-HPLC method is as described in Example 1.

[0132] In some embodiments, the HIC-HPLC method is as described in Example 2.

[0133] In some embodiments, the sum of the peak areas of D0, D2, D4, D6, and D8 for the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method disclosed herein refers to the sum of the peak areas of unpurified D0, D2, D4, D6, and D8 obtained after the conjugation reaction.

[0134] In some embodiments, the sum of the peak areas of D0, D2, D4, D6, and D8 for the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods disclosed herein refers to the sum of the peak areas of D0, D2, D4, D6, and D8 obtained after purification to remove only small molecule process impurities (e.g., free toxins and organic solvents, etc.), wherein the purification step does not affect the different drug-loaded components of the prepared ADC.

[0135] In some embodiments, the average drug loading of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is selected from about 3.0 to about 5.0, about 3.5 to about 4.5, for example, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or any value or range between any two values; in some embodiments, the average drug loading is greater than 3.0 and less than 5.0; in some embodiments, the average drug loading is selected from about 3.8 to about 4.4; in some embodiments, the average drug loading is selected from about 3.9 to about 4.1.

[0136] In some embodiments, the antibody-drug conjugate (ADC) of the present disclosure is selected from: Ab-(LD) k (I)

[0137] Wherein, Ab is an antibody or its antigen-binding fragment, and LD is a drug-linker intermediate;

[0138] L is a linker that covalently links Ab to D, and k is selected from 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any number between any two numbers),

[0139] D is a drug, as shown in the following formula (II-A) or (II-B):

[0140] in,

[0141] represents a single bond or a double bond;

[0142] R 1ais selected from hydrogen, alkyl and alkoxy, wherein the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy and hydroxyalkyl;

[0143] Ring A is an aryl or heteroaryl group optionally substituted by one or more substituents Q1;

[0144] Ring B is an aryl or heteroaryl group optionally substituted by one or more substituents Q1;

[0145] X1 is -(CR 5a R 5b ) m - and aryl or heteroaryl optionally substituted by one or more substituents Q1;

[0146] R 5a and R 5b are independently selected from hydrogen, halogen, hydroxy, mercapto, deuterium, nitro, cyano and the following groups optionally substituted by one or more substituents Q1: alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)OR k 、-S(O)(O)R k 、-S(O)(O)OR k 、-C(S)R k , alkoxy, alkylthio, alkenyl and alkynyl, or R 5a and R 5b Together they form oxo or thio;

[0147] Ring C and Ring D are each independently selected from aryl and heteroaryl groups optionally substituted with one or more substituents Q1, and at least one of Ring C and Ring D is selected from fused ring aryl or fused heteroaryl groups optionally substituted with one or more substituents Q1;

[0148] X2 is selected from -(CR 6a R 6b ) n -, aryl or heteroaryl optionally substituted by one or more substituents Q1, -O-, -S-, -S(O)-, -S(O)(O)-, -NR 6c -、-CH2S-、-CH2O-、-NHCR 6d R 6e -、-CR 6f =CR 6g - and -C≡C-, or X2 does not exist;

[0149] R 6aand R 6b are independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano or the following groups optionally substituted by one or more substituents Q1: alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)OR k 、-S(O)(O)R k 、-S(O)(O)OR k 、-C(S)R k , alkoxy, alkylthio, alkenyl and alkynyl, or R 6a 、R 6b Together with the carbon atom to which it is attached, it forms a 3- to 10-membered cycloalkyl group, or R 6a and R 6b Together they form oxo or thio;

[0150] R 6c 、R 6d 、R 6e 、R 6f and R 6g Each independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl and C1-C6 alkoxy;

[0151] R1 is each independently selected from hydrogen, alkyl and alkoxy, wherein the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy and hydroxyalkyl;

[0152] R2 is each independently selected from -CH2OH, -CH2SH, -CH2Cl, -SCH2Cl, -SCH2F, -SCH2CF3, -OH, -OCH2CN, -OCH2Cl, -OCH2F, -OCH3, -OCH2CH3, -SCH2CN,

[0153] R 2a Each is independently hydrogen or C1-C6 alkyl;

[0154] R 2b Each is independently a C1-C6 alkyl group or a C1-C6 alkoxy group;

[0155] R 2c Each independently selected from hydrogen, C1-C6 alkyl, -CH2OH and C1-C6 alkoxy;

[0156] R 2d and R 2eEach is independently hydrogen or C1-C6 alkyl;

[0157] R3 are each independently hydrogen or halogen;

[0158] R4 are each independently selected from hydrogen, halogen and hydroxy;

[0159] m and n are each independently an integer from 1 to 6;

[0160] The substituent groups Q1 are each independently selected from C1-C6 alkyl, halogen, deuterium, hydroxyl, mercapto, -NR i R j , oxo, thio, -C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)OR k 、-S(O)(O)R k 、-S(O)(O)OR k 、-C(S)R k , nitro, cyano, C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, C2-C6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused ring aryl, and 5- to 12-membered fused heteroaryl;

[0161] R i and R j Each independently selected from a hydrogen atom, a hydroxyl group, a C1-C6 alkyl group and a C1-C6 alkoxy group;

[0162] R k independently selected from hydrogen atoms, C1-C6 alkyl groups, C1-C6 haloalkyl groups, C1-C6 alkoxy groups, hydroxyl groups and -NR i R j wherein the alkyl, alkoxy and haloalkyl are each independently selected from C1-C6 alkyl, halogen, hydroxyl, mercapto, -NR i R j , oxo, thioxo, carboxyl, nitro, cyano, C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, C2-C6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, and 5- to 10-membered heteroaryl; and

[0163] The condition is that when R 5a When R is hydrogen or alkyl, 5b Not hydrogen or alkyl.

[0164] In certain embodiments, R 1aEach is independently selected from hydrogen, C1-C6 alkyl and C1-C6 alkoxy, and the alkyl and alkoxy are each independently optionally substituted by one or more substituents selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, deuterium, amino, cyano and hydroxy.

[0165] In certain embodiments, R 1a Each is independently selected from hydrogen, C1-C6 alkyl and C1-C6 alkoxy.

[0166] In certain embodiments, Ring A is a 6- to 10-membered aryl or 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q1, wherein the heteroaryl group comprises at least one nitrogen atom.

[0167] In certain embodiments, Ring A is optionally substituted with one or more substituents Q1.

[0168] In certain embodiments, Ring B is a 6- to 10-membered aryl or 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q1, wherein the heteroaryl group comprises at least one nitrogen atom.

[0169] In certain embodiments, Ring B is optionally substituted with one or more substituents Q1.

[0170] In certain embodiments, X1 is -(CR 5a R 5b ) m - or a 6- to 10-membered aryl or 5- to 10-membered heteroaryl group optionally substituted by one or more substituents Q1, wherein the heteroaryl group contains at least one nitrogen atom.

[0171] In certain embodiments, R 5a and R 5b are independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano and the following groups optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)OR k 、-S(O)(O)R k 、-S(O)(O)OR k 、-C(S)R k , C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl and C2-C6 alkynyl, or R 5a and R 5bTogether they form oxo or thio.

[0172] In certain embodiments, R 5a and R 5b are independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)OR k 、-S(O)(O)R k 、-S(O)(O)OR k , C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl and C2-C6 alkynyl, or R 5a and R 5b Together they form oxo or thio.

[0173] In certain embodiments, R 5a and R 5b are independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)(O)R k , C1-C6 alkoxy, C2-C6 alkenyl and C2-C6 alkynyl, or R 5a and R 5b Together they form oxo or thio.

[0174] In certain embodiments, R 5a and R 5b are independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k and C1-C6 alkoxy, or R 5a and R 5b Together they form oxo or thio.

[0175] In certain embodiments, Ring C and Ring D are each independently selected from a 6- to 10-membered aryl, a 5- to 10-membered heteroaryl, an 8- to 12-membered fused ring aryl, or a 5- to 12-membered fused heteroaryl, optionally substituted with one or more substituents Q1, wherein the heteroaryl or fused heteroaryl comprises at least one nitrogen atom.

[0176] In certain embodiments, Ring C and Ring D are each independently selected from the following groups optionally substituted with one or more substituents Q1:

[0177] In certain embodiments, Ring C is selected from the group consisting of:

[0178] In certain embodiments, Ring D is a 6- to 10-membered aryl or 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q1, wherein the heteroaryl group comprises at least one nitrogen atom.

[0179] In certain embodiments, ring D is optionally substituted with one or more substituents Q1.

[0180] In certain embodiments, X2 is selected from -(CR 6a R 6b )n-、-O-、-S-、-NR 6c -、-CH2S-、-CH2O-、-NHCR 6d R 6e - and a 6- to 10-membered aryl or 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q1, said heteroaryl group containing at least one nitrogen atom.

[0181] In certain embodiments, R 6a and R 6b are independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)OR k 、-S(O)(O)R k 、-S(O)(O)OR k , C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl and C2-C6 alkynyl, or R 6a 、R 6b Together with the carbon atom to which it is attached, it forms a 3- to 10-membered cycloalkyl group, or R 6a and R6b Together they form oxo or thio.

[0182] In certain embodiments, R 6a and R 6b are independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)(O)R k , C1-C6 alkoxy, C2-C6 alkenyl and C2-C6 alkynyl, or R 6a 、R 6b Together with the carbon atom to which it is attached, it forms a 3- to 10-membered cycloalkyl group, or R 6a and R 6b Together they form oxo or thio.

[0183] In certain embodiments, R 6a and R 6b are independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k and C1-C6 alkoxy, or R 6a and R 6b Together they form oxo or thio.

[0184] In certain embodiments, R1 is each independently selected from hydrogen, C1-C6 alkyl and C1-C6 alkoxy, preferably hydrogen.

[0185] In certain embodiments, each R4 is independently hydrogen.

[0186] In certain embodiments, the substituent groups Q1 are each independently selected from halogen, hydroxy, thiol, deuterium, oxo, thioxo, cyano, amino, carboxyl, C1-C6 alkyl, and C1-C6 alkoxy.

[0187] In certain embodiments, R k independently selected from hydrogen atoms, C1-C6 alkyl groups, C1-C6 haloalkyl groups, C1-C6 alkoxy groups, hydroxyl groups and -NR i R j .

[0188] In certain embodiments, D is represented by the following formula (II-A') or (II-B'):

[0189] in,

[0190] R 1a Each independently selected from hydrogen, C1-C6 alkyl and C1-C6 alkoxy;

[0191] Ring A is The ring A is optionally substituted with one or more substituents Q1;

[0192] Ring B is The ring B is optionally substituted with one or more substituents Q1;

[0193] X1 is -(CR 5a R 5b ) m - or a 6- to 10-membered aryl or 5- to 10-membered heteroaryl group optionally substituted by one or more substituents Q1, wherein the heteroaryl group contains at least one nitrogen atom;

[0194] R 5a and R 5b are independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k and C1-C6 alkoxy, or R 5a and R 5b Together they form oxo or thio;

[0195] Ring C is selected from The ring C is optionally substituted with one or more substituents Q1;

[0196] Ring D is The ring D is optionally substituted with one or more substituents Q1;

[0197] X2 is selected from -(CR 6a R 6b )n-、-O-、-S-、-NR 6c -、-CH2S-、-CH2O-、-NHCR 6d R 6e - and 6 to 10 membered aryl or 5 to 10 membered heteroaryl, optionally substituted by one or more substituents Q1;

[0198] R 6a and R 6bare independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j 、-C(O)R k 、-C(O)OR k and C1-C6 alkoxy, or R 6a and R 6b Together they form oxo or thio;

[0199] R 6c 、R 6d and R 6e Each independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl and C1-C6 alkoxy;

[0200] R2 is each independently selected from -CH2OH, -CH2SH, -CH2Cl, -SCH2Cl, -SCH2F, -SCH2CF3, -OH, -OCH2CN, -OCH2Cl, -OCH2F, -OCH3, -OCH2CH3, -SCH2CN,

[0201] R 2a Each is independently hydrogen or C1-C6 alkyl;

[0202] R 2b Each is independently a C1-C6 alkyl group or a C1-C6 alkoxy group;

[0203] R 2c Each independently selected from hydrogen, C1-C6 alkyl, -CH2OH and C1-C6 alkoxy;

[0204] R 2d and R 2e Each is independently hydrogen or C1-C6 alkyl;

[0205] R3 are each independently hydrogen or halogen;

[0206] m and n are each independently an integer from 1 to 6;

[0207] The substituent groups Q1 are each independently selected from halogen, hydroxy, mercapto, deuterium, oxo, thioxo, cyano, amino, carboxyl, C1-C6 alkyl and C1-C6 alkoxy;

[0208] R i and R j Each independently selected from a hydrogen atom, a hydroxyl group, a C1-C6 alkyl group and a C1-C6 alkoxy group;

[0209] R kindependently selected from hydrogen atoms, C1-C6 alkyl groups, C1-C6 haloalkyl groups, C1-C6 alkoxy groups, hydroxyl groups and -NR i R j ;and

[0210] The condition is that when R 5a When R is hydrogen or alkyl, 5b Not hydrogen or alkyl.

[0211] In certain embodiments, R 1a For hydrogen.

[0212] In certain embodiments, X1 is selected from -(CR 5a R 5b ) m -, optionally substituted by one or more substituents Q1:

[0213] In certain embodiments, R 5a and R 5b All are fluorine.

[0214] In certain embodiments, R 5a and R 5b Together they form oxo or thio, preferably oxo.

[0215] In certain embodiments, X2 is selected from -(CR 6a R 6b ) n -, optionally substituted by one or more substituents Q1:

[0216] In certain embodiments, R 6a and R 6b are each independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, C1-C6 alkyl, -NR i R j 、-C(O)OR k and C1-C6 alkoxy, or R 6a and R 6b Together they form oxo or thio.

[0217] In certain embodiments, each R2 is independently selected from -CH2OH, -CH2SH, -OH and

[0218] In certain embodiments, R3 is hydrogen.

[0219] In certain embodiments, R3 is fluoro.

[0220] In certain embodiments, k is any value between 1 and 10, and in certain embodiments, k is any value between 2 and 5. k can be an integer or a decimal.

[0221] In certain embodiments, k is selected from 3.0 to 5.0 (e.g., 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9).

[0222] In certain embodiments, the antibody-drug conjugate is selected from:

[0223] Wherein, Ab, D, and k are as defined above, and p is independently 1, 2, 3, 4, 5, or 6. In certain embodiments, the antibody-drug conjugate is selected from:

[0224] Wherein, k is selected from 1 to 10 and can be an integer or a decimal.

[0225] In certain embodiments, k is any value between 2 and 5. k can be an integer or a decimal.

[0226] In certain embodiments, k is selected from 3.0 to 5.0 (e.g., 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or any number or range between any two numbers).

[0227] The antibodies disclosed herein are not particularly limited. The choice of antibody depends on the disease or condition (e.g., cancer) being treated by the antibody-drug conjugate (ADC). The antibody can specifically bind to the corresponding antigen (also known as tumor-associated antigen (TAA)), viral antigen, or microbial antigen expressed on cancer cells, and has antibody-dependent cell-mediated phagocytosis (ADCP) activity and in vivo anti-tumor, antiviral, or antimicrobial activity. The interchain SS bond in the antibody is the site for connecting the drug-linker complex.

[0228] Antibodies suitable for use in the methods of the present disclosure can be made by any method known in the art for synthesizing antibodies, such as by chemical synthesis or by recombinant expression, such as by recombinant expression techniques.

[0229] In some embodiments, the antibodies disclosed herein are selected from monoclonal antibodies or polyclonal antibodies.

[0230] In some embodiments, the antibodies disclosed herein are selected from murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, or antigen-binding fragments thereof.

[0231] In some embodiments, the isotype of the antibodies of the present disclosure is selected from IgG (IgG1, IgG2, IgG3, or IgG4). In some embodiments, the antibodies are selected from IgG1 or IgG4 isotypes. In some specific embodiments, the antibodies are selected from IgG1 monoclonal antibodies or IgG4 monoclonal antibodies.

[0232] In some embodiments, the antibodies disclosed herein are selected from anti-HER2 (ErbB2) antibodies, anti-EGFR antibodies, anti-B7-H3 antibodies, anti-c-Met antibodies, anti-HER3 (ErbB3) antibodies, anti-HER4 (ErbB4) antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD44 antibodies, anti-CD56 antibodies, anti-CD70 antibodies, anti-CD73 antibodies, anti-CD105 antibodies, anti-CEA antibodies, anti-A33 antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, anti-G250 antibodies, anti-MUCl antibodies, anti-Lewis Y antibodies, anti-VEGFR antibodies, anti-GPNMB antibodies, anti-Integrin antibodies, anti-PSMA antibodies, anti-Tenascin-C antibodies, anti-SLC44A4 antibodies, anti-CD79 antibodies, anti-TROP2 antibodies, anti-CD79B antibodies, anti-Mesothelin antibodies, anti-TNF-α antibodies, anti-folate receptor α antibodies (anti-human folate receptor alpha (FRA) antibody), anti-nectin-4 antibody, anti-AXL antibody, anti-CD19 antibody or antigen-binding fragments thereof.

[0233] In some embodiments, the antibody is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, adalimumab, Farletuzumab, Glematumamab and Enfortumab, or an antigen-binding fragment thereof.

[0234] In certain embodiments, the antibody or antigen-binding fragment thereof binds to human and / or mouse TNFα.Antibodies and antigen-binding fragments that bind to TNFα are known in the art.

[0235] In certain embodiments, the anti-TNFα antibody or antigen-binding fragment does not bind to TNF-β.

[0236] Anti-TNFα antibodies and antigen-binding fragments thereof include, for example, adalimumab, infliximab, certolizumab pegol, afelimomab, nerelimomab, ozoralizumab, placulumab, and golimumab, or antigen-binding fragments thereof. Additional anti-TNFα antibodies and antigen-binding fragments are provided, for example, in WO 2013 / 087912, WO 2014 / 152247, and WO 2015 / 073884, each of which is incorporated herein by reference in its entirety.

[0237] Anti-TNFα antibodies and antigen-binding fragments thereof also include antibodies and antigen-binding fragments thereof that competitively inhibit the binding of adalimumab, infliximab, certolizumab plucatum, afelimomab, nerimumab, ozolazumab, prukumab, or golimumab to TNFα. Anti-TNFα antibodies and antigen-binding fragments thereof also include antibodies and antigen-binding fragments that bind to the same TNFα epitope as adalimumab, infliximab, certolizumab plucatum, afelimomab, nerimumab, ozolazumab, prukumab, or golimumab.

[0238] In certain embodiments, the anti-TNFα antibody or antigen-binding fragment thereof competitively inhibits the binding of adalimumab to TNFα. In certain embodiments, the anti-TNFα antibody or antigen-binding fragment thereof binds to the same TNFα epitope as adalimumab. In certain embodiments, the anti-TNFα antibody or antigen-binding fragment thereof is adalimumab or an antigen-binding fragment thereof. In certain embodiments, the anti-TNFα antibody or antigen-binding fragment thereof is adalimumab.

[0239] In certain embodiments, the anti-TNFα antibody or antigen-binding fragment thereof comprises the sequence of adalimumab, infliximab, certolizumab plucatumumab, afelimomab, nerimumab, ozolazumab, prukumab, or golimumab, e.g., the complementarity determining regions (CDRs), variable heavy chain domain (VH), and / or variable light chain domain (VL).

[0240] The drugs used in the present disclosure are not particularly limited, as long as the drug molecule has the desired (e.g., cytotoxic, anti-tumor, or labeling agent, etc.) effect and has at least one substituent group or partial structure that allows connection to the linker structure. In some embodiments, the drug is selected from a diagnostic agent, a therapeutic agent, or a labeling agent; in some embodiments, the drug includes but is not limited to a cytotoxic agent, such as a chemotherapeutic agent, an immunotherapeutic agent, an antiviral agent, or an antimicrobial agent. In some embodiments, the drug can be selected from but is not limited to maytansinoids, auristatins (e.g., MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), MMAF (monomethyl auristatin F), etc.), calicheamicins, doxorubicins, benzodipyrroles (duocarmycins and CC-1065), camptothecins, pyrrolobenzodiazepines, and pyrrolobenzodiazepine dimers (PBD Dimmers), etc. In some embodiments, the drug is selected from glucocorticoid receptor agonists.

[0241] In certain embodiments, the drug is as defined for D in the antibody-drug conjugate (ADC) of formula (I).

[0242] In some embodiments, the present disclosure provides a method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0243] (1) Add an appropriate amount of ZnCl2 and the reducing agent diphenylphosphinoacetic acid to the antibody solution to be reduced;

[0244] (2) Add metal chelating agents to chelate Zn 2+ ion;

[0245] (3) adding an appropriate amount of the dissolved drug-linker intermediate or a pharmaceutically acceptable salt thereof and reacting for a certain period of time;

[0246] (4) Obtaining an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof.

[0247] In some embodiments, the final concentration of the antibody in step (1) is as described in the present disclosure. In some embodiments, the final concentration of the antibody is selected from about 0.10 mM to about 0.20 mM. In some embodiments, the final concentration of the antibody is selected from about 0.12 mM to about 0.14 mM.

[0248] In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the reducing agent in step (1) is as described in the present disclosure; in some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the reducing agent in step (1) is about 1:2.5 to about 1:3.5.

[0249] In some embodiments, in step (1), the antibody is combined with Zn 2+ The final concentration equivalent ratio (mM / mM) of the ions was approximately 1:2.

[0250] In some embodiments, the reaction conditions of step (1) are standing at 0-4°C overnight or reacting at 25°C for 2 hours.

[0251] In some embodiments, step (1) is carried out in a buffer system, and the buffer system is a histidine buffer with a pH of 6.0-7.0.

[0252] In some embodiments, the metal chelator of step (2) is selected from EDTA; in some embodiments, the metal chelator of step (2) is selected from ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetic acid, dicalcium ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid or a mixture thereof.

[0253] In some embodiments, Zn 2+ The final concentration equivalent ratio (mM / mM) of ion to metal chelator was approximately 1:2.

[0254] In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody and the drug-linker intermediate or a pharmaceutically acceptable salt thereof (calculated as the drug-linker intermediate) in step (3) is about 1:6.

[0255] In some embodiments, the reaction conditions of step (3) are 0°C or room temperature for 1 hour.

[0256] In some embodiments, step (4) further comprises a step of purifying the reaction solution, wherein the purification is selected from the step of using a desalting column or an ultrafiltration centrifuge tube to remove impurities such as free toxins and organic solvents.

[0257] The present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, which is prepared by the method described in the present disclosure.

[0258] The present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is obtained without a quenching step and / or a reoxidation step.

[0259] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure comprises D4 in an amount greater than about 50 wt %, such as greater than about 50 wt %, greater than about 55 wt %, greater than about 60 wt %, greater than about 61 wt %, greater than about 62 wt %, greater than about 63 wt %, greater than about 64 wt %, greater than about 65 wt %, greater than about 66 wt %, greater than about 67 wt %, greater than about 68 wt %, greater than about 69 wt %, greater than about 70 wt %, greater than about 71 wt %, greater than about 72 wt %, greater than about 73 wt %, greater than about 74 wt %, greater than about 75 wt %, greater than about 76 wt %, greater than about 77 wt %, greater than about 78 wt %, greater than about 79 wt %, greater than about 80 wt %, greater than about 81 wt %, greater than about 82 wt %, greater than about 83 wt %, greater than about 84 wt %, greater than about 85 wt %, greater than about 86 wt %, greater than about 87 wt %, greater than about 88 wt %, greater than about 89 wt %, greater than about 90 wt %, greater than about 91 wt %, greater than about 92 wt %, greater than about 93 wt %, greater than about 94 wt %, greater than about 95 wt %, greater than about 96 wt %, greater than about 97 wt %, greater than about 98 wt %, greater than about 99 wt %, greater than about In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains D4 in an amount selected from about 55 wt% to about 75 wt%, about 60 wt% to about 70 wt%, about 55 wt% to about 65 wt%, based on the total weight of D0, D2, D4, D6 and D8.

[0260] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure comprises a D4 content higher than that obtained using TCEP, based on the total weight of D0, D2, D4, D6, and D8.

[0261] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method described in the present disclosure comprises D4a, wherein D4a is selected from an antibody-drug conjugate or its pharmaceutically acceptable salt having only four drug linkers, and the four drug linkers are bound to the heavy-light chain intersulfhydryl group; based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt comprises D4a in an amount greater than about 50 wt%, for example, greater than about 50 wt%, greater than about 55 wt%, greater than about 56 wt%, greater than about 58 wt%, greater than about 59 wt%, greater than about 60 wt%, greater than about 61 wt%, greater than about 62 wt%, greater than about 64 wt%, greater than about 65 wt%, greater than about 66 wt%, greater than about 67 wt%, greater than about 68 wt%, greater than about 69 wt%, greater than about 70 wt%, greater than about 71 wt%, greater than about 72 wt%, greater than about 73 wt%, greater than about 74 wt%, greater than about 75 wt%, greater than about 76 wt%, greater than about 77 wt%, greater than about 78 wt%, greater than about 79 wt%, greater than about 80 wt%, greater than about 81 wt%, greater than about 82 wt%, greater than about 83 wt%, greater than about 84 wt%, greater than about 85 wt%, greater than about 86 wt%, greater than about 87 wt%, greater than about 88 wt%, greater than about 89 wt%, greater than about 90 wt%, greater than about 91 wt%, greater than about 92 wt%, greater than about 93 wt%, greater than about 94 wt%, greater than about %, higher than about 60 wt %, higher than about 61 wt %, higher than about 62 wt %, higher than about 63 wt %, higher than about 64 wt %, higher than about 65 wt %, higher than about 66 wt %, higher than about 67 wt %, higher than about 68 wt %, higher than about 69 wt %, higher than about 70 wt %, higher than about 71 wt %, higher than about 72 wt %, higher than about 73 wt %, higher than about 74 wt %, higher than about 75 wt %; in some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains D4a in an amount selected from about 55 wt % to about 75 wt %, about 60 wt % to about 70 wt %, about 55 wt % to about 65 wt %.

[0262] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure comprises D4b, wherein D4b is selected from an antibody-drug conjugate or a pharmaceutically acceptable salt thereof having and only having 4 drug linkers, and the 4 drug linkers are bound to the heavy-heavy interchain sulfhydryl group; based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises D4b in an amount of less than about 40 wt%, such as less than about 40 wt%, less than about 30 wt%, less than about 20 wt%, less than about 15 wt%, less than about 10 wt%, or less than about 5 wt%; in some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises D4b in an amount selected from about 5 wt% to about 30 wt%, about 5 wt% to about 20 wt%, or about 5 wt% to about 10 wt%.

[0263] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure has a D0+D8 content of less than about 20 wt %, such as less than about 19 wt %, less than about 18 wt %, less than about 17 wt %, less than about 16 wt %, less than about 15 wt %, less than about 14 wt %, less than about 13 wt %, less than about 12 wt %, less than about 13 wt %, less than about 14 wt %, less than about 15 wt %, less than about 16 wt %, less than about 17 wt %, less than about 18 wt %, less than about 19 ... Less than about 11 wt%, less than about 10 wt%, less than about 9 wt%, less than about 8 wt%, less than about 7 wt%, less than about 6 wt%, less than about 5 wt%, less than about 4 wt%, less than about 3 wt%; in some embodiments, based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains D0+D8 in an amount selected from about 3 wt% to about 20 wt%, about 5 wt% to about 15 wt%, about 3 wt% to about 10 wt%.

[0264] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described herein comprises less than about 20 wt% of D6, such as less than about 19 wt%, less than about 18 wt%, less than about 17 wt%, less than about 16 wt%, less than about 15 wt%, less than about 14 wt%, less than about 13 wt%, less than about 12 wt%, less than about 11 wt%, less than about 10 wt%, less than about 9 wt%, less than about 8 wt%, less than about 7 wt%, less than about 6 wt%, or less than about 5 wt%, based on the total weight of D0, D2, D4, D6, and D8; in some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises D6 in an amount selected from about 5 wt% to about 20 wt%, about 5 wt% to about 15 wt%, or about 5 wt% to about 10 wt%, based on the total weight of D0, D2, D4, D6, and D8.

[0265] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure, based on the total weight of D0, D2, D4, D6 and D8, refers to the total weight of unpurified D0, D2, D4, D6 and D8 obtained after the conjugation reaction.

[0266] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described herein, based on the total weight of D0, D2, D4, D6 and D8, refers to the total weight of D0, D2, D4, D6 and D8 obtained after purification to remove only small molecule process impurities (e.g., free toxins and organic solvents, etc.), wherein the purification step does not affect the different drug-loaded components of the prepared ADC.

[0267] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods described herein comprises a peak area percentage of D4 greater than about 50%, such as greater than about 50%, greater than about 55%, greater than about 60%, greater than about 61%, greater than about 62%, greater than about 63%, greater than about 64%, greater than about 65%, greater than about 66%, greater than about 67%, greater than about 68%, greater than about 69%, greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, or greater than about 75%, based on the sum of peak areas. In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises a peak area percentage of D4 selected from about 55% to about 75%, about 60% to about 70%, or about 55% to about 65%, based on the sum of peak areas.

[0268] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods described herein comprises a peak area percentage of D4 that is greater than the peak area percentage of D4 obtained using TCEP, based on the sum of peak areas.

[0269] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method of the present disclosure comprises D4a, wherein D4a is selected from an antibody-drug conjugate or its pharmaceutically acceptable salt having and only having 4 drug linkers, and the 4 drug linkers are bound to the sulfhydryl group between the heavy and light chains; based on the sum of the peak areas, the peak area percentage of D4a contained in the antibody-drug conjugate or its pharmaceutically acceptable salt is greater than about 50%, for example, greater than about 50%, greater than about 55%, greater than about 6%. 0%, greater than about 61%, greater than about 62%, greater than about 63%, greater than about 64%, greater than about 65%, greater than about 66%, greater than about 67%, greater than about 68%, greater than about 69%, greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, greater than about 75%; in some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises a peak area percentage of D4a selected from about 55% to about 75%, about 60% to about 70%, about 55% to about 65%, based on the sum of the peak areas.

[0270] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described herein comprises D4b, wherein D4b is selected from an antibody-drug conjugate or a pharmaceutically acceptable salt thereof having and only having 4 drug linkers, and the 4 drug linkers are bound to the heavy-heavy chain intersulfhydryl group; based on the sum of peak areas, the peak area percentage of D4b contained in the antibody-drug conjugate or the pharmaceutically acceptable salt thereof is less than about 40%, for example, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%; in some embodiments, based on the sum of peak areas, the peak area percentage of D4b contained in the antibody-drug conjugate or the pharmaceutically acceptable salt thereof is selected from about 5% to about 30%, about 5% to about 20%, or about 5% to about 10%.

[0271] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods described herein comprises a peak area percentage of D0+D8 of less than about 20%, such as less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3%, based on the sum of the peak areas. In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises a peak area percentage of D0+D8 selected from about 3% to about 20%, about 5% to about 15%, or about 3% to about 10%, based on the sum of the peak areas.

[0272] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods described herein has a peak area percentage of D6 of less than about 20%, such as less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, or less than about 5%, based on the sum of peak areas. In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof has a peak area percentage of D6 selected from about 5% to about 20%, about 5% to about 15%, or about 5% to about 10%, based on the sum of peak areas.

[0273] The peak area percentage based on the sum of peak areas refers to: the antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof prepared by the present disclosure is tested by the HIC-HPLC method, and the sum of the areas of each chromatographic peak after deducting the blank solution is calculated by comparing the chromatogram with the blank solution. The chromatogram is integrated to obtain the sum of the peak areas, and the peak area of ​​each component (e.g., D0, D2, D4, D6 or D8) is calculated as a percentage of the total peak area using the area normalization method.

[0274] In some embodiments, the HIC-HPLC method is as described in Example 1.

[0275] In some embodiments, the HIC-HPLC method is as described in Example 2.

[0276] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described herein comprises a peak area percentage of D4 greater than about 50%, such as greater than about 50%, greater than about 55%, greater than about 60%, greater than about 61%, greater than about 62%, greater than about 63%, greater than about 64%, greater than about 65%, greater than about 66%, greater than about 67%, greater than about 68%, greater than about 69%, greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, greater than about 75%, greater than about 76%, greater than about 77%, greater than about 78%, greater than about 79%, greater than about 80%, greater than about 81%, greater than about 82%, greater than about 83%, greater than about 84%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 100%, greater than about 101%, greater than about 102%, greater than about 103%, greater than about 104%, greater than about 105%, greater than about 106%, greater than about 107%, greater than about 108%, greater than about 109%, greater than about 110%, greater than about 111%, greater than about 112%, greater than about 113%, greater than about 114%, greater than about 115%, greater than about 116%, greater than about 117%, greater than about 118%, greater than about 119%, greater than about 120%, greater than about In some embodiments, the peak area percentage of D4 contained in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is selected from about 55% to about 75%, about 60% to about 70%, about 55% to about 65%, based on the sum of the peak areas of D0, D2, D4, D6 and D8.

[0277] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods described herein comprises a peak area percentage of D4 that is greater than the peak area percentage of D4 obtained using TCEP, based on the sum of the peak areas of D0, D2, D4, D6, and D8.

[0278] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure comprises D4a, wherein D4a is selected from an antibody-drug conjugate or a pharmaceutically acceptable salt thereof having and only having 4 drug linkers, and the 4 drug linkers are bound to the sulfhydryl group between the heavy and light chains; based on the sum of the peak areas of D0, D2, D4, D6 and D8, the peak area percentage of D4a contained in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is greater than about 50%, for example, greater than about 50%, greater than about 55%, greater than about 6 0%, greater than about 61%, greater than about 62%, greater than about 63%, greater than about 64%, greater than about 65%, greater than about 66%, greater than about 67%, greater than about 68%, greater than about 69%, greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, greater than about 75%; in some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises a peak area percentage of D4a selected from about 55% to about 75%, about 60% to about 70%, about 55% to about 65%, based on the sum of the peak areas of D0, D2, D4, D6 and D8.

[0279] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described herein comprises D4b, wherein D4b is selected from an antibody-drug conjugate or a pharmaceutically acceptable salt thereof having and only having 4 drug linkers, and the 4 drug linkers are bound to the heavy-heavy chain intersulfhydryl group; based on the sum of the peak areas of D0, D2, D4, D6 and D8, the peak area percentage of D4b contained in the antibody-drug conjugate or the pharmaceutically acceptable salt thereof is less than about 40%, for example, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%; in some embodiments, based on the sum of the peak areas of D0, D2, D4, D6 and D8, the peak area percentage of D4b contained in the antibody-drug conjugate or the pharmaceutically acceptable salt thereof is selected from about 5% to about 30%, about 5% to about 20%, or about 5% to about 10%.

[0280] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods described herein comprises a peak area percentage of D0+D8 of less than about 20%, such as less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3%, based on the sum of the peak areas of D0, D2, D4, D6, and D8; and in some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises a peak area percentage of D0+D8 selected from about 3% to about 20%, about 5% to about 15%, or about 3% to about 10%, based on the sum of the peak areas of D0, D2, D4, D6, and D8.

[0281] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described herein comprises less than about 20% by peak area percentage of D6, such as less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, or less than about 5%, based on the sum of the peak areas of D0, D2, D4, D6, and D8; and in some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises a peak area percentage of D6 selected from about 5% to about 20%, about 5% to about 15%, or about 5% to about 10%, based on the sum of the peak areas of D0, D2, D4, D6, and D8.

[0282] In some embodiments, the sum of the peak areas of D0, D2, D4, D6, and D8 for the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure refers to the sum of the peak areas of D0, D2, D4, D6, and D8 obtained without purification after the conjugation reaction.

[0283] In some embodiments, the sum of the peak areas of D0, D2, D4, D6, and D8 for the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods described herein refers to the sum of the peak areas of D0, D2, D4, D6, and D8 obtained after purification to remove only small molecule process impurities (e.g., free toxins and organic solvents, etc.), wherein the purification step does not affect the different drug-loaded components of the prepared ADC.

[0284] The peak area percentage based on the sum of the peak areas of D0, D2, D4, D6, and D8 refers to: the antibody-drug conjugate (ADC) prepared by the present disclosure or a pharmaceutically acceptable salt thereof is detected by the HIC-HPLC method, and the sum of the areas of D0, D2, D4, D6, and D8 after deducting the blank solution is calculated by comparing the chromatogram with the blank solution, and the sum of the peak areas based on D0, D2, D4, D6, and D8 is obtained by integrating the chromatogram. The peak area percentage of each component (e.g., D0, D2, D4, D6, or D8) is calculated using the area normalization method to the sum of the peak areas based on D0, D2, D4, D6, and D8.

[0285] In some embodiments, the HIC-HPLC method is as described in Example 1.

[0286] In some embodiments, the HIC-HPLC method is as described in Example 2.

[0287] In some embodiments, the sum of the peak areas of D0, D2, D4, D6, and D8 for the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure refers to the sum of the peak areas of D0, D2, D4, D6, and D8 obtained without purification after the conjugation reaction.

[0288] In some embodiments, the sum of the peak areas of D0, D2, D4, D6, and D8 for the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods described herein refers to the sum of the peak areas of D0, D2, D4, D6, and D8 obtained after purification to remove only small molecule process impurities (e.g., free toxins and organic solvents, etc.), wherein the purification step does not affect the different drug-loaded components of the prepared ADC.

[0289] In some embodiments, the average drug loading of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is selected from about 3.0 to about 5.0, about 3.5 to about 4.5, for example, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9; in some embodiments, the average drug loading of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is greater than 3.0 and less than 5.0; in some embodiments, the average drug loading is selected from about 3.8 to about 4.4; in some embodiments, the average drug loading is selected from about 3.9 to about 4.1.

[0290] The aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof disclosed herein can be prepared into a kit.

[0291] On the other hand, the present disclosure also provides a pharmaceutical composition comprising an effective amount of the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0292] The present disclosure also provides a use of the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in the preparation of a medicament for treating and / or preventing a tumor, cancer, autoimmune disease, or infectious disease. In some embodiments, the cancer is a cancer associated with the expression of HER2, HER3, B7H3, CD19, CD30, CD33, Trop2, CD79b, Nectin-4, TNF-α, folate receptor α, or EGFR. In some embodiments, the infectious disease is a viral or microbial infection. In some embodiments, the autoimmune disease is selected from rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, hidradenitis suppurativa, uveitis, Behçet's disease, spondyloarthropathy, and psoriasis.

[0293] The present disclosure also provides a use of the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition for preparing a medicament for treating and / or preventing cancer. In some embodiments, the cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma.

[0294] The active compound, such as an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, can be formulated for administration by any suitable route, preferably in a unit dosage form, or in a form that a patient can self-administer as a single dose. A unit dosage form of the disclosed compound or composition can be in the form of a tablet, capsule, cachet, bottled solution, powder, granules, lozenge, suppository, reconstituted powder, or liquid formulation.

[0295] The dosage of the antibody-drug conjugate or pharmaceutically acceptable salt thereof or compound or composition used in the treatment methods disclosed herein will generally vary with the severity of the disease, the patient's weight and the relative efficacy of the compound. However, as a general guide, a suitable unit dose may be 0.1 to 1000 mg.

[0296] The pharmaceutical compositions of the present disclosure may contain, in addition to the active compound, such as an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, one or more excipients selected from the following: fillers (diluents), binders, wetting agents, disintegrants, or excipients. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound, such as an antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

[0297] On the other hand, without specifying the configuration, the disclosed compounds, drugs, intermediates, and conjugates may exist in specific isomeric forms, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers. Without specifying the configuration, the disclosure contemplates all such compounds and conjugates, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. Unless otherwise indicated, all of these isomers and their mixtures are included within the scope of the disclosure.

[0298] The compounds, drugs, intermediates, and conjugates of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents. Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound, drug, intermediate, or conjugate of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary groups are cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, separation of enantiomers and diastereomers is often accomplished by the use of chromatography employing chiral stationary phases, optionally coupled with chemical derivatization (eg, formation of carbamates from amines).

[0299] Any isotope-labeled derivatives of the compounds, drugs, intermediates, conjugates, or pharmaceutically acceptable salts thereof, or isomers thereof, described herein are covered by the present disclosure. Atoms that can be isotopically labeled include, but are not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, iodine, and the like. They can be labeled with isotopes of different species. 2 H(D), 3 H. 11 C. 13 C. 14 C. 15 N. 18 F. 31 P. 32 P. 35 S. 36 Cl and 125 I etc. instead.

[0300] Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have deuterium (i.e., at least 10% deuterium incorporation) at least 1000 times greater than the natural abundance of deuterium (which is 0.015%). In the example, the compound has a natural abundance greater than deuterium of at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium (i.e., at least 45% deuterium incorporation), at least 4000 times the abundance of deuterium, at least 5000 times the abundance of deuterium, at least 6000 times the abundance of deuterium or more. The present disclosure also includes various deuterated compounds. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated compounds with reference to relevant literature. In preparing deuterated forms of the compounds, commercially available deuterated starting materials may be used, or they may be synthesized using conventional techniques with deuterated reagents including, but not limited to, deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

[0301] On the other hand, the present disclosure also provides a method for selectively reducing an antibody, comprising the step of reacting a reducing agent and an antibody in a buffer system in the presence of an effective amount of transition metal ions to selectively reduce interchain disulfide bonds within the antibody to sulfhydryl groups.

[0302] In some embodiments, the transition metal ion in the method for selectively reducing antibodies is selected from Zn 2+ 、Cd 2+ 、Hg 2+ or a combination thereof; in some embodiments, the transition metal ion is selected from Zn 2+ .

[0303] In some embodiments, the transition metal ions are derived from transition metal salts, as long as they are soluble in the reaction solution so that free transition metal ions can be released into the reaction solution.

[0304] In some embodiments, suitable zinc salts include, but are not limited to, ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, zinc formate, and zinc tetrafluoroborate.

[0305] In some embodiments, the suitable Cd is soluble in the reaction solution and can release free Cd. 2+ or Hg 2+ Other transition metal salts of ions include, but are not limited to: CdCl2, Cd(NO3)2, CdSO4, Cd(CH3COO)2, CdI2, CdBr2, cadmium formate and cadmium tetrafluoroborate; HgCl2, Hg(NO3)2, HgSO4, Hg(CH3COO)2, HgBr2, mercury (II) formate, and mercury (II) tetrafluoroborate, etc.

[0306] In some embodiments, the reducing agent in the method for selectively reducing antibodies is a reducing agent containing a diphenylphosphine group, or a salt thereof; in some embodiments, the reducing agent is selected from diphenylphosphinoacetic acid, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(diphenylphosphino)propionic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, or a salt thereof; in some embodiments, the reducing agent is selected from diphenylphosphinoacetic acid, or a salt thereof.

[0307] In some embodiments, the buffer system used in the method for selectively reducing antibodies is selected from: Hepes buffer, histidine buffer, PBS buffer, or MES buffer, citrate buffer, tris buffer, gluconate buffer, adipic acid buffer, lactate buffer, acetate buffer, or succinate buffer; in some embodiments, the buffer system is selected from histidine buffer. In some embodiments, the buffer system depends on a transition metal ion.

[0308] In some embodiments, the buffer system may or may not contain a metal chelator. In some embodiments, the buffer system does not contain a metal chelator.

[0309] In some embodiments, the pH of the buffer system used in the method of selectively reducing antibodies is selected from about 4 to about 10, for example, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0; in some embodiments, selected from about 5.5 to about 8, about 6 to about 7.5, in some embodiments, selected from 6.0 to 7.0.

[0310] In some embodiments, the method of selectively reducing antibodies is performed at about -10°C to about 40°C, for example, about -5°C, about -3°C, about -2°C, about -1°C, about 0°C, about 2°C, about 3°C, about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 37°C; in some embodiments, at about -5°C to about 37°C; in some embodiments, at about 0°C to about 4°C; in some embodiments, at about 0°C to about 37°C; in some embodiments, at about 10°C to about 25°C; in some embodiments, at about -5°C to about 5°C.

[0311] In some embodiments, the reaction time of the method for selectively reducing antibodies is selected from 1 h to 24 h, such as 2 h, 4 h, 8 h, 12 h, 16 h; in some embodiments, the reaction time of the method for selectively reducing antibodies is 16 h.

[0312] In some embodiments, the method for selectively reducing antibodies is selected from the group consisting of standing at 0-4°C overnight or reacting at 25°C for 2 hours;

[0313] In some embodiments, the reaction conditions depend on the specific antibody to be reduced. The determination of the incubation period and temperature based on the specific antibody is within the ability of those of ordinary skill in the art. For example, the antibody to be conjugated is typically incubated with a reducing agent in the presence of a transition metal ion at 4°C overnight.

[0314] In some embodiments, the final concentration of the antibody is selected from about 0.01 mM to about 0.50 mM, such as about 0.01 mM, about 0.02 mM, about 0.03 mM, about 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.09 mM, about 0.10 mM, about 0.11 mM, about 0.12 mM, about 0.13 mM. , about 0.14 mM, about 0.15 mM, about 0.20 mM, about 0.25 mM, about 0.30 mM, about 0.35 mM, about 0.40 mM, about 0.45 mM, about 0.50 mM; in some embodiments, the final concentration of the antibody is selected from about 0.10 mM to about 0.20 mM; in some embodiments, the final concentration of the antibody is selected from about 0.12 mM to about 0.14 mM.

[0315] In some embodiments, the final concentration of the transition metal ion is selected from about 0.01 mM to about 0.50 mM, such as about 0.01 mM, about 0.02 mM, about 0.03 mM, about 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.09 mM, about 0.10 mM, about 0.15 mM, about 0.20 mM, about 0.21 mM, about 0.22 mM, about 0.23 mM. , about 0.24 mM, about 0.25 mM, about 0.26 mM, about 0.27 mM, about 0.28 mM, about 0.29 mM, about 0.30 mM, about 0.31 mM, about 0.32 mM, about 0.33 mM, about 0.34 mM, about 0.35 mM, about 0.40 mM, about 0.45 mM, about 0.50 mM; in some embodiments, the final concentration of the transition metal ion is selected from about 0.27 mM to about 0.28 mM.

[0316] In some embodiments, the final concentration of the reducing agent is selected from about 0.20 mM to about 1.00 mM, for example, about 0.20 mM, about 0.25 mM, about 0.30 mM, about 0.35 mM, about 0.40 mM, about 0.45 mM, about 0.50 mM, about 0.55 mM, about 0.60 mM, about 0.65 mM, about 0.70 mM, about 0.75 mM, about 0.80 mM, about 0.85 mM, about 0.90 mM, about 0.95 mM, about 1.00 mM; in some embodiments, the final concentration of the reducing agent is selected from about 0.35 mM to about 0.50 mM, about 0.38 mM to about 0.45 mM.

[0317] In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the reducing agent is about 3:1 to about 1:10, for example, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1: 10. In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the reducing agent is about 1:2.5 to about 1:3.5.

[0318] In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the transition metal ion is 5:1 to about 1:5, for example, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5. In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the transition metal ion is about 1:2.

[0319] In some embodiments, the method for selectively reducing antibodies described herein further comprises the step of adding a metal chelator. The metal chelator is used to chelate transition metal ions. In some embodiments, the metal chelator is selected from EDTA; in some embodiments, the metal chelator is selected from ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetic acid, dicalcium ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, or mixtures thereof.

[0320] In some embodiments, the final concentration equivalent ratio (mM / mM) of the transition metal ion to the metal chelator is 1:1 to about 1:5, for example, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5. In some embodiments, the final concentration equivalent ratio (mM / mM) of the transition metal ion to the metal chelator is about 1:2.

[0321] In some embodiments, the antibodies disclosed herein are selected from monoclonal antibodies or polyclonal antibodies.

[0322] In some embodiments, the antibodies disclosed herein are selected from murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, or antigen-binding fragments thereof.

[0323] In some embodiments, the isotype of the antibodies of the present disclosure is selected from IgG (IgG1, IgG2, IgG3, or IgG4). In some embodiments, the antibodies are selected from IgG1 or IgG4 isotypes. In some specific embodiments, the antibodies are selected from IgG1 monoclonal antibodies or IgG4 monoclonal antibodies.

[0324] In some embodiments, the present disclosure provides a method for selectively reducing an antibody, comprising the following steps: (1a) adding an appropriate amount of ZnCl2 and a reducing agent, diphenylphosphinoacetic acid, to an antibody solution to be reduced.

[0325] In some embodiments, the final concentration of the antibody in step (1a) is as described herein. In some embodiments, the final concentration of the antibody is selected from about 0.10 mM to about 0.20 mM. In some embodiments, the final concentration of the antibody is selected from about 0.12 mM to about 0.14 mM.

[0326] In some embodiments, the final concentration equivalent ratio (mM / mM) of the antibody to the reducing agent in step (1a) is about 1:2.5 to about 1:3.5.

[0327] In some embodiments, the antibody in step (1a) is combined with Zn 2+ The final concentration equivalent ratio (mM / mM) of the ions was approximately 1:2.

[0328] In some embodiments, the reaction conditions of step (1a) are standing at 0-4°C overnight or reacting at 25°C for 2 hours.

[0329] In some embodiments, step (1a) is carried out in a buffer system, and the buffer system is a histidine buffer, pH 6.0 to 7.0.

[0330] In some embodiments, the method for selectively reducing antibodies disclosed herein further comprises (2a) adding a metal chelator to chelate Zn 2+ ion.

[0331] In some embodiments, the metal chelator of step (2a) is selected from EDTA. In some embodiments, the metal chelator of step (2a) is selected from ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetic acid, calcium ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid or a mixture thereof.

[0332] In some embodiments, Zn 2+ The final concentration equivalent ratio (mM / mM) of ion to metal chelator was approximately 1:2.

[0333] In some embodiments, based on the total number of antibody interchain disulfide bonds and intrachain disulfide bonds, the method for selectively reducing antibodies described in the present disclosure selectively reduces the proportion of heavy-light chain interchain disulfide bonds higher than about 50 wt%, such as higher than about 50 wt%, higher than about 55 wt%, higher than about 60 wt%, higher than about 61 wt%, higher than about 62 wt%, higher than about 63 wt%, higher than about 64 wt%, higher than about 65 wt%, higher than about 66 wt%, higher than about 67 wt%, higher than about 68 wt%, higher than about 69 wt%, higher than about 70 wt%, higher than about 71 wt%, higher than about 72 wt%, higher than about 73 wt%, higher than about 74 wt%, higher than about 75 wt%; in some embodiments, the proportion is selected from about 55 wt% to about 75 wt%, about 60 wt% to about 70 wt%, about 55 wt% to about 65 wt%.

[0334] The present disclosure incorporates the full text of WO2022166779A1.

[0335] Terminology

[0336] Unless otherwise defined, all technical and scientific terms used in this disclosure are consistent with the common understanding of one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described in this disclosure can be used to practice or test the present disclosure, this disclosure describes preferred methods and materials. When describing and claiming this disclosure, the following terms are used in accordance with the following definitions.

[0337] When a trade name is used in this disclosure, applicants intend to include the formulations of the trade name product, the generic version, and the active drug portion of the trade name product.

[0338] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0339] The term "antibody-drug conjugate" (ADC), also known as antibody-drug conjugate or antibody-drug conjugate, refers to an antibody linked to a drug via a linker. In this disclosure, "antibody-drug conjugate" refers to an antibody or antigen-binding fragment linked to a biologically active toxic drug via a stable linker. In this disclosure, the terms "antibody-drug conjugate," "antibody-drug conjugate," and "antibody-drug conjugate" are used interchangeably.

[0340] The terms "linker," "connector," "linking unit," "linker unit," or "linking fragment" refer to a chemical structure fragment or bond that is connected to an antibody or antigen-binding fragment at one end and to a drug at the other end, and may also be connected to other linkers before being connected to an antibody or drug.

[0341] The joint can include one or more joint components. Exemplary joint components include 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with joint reagents: N-succinimidyl 4-(2-pyridylthio) valerate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC, also known as MCC) and N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB). The joint can include a stretching unit, a spacer unit, an amino acid unit and an extension unit. It can be synthesized by methods known in the art, such as described in US2005-0238649A1. The joint can be a "cleavable joint" for facilitating release of the drug in the cell. For example, an acid-labile linker (eg, a hydrazone), a protease-sensitive (eg, a peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0342] The term "drug loading" (also expressed as DAR, DAR value, drug:antibody ratio) refers to the average amount of drug loaded per antibody-drug conjugate molecule in a population of antibody-drug conjugates, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading can range from 1 to 20 linked per antibody (Ab). In embodiments of the present disclosure, the drug loading can be illustratively 3, 4, 5, or the average of any two values. The average amount of drug per ADC molecule after the conjugation reaction can be characterized using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, monoclonal antibody size variant assay (CE-SDS), and HPLC. For example, in one embodiment, HIC is used to determine the yield and isomeric mixture of the resulting antibody-drug conjugate (e.g., for D4 conjugates). This technique is capable of separating antibodies loaded with various amounts of drug. The drug loading level can be determined based on, for example, the ratio of absorbance at 250 nm and 280 nm. For example, if the drug absorbs at 250 nm and the antibody absorbs at 280 nm, the drug loading level can be determined. Thus, the 250 / 280 ratio increases with drug loading.Using the bioconjugation methods described herein, antibodies with an even number of drugs are generally observed to be conjugated to the antibody, as reduction of disulfide bonds generates an even number of free cysteine ​​thiols.

[0343] Typically, an antibody molecule belonging to an IgG1 or IgG4 subclass has four interchain SS bonds, each formed by two -SH groups. The antibody molecule can undergo partial or complete reduction of one or more interchain SS bonds to form 2n (n is an integer selected from 1, 2, 3, or 4) reactive -SH groups, so the number of drugs coupled to a single antibody molecule is 2, 4, 6, or 8. Depending on the number of drugs coupled to a single antibody molecule, different conjugates containing different numbers of drug molecules are named D0, D2, D4, D6, and D8. If the number of drugs coupled to a single antibody molecule is 0, the product is called D0.

[0344] Thus, D2 refers to an ADC in which two drug molecules are coupled to a single antibody molecule, wherein the two drug molecules can be coupled via a linker to a -SH group generated by reducing the SS bond between the heavy and light chains, or can be coupled via a linker to a -SH group generated by reducing the SS bond between the heavy and heavy chains.

[0345] D4 refers to an ADC in which four drug molecules are coupled to a single antibody molecule, wherein the four drug molecules can be coupled to four -SH groups generated by reducing two SS bonds between the heavy chain and the light chain via a linker (this ADC is referred to as D4a), or four drug molecules can be coupled to four -SH groups generated by reducing two SS bonds between the heavy chain and the light chain via a linker (this ADC is referred to as D4b), or two drug molecules can be coupled to two -SH groups generated by reducing one SS bond between the heavy chain and the light chain via a linker and the other two drug molecules can be coupled to two -SH groups generated by reducing one SS bond between the heavy chain and the light chain via a linker (this ADC is referred to as D4c).

[0346] D6 refers to an ADC in which six drug molecules are coupled to a single antibody molecule, wherein four drug molecules can be coupled to four -SH groups generated by reducing two SS bonds between the heavy chain and the light chain via a linker and two drug molecules can be coupled to two -SH groups generated by reducing one SS bond between the heavy chain and the light chain via a linker (this ADC is referred to as D6a), or four drug molecules can be coupled to four -SH groups generated by reducing two SS bonds between the heavy chain and the light chain via a linker and two drug molecules can be coupled to two -SH groups generated by reducing one SS bond between the heavy chain and the light chain via a linker (this ADC is referred to as D6b).

[0347] D8 refers to an ADC in which eight drug molecules are coupled to a single antibody molecule, i.e., all four SS bonds in one antibody molecule are reduced to eight -SH groups and each -SH group is linked to one drug molecule.

[0348] Typically, a heterogeneous mixture of ADC molecules produced by conventional conjugation methods or the methods of the present disclosure is a mixture of D0, D2, D4, D6, and D8. Therefore, the "homogeneity" or "uniformity" of an antibody-drug conjugate is a property used to describe the predominance of a particular type of antibody-drug conjugate (i.e., a type selected from D0, D2, D4, D6, and D8 conjugates) in a given mixture of antibody-drug conjugates. Generally, if the content of D4 in the mixture is high, the ADC is considered to have high homogeneity or high uniformity.

[0349] In this disclosure, "homogeneity" or "homogeneity" of an antibody-drug conjugate refers to having a high level of D4 in a mixture of antibody-drug conjugates.

[0350] The term "interchain disulfide" is used to refer to the disulfide between two heavy chains in an antibody (heavy-heavy interdisulfide) or the disulfide between a heavy and a light chain in an antibody (heavy-light interdisulfide).

[0351] The term "interchain thiol" or "interchain sulfhydryl" is used to refer to a thiol group obtained by reducing the interchain disulfides of an antibody.

[0352] The term "heavy-heavy chain inter-thiol" is used to refer to the sulfhydryl groups obtained by reducing the heavy-heavy chain inter-disulfides of an antibody.

[0353] The term "heavy-light chain inter-thiol" is used to refer to the sulfhydryl group obtained by reducing the inter-heavy-light chain disulfides of an antibody.

[0354] Unless otherwise indicated, the use of the term "add" does not limit the order, method, or how the added materials are combined. For example, "adding A to B" can also describe "adding B to A." Furthermore, "adding A and B to C" can also describe other different combinations, such as "adding A to B and C," "adding A and C to B," "adding B to A and C," "adding B and C to A," and "adding C to A and B."

[0355] The terms "mercapto" and "thiol" are used interchangeably to refer to -SH.

[0356] The term "diphenylphosphino" refers to

[0357] The term "diphenylphosphinoacetic acid" also known as "DPA" or "diphenylphosphinoacetic acid" refers to

[0358] In the structure described in this disclosure, Indicates a single bond or a double bond.

[0359] It will be understood by those skilled in the art that, for example, when R 5aand R 5b When one of the is selected from oxo or thio, the other is absent.

[0360] The term "stretcher" refers to a chemical structural segment that is covalently linked to an antibody through a carbon atom at one end and is linked to an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptide chemical moiety at the other end.

[0361] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. More preferred are alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

[0362] The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same or two different carbon atoms of an alkane radical by removing two hydrogen atoms, preferably from 1 to 20 carbon atoms, more preferably from 1 to 12 carbon atoms, and more preferably from 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and the like. Alkylene groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment.

[0363] The term "alkenylene" refers to a linear alkenyl group having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms and having at least one double bond at any position, including, for example, vinylene, allylene, propenylene, butenylene, prenylene, butadienylene, pentenylene, pentadienylene, hexenylene, hexadienylene, etc.

[0364] The term "alkynylene" includes linear alkynylene groups having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, and having at least one triple bond at any position, including, for example, ethynylene, propynylene, butynylene, pentynylene, hexynylene, and the like.

[0365] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls. "Carbocycle" refers to the ring system within a cycloalkyl group.

[0366] The term "spiroalkyl" refers to a polycyclic group that shares a carbon atom (called a spiro atom) between 5 to 20 monocyclic rings, which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of spiro atoms shared between the rings, the spiroalkyl is divided into a single spiroalkyl, a double spiroalkyl or a multi-spiroalkyl, preferably a single spiroalkyl and a double spiroalkyl. More preferably, it is 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6 yuan single spiroalkyl. "Spiro carbocycle" refers to the ring system in the spiroalkyl. Non-limiting examples of spiroalkyl include:

[0367] The term "fused cycloalkyl" refers to a 5 to 20-membered, all-carbon polycyclic group in which each ring in the system shares a pair of adjacent carbon atoms with the other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl. "Fused carbocycle" refers to the ring system in a fused cycloalkyl. Non-limiting examples of fused cycloalkyls include:

[0368] The term "bridged cycloalkyl" refers to a 5-20 membered, all-carbon polycyclic group in which any two rings share two carbon atoms that are not directly connected, which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6-14 members, more preferably 7-10 members. Depending on the number of constituent rings, it can be classified as a bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl group, preferably a bicyclic, tricyclic or tetracyclic group, more preferably a bicyclic or tricyclic group. Non-limiting examples of bridged cycloalkyl groups include:

[0369] The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring attached to the parent structure is a cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc. The cycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

[0370] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m(wherein m is an integer from 0 to 2) heteroatoms, but excluding the ring portion of -OO-, -OS- or -SS-, the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 6 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl and pyrrolidinyl. Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups. "Heterocycle" refers to the ring system in the heterocyclic group.

[0371] The term "spiroheterocyclyl" refers to a polycyclic heterocyclic group in which the monocyclic rings of 5 to 20 members share one atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer 0 to 2) heteroatom, and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. According to the number of shared spiral atoms between the rings, the spiro heterocyclic group is divided into a monospiro heterocyclic group, a dispiro heterocyclic group or a polyspiro heterocyclic group, preferably a monospiro heterocyclic group and a dispiro heterocyclic group. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered or 5-membered / 6-membered monospiro heterocyclic group. "Spiro heterocycle" refers to the ring system in the spiro heterocyclic group. Non-limiting examples of spiro heterocyclic groups include:

[0372] The term "fused heterocyclyl" refers to a polycyclic heterocyclic group of 5 to 20 members, wherein each ring in the system shares a pair of adjacent atoms with other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, the remaining ring atoms being carbon. Preferably 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic group, preferably a bicyclic or tricyclic, more preferably a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic group. "Fused heterocycle" refers to the ring system in a fused heterocyclic group. Non-limiting examples of fused heterocyclic groups include:

[0373] The term "bridged heterocyclyl" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m(wherein m is an integer from 0 to 2) heteroatoms, the remaining ring atoms being carbon. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it can be classified as a bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic group, preferably a bicyclic, tricyclic or tetracyclic group, more preferably a bicyclic or tricyclic group. Non-limiting examples of bridged heterocyclic groups include:

[0374] The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:

[0375] wait.

[0376] The heterocyclyl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

[0377] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring. "Aromatic ring" refers to the ring system in an aryl group. Non-limiting examples of aryl groups include:

[0378] The aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate, preferably phenyl.

[0379] The term "fused aryl" may be an unsaturated aromatic fused ring structure containing 8-14 ring atoms formed by two or more cyclic structures sharing two adjacent atoms, preferably 8-12 ring atoms. For example, it includes all unsaturated fused aryl rings, such as naphthalene, phenanthrene, etc., and also includes partially saturated fused aryl rings, such as benzo 3-8 membered saturated monocyclic cycloalkyl and benzo 3-8 membered partially saturated monocyclic cycloalkyl. "Fused aromatic ring" refers to the ring system in the fused aryl ring. Specific examples of fused aryl rings include 2,3-dihydro-1H-indenyl, 1H-indenyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl, etc.

[0380] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 12 members, such as imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. "Heteroaromatic ring" refers to the ring system in a heteroaryl group. Non-limiting examples of heteroaryl groups include:

[0381] The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0382] The term "fused heteroaryl" may be an unsaturated aromatic fused ring structure containing 5-14 ring atoms (including at least one heteroatom) formed by two or more ring structures sharing two adjacent atoms, wherein the carbon atoms, nitrogen atoms and sulfur atoms may be oxidized, preferably "5-12 membered fused heteroaryl", "7-12 membered fused heteroaryl", "9-12 membered fused heteroaryl" and the like, such as benzofuranyl, benzisofuranyl, benzothiophenyl, indolyl, isoindole, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, phenolazinyl, pteridinyl, purinyl, naphthyridinyl, phenazine, phenothiazine and the like. "Fused heteroaryl ring" refers to the ring system within a fused heteroaryl group.

[0383] The fused heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0384] The term "alkoxy" refers to-O-(alkyl) and-O-(unsubstituted cycloalkyl), wherein the definition of alkyl is as described above. The limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0385] The term "alkylthio" refers to -S-(alkyl) and -S-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkylthio include: methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio. Alkylthio can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0386] The term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

[0387] The term "haloalkyl" refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.

[0388] The term "deuterated alkyl" refers to an alkyl group substituted with a deuterium atom, wherein alkyl is as defined above.

[0389] The term "hydroxy" refers to an -OH group.

[0390] The term "oxo" refers to a =0 group, for example, a carbon atom and an oxygen atom connected by a double bond, wherein a ketone or aldehyde group is formed.

[0391] The term "thio" refers to a =S group. For example, a carbon atom is linked to a sulfur atom via a double bond to form a thiocarbonyl group -C(S)-.

[0392] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0393] The term "amino" refers to -NH2.

[0394] The term "cyano" refers to -CN.

[0395] The term "nitro" refers to -NO2.

[0396] The term "carboxy" refers to -C(O)OH.

[0397] The term "aldehyde" refers to -CHO.

[0398] The term "carboxylate" refers to -C(O)O(alkyl) or -C(O)O(cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0399] The term "acyl halide" refers to a compound containing a -C(O)-halogen group.

[0400] The term "sulfonyl" refers to -S(O)(O)-.

[0401] The term "sulfinyl" refers to -S(O)-.

[0402] "Amino protecting group" is a suitable amino protecting group known in the art, see the literature ("Protective Groups in Organic Synthesis", 5 Th .Ed.TWGreene & P.GMWuts) in the amino protecting group, preferably, the amino protecting group can be (C 1-10 Alkyl or aromatic) acyl, for example: formyl, acetyl, benzoyl, etc.; can be (C 1-6 Alkyl or C 6-10 aryl)sulfonyl; can also be (C 1-6 Alkoxy or C 6-10 It can also be a substituted or unsubstituted alkyl group, such as trityl (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).

[0403] The term "transition metal" refers to elements of Groups 4-11, which are distinguished by their typical chemical characteristics, namely, complex ions with a wide range of oxidation states, colored complexes, and catalytic properties in either the elemental or ionic state (or both). Sc and Y of Group 3 are also generally considered transition metals.

[0404] The terms "buffer" and "buffer system" refer to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0405] A "histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, and histidine-sulfate buffers. A preferred histidine buffer is histidine-hydrochloride buffer. Histidine-hydrochloride buffer is a mixture of histidine and hydrochloric acid or histidine and histidine hydrochloride.

[0406] "Succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid histidine salt, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate buffer.

[0407] "Phosphate buffered saline," also known as PBS buffer, is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and the like. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0408] "Acetate buffer," also known as "acetate buffer," is a buffer containing acetate ions. Examples of acetate buffers include acetate-sodium acetate, histidine acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, and the like. A preferred acetate buffer is acetate-sodium acetate buffer.

[0409] The terms "about" and "approximately" refer to values ​​within an acceptable error range for a specific value as determined by one of ordinary skill in the art, which depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean a standard deviation within or exceeding 1. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%. In this disclosure, each case in which a number or numerical range is preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in this disclosure, the meaning of "about" or "substantially comprising" should be within an acceptable error range for that specific value.

[0410] The disclosed values ​​are instrumentally measured or calculated, and are subject to a certain degree of error. Generally speaking, a value within a reasonable error range of plus or minus 10% is considered. The context in which the value is used must be considered. For example, the total impurity content, where the error after measurement does not exceed plus or minus 10%, may be plus or minus 9%, plus or minus 8%, plus or minus 7%, plus or minus 6%, plus or minus 5%, plus or minus 4%, plus or minus 3%, plus or minus 2%, or plus or minus 1%, with plus or minus 5% being preferred.

[0411] The term "optionally" or "optionally" is intended to mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not.

[0412] The disclosed monoclonal antibody size variant determination method (CE-SDS) can employ capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) ultraviolet detection to quantitatively determine the purity of recombinant monoclonal antibody products based on molecular weight under reducing and non-reducing conditions according to capillary electrophoresis (Chinese Pharmacopoeia 2015 edition, 0542).

[0413] The term "pharmaceutical composition" refers to a mixture containing one or more compounds, conjugates, or physiologically / pharmaceutically acceptable salts or prodrugs thereof described herein, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. In this disclosure, the terms "pharmaceutical composition" and "preparation" are not mutually exclusive.

[0414] Pharmaceutical compositions can be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. Such suspensions can be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions prepared in a nontoxic, parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, sterile fixed oils can conveniently be used as solvents or suspending media. For this purpose, any blended fixed oil, including synthetic mono- or diglycerides, can be used. Furthermore, fatty acids, such as oleic acid, can also be used to prepare injectable formulations.

[0415] The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of the ligand-drug conjugates of the present disclosure, or a salt of a compound described herein, which salt is safe and effective when used in mammals and exhibits the desired biological activity. The antibody-drug conjugates of the present disclosure contain at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0416] The term "carrier" as used in the context of the present invention refers to a system that can alter the way a drug enters the human body and its distribution within the body, control the rate of drug release, and deliver the drug to a targeted organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, preferably micelles, microemulsions, gels, liquid crystals, vesicles, and the like. These aggregates have the ability to encapsulate drug molecules while also having good membrane permeability, making them excellent drug carriers.

[0417] The term "excipient" refers to any additive in a pharmaceutical preparation other than the main drug, also known as an adjuvant. Examples include binders, fillers, disintegrants, and lubricants in tablets; the base component of semisolid ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0418] The term "diluent," also known as filler, primarily increases the weight and volume of a tablet. The addition of a diluent not only maintains a certain volume, but also reduces dosage variations of the primary ingredient and improves the drug's compressibility. When the tablet contains an oily component, an absorbent is added to absorb the oil and maintain a "dry" state, facilitating tablet production.

[0419] The terms "connection" and "binding" are used interchangeably and, when referring to a connection between two molecules, refer to the connection of two molecules by a covalent bond or the association of two molecules via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). Connection includes direct connection and indirect connection, direct binding and indirect binding. The terms "direct connection" and "direct binding" refer to the connection of a first compound or group to a second compound or group without any intervening atoms or atomic groups. The terms "indirect connection" and "indirect binding" refer to the connection of a first compound or group to a second compound or group through an intermediate group, compound or molecule (e.g., a linking group). "Connection" covers the connection of amino acid residues by covalent bonding, including but not limited to amide bonds, disulfide bonds; methylene bonds (also known as methylene bridge connections), thioether bonds, hydrogen bonds and electrostatic bonds.

[0420] The terms "subject," "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0421] The term "effective amount" or "effective dose" refers to the amount of a drug, compound, conjugate, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that present during the progression of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various conditions associated with the disclosed target genes, target mRNAs, or target proteins, or ameliorating one or more symptoms of the condition, reducing the dose of another agent required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of a condition associated with the disclosed target genes, target mRNAs, or target proteins in a patient.

[0422] The term "antibody" refers to immunoglobulins. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Immunoglobulins are divided into five classes, or isotypes, based on the amino acid composition and order of arrangement of the constant regions of their heavy chains: IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Within the same class, Igs are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0423] The sequence of approximately 110 amino acids near the N-terminus of the full-length antibody heavy and light chains varies greatly and is the variable region (Fv region); the amino acid sequence near the C-terminus is relatively stable and is the constant region. The variable region includes three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also called complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments described in the present disclosure conform to the known IMGT rules.

[0424] In the embodiments of the present disclosure, the antibody may form a linker bond with the linker through a heteroatom on the antibody.

[0425] In this disclosure, the term "murine antibody" refers to antibodies prepared in mice according to the knowledge and skills in the art. During preparation, a test subject is injected with a specific antigen and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated.

[0426] The term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies. The variable region genes are then cloned from the mouse hybridoma cells. Furthermore, the constant region genes of the human antibody are cloned as needed. The mouse variable region genes and the human constant region genes are then linked to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0427] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the antibody variable region framework of a human, i.e., different types of human germline antibody framework sequences. This can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of mouse protein components they carry. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk / vbase), as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid a decrease in immunogenicity and a decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies after CDRs have been affinity matured by phage display. References further describing methods for humanizing mouse antibodies include, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and the methods of Winter and colleagues [Jones et al., Nature, 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].

[0428] The terms "fully human antibody," "whole human antibody," or "completely human antibody," also known as "fully human monoclonal antibody," refer to antibodies whose variable and constant regions are all human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. The main technologies related to human antibody production include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody production technology, and single B cell antibody production technology.

[0429] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments included in "antigen-binding fragments" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a single domain or dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) a combination of two or more isolated CDRs, optionally connected by a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulins. The antibodies can be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

[0430] Fab is an antibody fragment having a molecular weight of approximately 50,000 and antigen-binding activity, among fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which approximately half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.

[0431] F(ab')2 is an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin, and comprises two Fab regions linked at the hinge position.

[0432] Fab' is an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2.

[0433] Furthermore, the Fab' fragment of the antibody can be produced by inserting a DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab'.

[0434] "Fc" refers to the portion of an antibody consisting of the second constant region (CH2) and the third constant region (CH3) of the first heavy chain, which are bound to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc portion of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not play a role in antigen binding.

[0435] The "hinge region" of an antibody comprises a portion of a heavy chain molecule connecting the CH1 and CH2 domains. The hinge region comprises approximately 25 amino acid residues and is flexible, thereby allowing the two N-terminal antigen-binding regions to move independently.

[0436] The term "single-chain antibody", "single-chain Fv" or "scFv" refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example, variants using 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0437] The term "CDR" refers to one of the six hypervariable regions that primarily contribute to antigen binding in the variable domains of an antibody. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR is applied only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3, or L1, L2, L3) of a light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3, or H2, H3) of a heavy chain variable domain. Typically, three CDRs (HCDR1, HCDR2, HCDR3) are present in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) are present in each light chain variable region. The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), among others. For example, for the classical format, following the Kabat convention, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following the Chothia convention, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).By combining the CDR definitions of Kabat and Chothia, CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Following the IMGT rules, the CDR amino acid residues in VH are numbered approximately as 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately as 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). Following the IMGT rules, the CDR regions of antibodies can be determined using the program IMGT / DomainGap Align.

[0438] The term "antibody framework" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

[0439] The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-continuous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0440] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than approximately 10 -8 M, 10 -9 M or 10 -10 Binds with an affinity (KD) of M or less.

[0441] The antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into host cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the N-terminal site of the Fc region. Positive clones are expanded in culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antigen-binding fragments are detected using SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product must be immediately frozen, such as at -70°C, or freeze-dried.

[0442] In the present disclosure, the CAS No. of mc-vc-pab-MMAE is: 646502-53-6, and has the following structure: BRIEF DESCRIPTION OF THE DRAWINGS

[0443] Figure 1 shows the HIC of adalimumab-MMAE-01 conjugate.

[0444] Figure 2 shows the HIC of the adalimumab-MMAE-02 conjugate.

[0445] FIG3 is the HIC of adalimumab-MMAE-03 conjugate.

[0446] FIG4 is the HIC of adalimumab-MMAE-04 conjugate.

[0447] FIG5 is the HIC of adalimumab-MMAE-05 conjugate.

[0448] FIG6 is the HIC of adalimumab-MMAE-06 conjugate.

[0449] FIG7 is the HIC of adalimumab-MMAE-07 conjugate.

[0450] FIG8 is the HIC of the adalimumab-MMAE-08 conjugate prepared without the addition of ZnCl2.

[0451] Figure 9 is the HIC of Farletuzumab-MMAE-01 conjugate.

[0452] FIG10 is the HIC of the Farletuzumab-MMAE-02 conjugate prepared without the addition of ZnCl2.

[0453] FIG11 is the HIC of Enoblituzumab-MMAE-01 conjugate.

[0454] FIG12 is the HIC of the Enoblituzumab-MMAE-02 conjugate prepared without the addition of ZnCl2.

[0455] FIG13 is a HIC of adalimumab-4-B00 conjugate prepared using the disclosed process.

[0456] FIG14 is a HIC of adalimumab-4-B00 conjugate prepared using conventional technology. DETAILED DESCRIPTION

[0457] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0458] Example

[0459] Example 1, HIC-HPLC method 1

[0460] The yield and isomeric mixture of the antibody-drug conjugate obtained (e.g., for D4 conjugate) were determined using the HIC-HPLC method:

[0461] 1.1 Main equipment and mobile phase

[0462] High performance liquid chromatograph: Agilent 1260.

[0463] Chromatographic column: TSK gel Butyl-NPR, 4.6 mm × 3.5 cm, 2.5 μm, TOSOH.

[0464] Mobile phase: Mobile phase A (MPA): 20 mM PB + 1.5 M (NH4)2SO4 (pH 7.00).

[0465] Preparation example of mobile phase A: Weigh 1.22 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O, MW: 156.01 g / mol), 4.37 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O, MW: 358.14 g / mol), and 198.21 g of ammonium sulfate ((NH4)2SO4, MW: 132.14 g / mol), dissolve in 950 ml of purified water, adjust the pH to 7.00±0.05 with 5 M NaOH solution, dissolve in purified water to 1000 ml, and filter with a 0.22 μm filter membrane. Store at 2-8°C with a shelf life of 1 month. Filter before use.

[0466] Mobile phase B (MPB): 20 mM PB (pH 7.00) + 25% IPA

[0467] Preparation example of mobile phase B: Weigh 0.97 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O, MW: 156.01 g / mol) and 3.50 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O, MW: 358.14 g / mol), dissolve in 700 ml of purified water, adjust the pH to 7.00±0.05 with 5 M NaOH solution, add purified water to 750 ml, add 250 ml of isopropanol, and then filter with a 0.22 μm filter membrane. Store at 2-8°C. The shelf life is 1 month. Filter before use.

[0468] 1.2 Chromatographic methods and calculations

[0469] The chromatographic method is shown in Table 1.

[0470] Sample treatment: Take an appropriate amount of ADC sample, centrifuge at 12000 rpm for 1 min, take the supernatant, and dilute it with 50% mobile phase A to a final concentration of 2.0 mg / ml.

[0471] Calculation formula: DAR = ∑ (DARn peak area percentage (%) * n) / 100

[0472] Note: n is the DAR value of each component, which are 0, 2, 4, 6, and 8 respectively.

[0473] The sum of peak areas refers to the sum of the peak areas of each component (D0, D2, D4, D6 and D8).

[0474] Peak area percentage refers to: the antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof prepared by the present disclosure is tested by HIC-HPLC, and the sum of the areas of each chromatographic peak after deducting the blank solution is calculated by comparing the chromatogram with the blank solution. The chromatogram is integrated to obtain the total peak area, and the peak area of ​​each component (e.g., D0, D2, D4, D6, or D8) is calculated as a percentage of the total peak area using the area normalization method.

[0475] Table 1. Chromatographic method 1

[0476] Example 2, HIC-HPLC method 2

[0477] The yield and isomeric mixture of the antibody-drug conjugate obtained (e.g., for D4 conjugate) were determined using the HIC-HPLC method:

[0478] 2.1 Main equipment and mobile phase

[0479] High performance liquid chromatograph: Agilent 1260.

[0480] Chromatographic column: Sepax Protemomix HIC Butyl NP5, 4.6 mm × 100 mm, 5 μm, Sepax.

[0481] Mobile phase: Mobile phase A (MPA): 100 mM Citrate + 1.5 M (NH4)2SO4 (pH 5.00).

[0482] Preparation example of mobile phase A: Weigh 8.62g of citric acid monohydrate (C6H8O7·H2O, MW: 210.14g / mol), 17.35g of sodium citrate dihydrate (Na3C6H5O7·2H2O, MW: 294.10g / mol), and 198.21g of ammonium sulfate ((NH4)2SO4, MW: 132.14g / mol), dissolve in 800ml of purified water, adjust the pH to 5.00±0.05 with 1M NaOH solution, dissolve in purified water to 1000ml, and filter through a 0.22μm filter membrane. Store at 2-8°C with a shelf life of 1 month. Filter before use.

[0483] Mobile phase B (MPB): 100 mM Citrate (pH 5.00) + 20% IPA

[0484] Preparation example of mobile phase B: Weigh 6.90g of citric acid monohydrate (C6H8O7·H2O, MW: 210.14g / mol), 13.88g of sodium citrate dihydrate (Na3C6H5O7·2H2O, MW: 294.10g / mol), and 198.21g of ammonium sulfate ((NH4)2SO4, MW: 132.14g / mol), dissolve in 700ml of purified water, adjust the pH to 5.00±0.05 with 1M NaOH solution, add purified water to 800ml, add 200ml of isopropanol, and then filter with a 0.22μm filter membrane. Store at 2-8°C. The shelf life is 1 month. Filter before use.

[0485] 2.2 Chromatographic methods and calculations

[0486] The chromatographic method is shown in Table 2.

[0487] Sample treatment: Take an appropriate amount of ADC sample, centrifuge at 12000 rpm for 1 min, take the supernatant, and dilute it with 50% mobile phase A to a final concentration of 5.0 mg / ml.

[0488] Calculation formula: DAR = ∑ (DARn peak area percentage (%) * n) / 100

[0489] Note: n is the DAR value of each component, which are 0, 2, 4, 6, and 8 respectively.

[0490] The sum of peak areas refers to the sum of the peak areas of each component (D0, D2, D4, D6 and D8).

[0491] Peak area percentage refers to: the antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof prepared by the present disclosure is tested by HIC-HPLC, and the sum of the areas of each chromatographic peak after deducting the blank solution is calculated by comparing the chromatogram with the blank solution. The chromatogram is integrated to obtain the total peak area, and the peak area of ​​each component (e.g., D0, D2, D4, D6, or D8) is calculated as a percentage of the total peak area using the area normalization method.

[0492] Table 2. Chromatographic method 2

[0493] Example 3: Investigating the effect of quenching agent on the preparation method

[0494] 3.1 With reference to WO2020164561A1, adalimumab-MMAE samples were prepared: adalimumab-MMAE-01, adalimumab-MMAE-02, and adalimumab-MMAE-03. See Table 3 for reaction conditions, and Table 4 and Figures 1-3 for specific results.

[0495] The specific steps are as follows:

[0496] (1) ZnCl2 (0.270 mM) and TCEP (purchased from Aldrich Sigma, 0.540 mM) were added to a solution of adalimumab (Shanghai Maijin Biopharmaceutical Technology Co., Ltd., 0.135 mM) (PB buffer, pH = 6.5) and allowed to stand at 0-4°C overnight;

[0497] (2) mc-vc-pab-MMAE (purchased from Lianning Biotechnology, 1.350 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) was added and reacted in a water bath at room temperature for 1 h;

[0498] (3) According to Table 3, appropriate amounts of N-acetamidocysteine ​​(NAC, 1.620 mM, as a quencher), EDTA solution (0.540 mM, as a metal chelator), and sodium ascorbate (DHAA, 1.080 mM, as an oxidant) solution were added or not added to the system and reacted at room temperature for 30 min;

[0499] (4) The reaction solution was purified using ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) (to remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC) and then characterized using the HIC-HPLC method. The HIC-HPLC method was referenced to Example 1.

[0500] Table 3. Reaction conditions

[0501] Note: “√” represents that this reagent was added to the reaction, and “×” represents that this reagent was not added to the reaction.

[0502] Table 4. Results

[0503] The HIC results show that the selectivity of the process in Example 3.1 mainly exists in the coupling reaction stage between the antibody and the toxin. Therefore, the coupling temperature of this process has a significant effect on the coupling reaction results. When the coupling temperature is increased, the selectivity deteriorates (the D4 ratio of the adalimumab-MMAE-01 sample is significantly reduced compared with the adalimumab-MMAE-01 sample), and a significant increase in odd peaks can be seen. In addition, if a quencher is not used to react with the remaining toxin, after the EDTA solution is added, the unreacted toxin will continue to react with the unreacted sulfhydryl groups on the antibody, resulting in a loss of selectivity (the main component of the adalimumab-MMAE-03 sample changes from D4 to D6).

[0504] 3.2 After replacing the reducing agent TCEP with diphenylphosphinoacetic acid (DPA), the coupling experiment was repeated using the same process as in Section 3.1 to prepare adalimumab-MMAE samples: adalimumab-MMAE-04, adalimumab-MMAE-05, and adalimumab-MMAE-06. Reaction conditions are shown in Table 5, and the results are shown in Table 6 and Figures 4-6.

[0505] The specific steps are as follows:

[0506] (1) ZnCl2 (0.270 mM) and diphenylphosphinothioacetic acid (purchased from Yuhan Chemical, 0.390 mM) were added to a solution of adalimumab (0.135 mM) (histidine buffer, pH = 6.5) and allowed to stand at 0-4°C overnight;

[0507] (2) mc-vc-pab-MMAE (purchased from Lianning Biotechnology, 1.350 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) was added and reacted in a water bath at room temperature for 1 h;

[0508] (3) According to Table 5, an appropriate amount of N-acetamidocysteine ​​(NAC, 1.620 mM, as a quencher) was added to the system, and EDTA solution (0.540 mM, as a metal chelator) and sodium ascorbate (DHAA, 1.080 mM, as an oxidant) solution were added and reacted at room temperature for 30 min;

[0509] (4) The reaction solution was purified using ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) (to remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC) and then characterized using the HIC-HPLC method. The HIC-HPLC method was referenced to Example 1.

[0510] Table 5. Process parameters

[0511] Note: “√” represents that this reagent was added to the reaction, and “×” represents that this reagent was not added to the reaction.

[0512] Table 6. Results

[0513] The HIC results in Table 6 show that after replacing the reducing agent with diphenylphosphinoacetic acid, the proportion of the D4 part is maintained, and the coupling reaction temperature has no effect on the reaction selectivity. At the same time, not quenching the reaction will not significantly affect the selectivity of the process.

[0514] From the results in Tables 4 and 6, it can be seen that the selectivity of the disclosed process mainly exists in the reduction stage of the antibody, which is different from the selective coupling process in Example 3.1. In addition, no reaction quenching reagent is required, and the coupling reaction is not limited by conditions such as temperature.

[0515] Example 4. Characterization of the prepared adalimumab-MMAE conjugate and its drug distribution

[0516] Based on Example 3.1, the reoxidation step was further omitted to optimize the preparation method to prepare adalimumab-MMAE-07. Specific results are shown in Table 7 and Figure 7.

[0517] The specific steps are as follows:

[0518] (1) ZnCl2 (0.270 mM) and diphenylphosphinoacetic acid (purchased from Yuhan Chemical, 0.412 mM) were added to a solution of adalimumab (0.135 mM) (histidine buffer, pH = 6.5) and allowed to stand at 0-4°C overnight;

[0519] (2) Add EDTA (0.540 mM) solution to chelate Zn 2+ ion;

[0520] (3) mc-vc-pab-MMAE (purchased from Lianning Biotechnology, 0.810 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) was added and reacted in a water bath at room temperature for 1 h;

[0521] (4) Purify the reaction solution using ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) to remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC.

[0522] Drug distribution characterization: HIC-HPLC was used to analyze drug-antibody ratio (DAR) and drug distribution. The HIC-HPLC method was described in Example 1.

[0523] Table 7. Results

[0524] Example 5. Preparation of Farletuzumab-MMAE Conjugate and Characterization of Its Drug Distribution

[0525] Based on Example 3.1, the reoxidation step was further omitted to optimize the preparation method. Farletuzumab-MMAE-01 was prepared. Specific results are shown in Table 8 and Figure 9. Farletuzumab is a humanized anti-human folate receptor alpha (FRA) antibody.

[0526] The specific steps are as follows:

[0527] (1) ZnCl2 (0.276 mM) and diphenylphosphinoacetic acid (purchased from Yuhan Chemical, 0.469 mM) were added to a Farletuzumab (Shanghai Maijin Biopharmaceutical Technology Co., Ltd., 0.138 mM) solution (histidine buffer, pH = 6.5) and allowed to stand at 0-4°C overnight;

[0528] (2) Add EDTA (0.552 mM) solution to chelate Zn 2+ ion;

[0529] (3) mc-vc-pab-MMAE (purchased from Lianning Biotechnology, 0.828 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) was added and reacted in a water bath at room temperature for 1 h;

[0530] (4) The reaction solution was purified using ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) to remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC. Drug distribution characterization: Drug-antibody ratio (DAR) and drug distribution were analyzed using HIC-HPLC. The HIC-HPLC method was similar to that in Example 1.

[0531] Table 8. Results

[0532] Example 6. Characterization of the Prepared Enoblituzumab-MMAE Conjugate and Its Drug Distribution

[0533] Based on Example 3.1, the reoxidation step was further omitted to optimize the preparation method. Enoblituzumab-MMAE-01 was prepared. Specific results are shown in Table 9 and Figure 11. Enoblituzumab is an anti-B7H3 antibody.

[0534] The specific steps are as follows:

[0535] (1) ZnCl2 (0.278 mM) and diphenylphosphinoacetic acid (purchased from Yuhan Chemical, 0.390 mM) were added to a solution of Enoblituzumab (Shanghai Maijin Biopharmaceutical Technology Co., Ltd., 0.139 mM) (histidine buffer, pH = 6.5) and allowed to stand at 0-4°C overnight;

[0536] (2) Add EDTA (0.556 mM) solution to chelate Zn 2+ ion;

[0537] (3) mc-vc-pab-MMAE (purchased from Lianning Biotechnology, 0.834 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) was added and reacted in a water bath at room temperature for 1 h;

[0538] (4) The reaction solution was purified using ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) to remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC. Drug distribution characterization: Drug-antibody ratio (DAR) and drug distribution were analyzed using HIC-HPLC. The HIC-HPLC method was similar to that in Example 1.

[0539] Table 9. Results

[0540] According to the results in Tables 7-9, adding a metal chelating agent after the reduction reaction is completed can further omit the reoxidation step and has almost no effect on the selectivity of the reaction.

[0541] Example 7: Investigating the influence of transition metal ions on the preparation method

[0542] The effect of not adding ZnCl2 on the preparation method was investigated. The preparation method of the sample with ZnCl2 added was referred to Examples 4-6.

[0543] Without adding ZnCl2, samples Adalimumab-MMAE-08 (without ZnCl2), Farletuzumab-MMAE-02 (without ZnCl2), and Enoblituzumab-MMAE-02 (without ZnCl2) were prepared. For specific results, see Table 10 and Figures 8, 10, and 12. The only difference from Examples 4-6 is that no ZnCl2 was added.

[0544] Without adding ZnCl2, the specific steps are as follows:

[0545] (1) Add an appropriate amount of diphenylphosphinoacetic acid (purchased from Yuhan Chemical, the concentration is the same as in Examples 4-6) to the antibody solution to be reduced (the antibody type and concentration are the same as in Examples 4-6), and let it stand at 0-4°C overnight;

[0546] (2) mc-vc-pab-MMAE (purchased from Lianning Biotechnology, the concentration is the same as in Examples 4-6) dissolved in 100% DMSO (purchased from Aldrich Sigma) was added and reacted in a water bath at room temperature for 1 h;

[0547] (3) The reaction solution was purified using ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) to remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC. Drug distribution characterization: Drug-antibody ratio (DAR) and drug distribution were analyzed using HIC-HPLC. The HIC-HPLC method was described in Example 1.

[0548] Table 10. Results

[0549] The HIC results in Table 10 show that the introduction of ZnCl2 can significantly improve the selectivity of the reduction reaction compared with using only the reducing agent DPA.

[0550] Example 8. Comparison of Adalimumab-4-B00 Conjugates Prepared Using the Disclosed Process and Traditional Processes and Their Drug Distribution

[0551] Adalimumab-4-B00 conjugates were prepared using the disclosed process and the traditional process (using TCEP as a reducing agent), respectively, wherein Adam represents adalimumab and n represents DAR.

[0552] 8.1 The specific steps of the disclosed process are as follows:

[0553] (1) ZnCl2 (0.270 mM) and diphenylphosphinoacetic acid (purchased from Yuhan Chemical, 0.412 mM) were added to a solution of adalimumab (0.135 mM) (histidine buffer, pH = 6.5) and allowed to stand at 0-4°C overnight;

[0554] (2) Add EDTA (0.540 mM) solution to chelate Zn 2+ ion;

[0555] (3) Add Tris to adjust the reaction pH to around 8.0;

[0556] (4) 4-B00 (0.81 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) was added and reacted in a water bath at room temperature for 3 h;

[0557] Among them, 4-B00 was prepared with reference to WO2022166779A1, and its structure is:

[0558] (5) The reaction solution was purified using ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) to remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC. The drug-antibody ratio (DAR) and drug distribution were analyzed using HIC-HPLC, using the same method as in Example 2. The results are shown in Table 11 and Figure 13.

[0559] 8.2 The specific steps of the traditional process are as follows:

[0560] (1) Tris(2-carboxyethyl)phosphine (TCEP, purchased from Aldrich Sigma, 0.297 mM) dissolved in water was added to a solution of adalimumab (0.135 mM) (histidine buffer, pH = 6.5) and reacted at room temperature for 2 h;

[0561] (2) Add Tris to adjust the reaction pH to around 8.0;

[0562] (3) 4-B00 (0.81 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) was added and reacted in a water bath at room temperature for 3 h;

[0563] (4) The reaction solution was purified using ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) to remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC. The drug-antibody ratio (DAR) and drug distribution were analyzed using HIC-HPLC, using the same method as in Example 2. The results are shown in Table 11 and Figure 14.

[0564] Table 11

[0565] The HIC results in Table 11 indicate that, compared with the conventional coupling process using TCEP as a reducing agent, the present disclosure can significantly improve the selectivity of the reaction and increase the proportion of the D4 component.

[0566] Although the above invention has been described in detail with the aid of drawings and examples for clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. A method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, comprising the following steps: (a) reacting a reducing agent with an antibody in the presence of a transition metal ion to selectively reduce interchain disulfide bonds within the antibody to sulfhydryl groups; (b) reacting the antibody having a thiol group obtained in step (a) with a drug linker intermediate or a pharmaceutically acceptable salt thereof; (c) Obtaining an antibody-drug conjugate or a pharmaceutically acceptable salt thereof by performing step (b) without the quenching step and / or the reoxidation step.

2. The method according to claim 1, wherein the transition metal ion in step (a) is selected from Zn 2+ 、Cd 2+ 、Hg 2+ or a combination thereof; preferably selected from Zn 2+ .

3. The method according to claim 1 or 2, wherein the reducing agent in step (a) is a reducing agent containing a diphenylphosphino group, or a salt thereof; preferably selected from diphenylphosphinoacetic acid, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(diphenylphosphino)propionic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, or a salt thereof; more preferably selected from diphenylphosphinoacetic acid, or a salt thereof.

4. The method according to any one of claims 1 to 3, wherein step (a) is carried out at -10°C to 37°C; preferably at 0°C to 30°C; more preferably at 0°C to 25°C.

5. The method according to any one of claims 1 to 4, wherein the step of adding a metal chelating agent is further included between step (a) and step (b); preferably, the metal chelating agent is selected from ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetic acid, dicalcium ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid or a mixture thereof. 6 . The method according to claim 1 , wherein the drug-linker intermediate or a pharmaceutically acceptable salt thereof contains a reactive group capable of reacting with a sulfhydryl group.

7. The method according to any one of claims 1 to 6, wherein the obtained antibody-drug conjugate or a pharmaceutically acceptable salt thereof has an average drug loading of 3.0 to 5.0; preferably greater than 3.0 and less than 5.

0.

8. The method according to any one of claims 1 to 7, wherein the antibody is selected from a monoclonal antibody or a polyclonal antibody.

9. The method according to any one of claims 1 to 8, wherein the antibody is selected from a murine antibody, a fully human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof.

10. The method according to any one of claims 1 to 9, wherein the antibody is selected from the IgG1 or IgG4 isotype.

11. The method according to any one of claims 1 to 10, wherein the drug is selected from a diagnostic agent, a therapeutic agent or a labeling agent. 12 . An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, prepared by the method according to claim 1 . 13 . A pharmaceutical composition comprising an effective amount of the antibody-drug conjugate according to claim 12 or a pharmaceutically acceptable salt thereof, and optionally an excipient, diluent or carrier.

14. A method for treating and / or preventing a condition or disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate according to claim 12 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 13; preferably, the condition or disease is selected from a tumor, a cancer, an autoimmune disease, or an infectious disease; more preferably, the autoimmune disease is selected from rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, hidradenitis suppurativa, uveitis, Behçet's disease, spondyloarthropathy, and psoriasis.

15. A method for selectively reducing an antibody, comprising step (a): reacting a reducing agent with an antibody in the presence of a transition metal ion to selectively reduce interchain disulfide bonds within the antibody to sulfhydryl groups.

16. The method for selectively reducing antibodies according to claim 15, wherein step (a) is as defined in any one of claims 2 to 10.

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