TW202411249A - Preparation method of antibody-drug conjugates - Google Patents

Abstract

The present disclosure provides a preparation method for antibody-drug conjugates. Specifically, the present disclosure provides a method for preparing an antibody-drug conjugates (ADCs) with improved homogeneity. Compared with the conventional method, the method of the present disclosure has simple process, does not require reaction quenching and oxidation steps, and coupling reaction is not limited by temperature and other conditions.

Description

Method for preparing antibody-drug conjugates

This disclosure claims the priority of Chinese patent application No. 202210553520.X filed on May 20, 2022, and this disclosure quotes the full text of the above Chinese patent application.

The present disclosure relates to a method for specific reduction of an antibody and a method for preparing an antibody-drug conjugate (ADC), as well as an antibody-drug conjugate (ADC) with improved uniformity.

Antibody drug conjugates (ADCs) are obtained by linking antibodies or antigen-binding fragments to biologically active toxins through stable chemical linker compounds. They have both the powerful lethality of small molecule drugs and the specificity of antibodies. They can have the specificity of antibodies binding to surface antigens of normal cells and tumor cells and the high efficiency of cytotoxins, while avoiding the defects of low efficacy of antibodies and excessive toxic side effects of cytotoxins. Antibody-drug conjugates can precisely bind to tumor cells, reduce the impact on normal cells, and thus reduce adverse reactions during treatment (Mullard A, (2013) Nature Reviews Drug Discovery, 12: 329-332; Di Joseph JF, Armellino DC, (2004) Blood, 103: 1807-1814).

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

ADC drugs often use two chemical strategies to link antibodies and toxins. One is based on the coupling of lysine on the antibody, such as the marketed drugs Mylotarg, Kadcyla, and Besponsa. They all use the succinimide group of the linker to bind to the amine group of the lysine side chain to connect the toxin to the single antibody. An antibody molecule contains 80 to 90 lysines, and the coupling may occur on nearly 40 different lysine residues, so the coupling sites are multiple and not fixed. The other is based on the coupling of cysteine on the antibody. Compared with the stable intrachain disulfide bonds on the antibody, the interchain disulfide bonds are easily partially reduced to free sulfhydryl groups, which then bind to the linker's linker group (such as the maleimide group). Both IgG1 and IgG4 antibodies contain four pairs of interchain disulfide bonds. Under appropriate reducing agent reduction and coupling, ADC drugs with a drug-to-antibody ratio (DAR) of 0-8 can be obtained, such as Adcetris, Polivy, Padcev and other seven drugs. Therefore, compared with lysine coupling, cysteine coupling contains fewer coupling sites and has become the preferred connection strategy for random coupling due to its simple coupling process.

However, the current cysteine technology still has shortcomings. For example, the ADC components of ADC samples with an average value of DAR 4 show a normal distribution, making the ADC drugs non-uniform. In addition, ADC samples with an average value of DAR4 also contain a certain proportion of ADC components connected to 6 and 8 toxins, making the ADC drugs easier to be cleared from the body (J Y, et al., 2021, Bioconjugate Chem, 18; 32(8): 1525-1534).

With the development of protein engineering technology, in order to obtain uniform and highly stable ADC drugs, a variety of technologies have been developed to try to introduce cysteine into the antibody structure, thereby introducing a hydroxyl group, and then realizing the site-specific coupling of the antibody and the drug molecule. For example, a literature pointed out that inserting cysteine after the 239th amino acid of the antibody recombinant chain, introducing a free hydroxyl group and coupling it with the toxin can obtain 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 mismatching, thereby affecting the stability of the ADC drug.

In addition, some scholars have chosen to mutate cysteine at specific sites on the antibody. The mutated antibody contains free cysteine 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 structure and function of the antibody, 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).

WO2020164561A1 relates to a method for preparing ADC, using TCEP as a reducing agent, using zinc ions to form chemical coordination bonds with the alkyl groups in the hinge region of the antibody, thereby increasing the probability of the reduced alkyl groups in the Fab region to bind to the toxin, and the method significantly increases the proportion of the DAR 4 component. However, in this method, because the excessive use of reducing agents in the reduction stage leads to a large amount of opening of the interchain disulfide bonds, the toxin quenching and reoxidation steps must be performed 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 ADC based on cysteine coupling.

The present disclosure provides a method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, and prepares an ADC with improved uniformity. The preparation method is simple in process, 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 hydroxyl 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, and has optimized safety and efficacy.

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

(a) reacting the reducing agent and the antibody in the presence of transition metal ions in a buffer system to selectively reduce the intrachain disulfide bonds of the antibody to hydroxyl groups;

(b) reacting the hydroxylated antibody obtained in step (a) with a drug linker intermediate or a pharmaceutically acceptable salt thereof.

In some embodiments, step (c) may or may not be included: 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.

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:

(a) reacting the reducing agent and the antibody in a buffer system in the presence of an effective amount of transition metal ions to selectively reduce the intrachain disulfide bonds of the antibody to hydroxyl groups;

(b) reacting the hydroxylated antibody obtained in step (a) with a drug linker intermediate or a pharmaceutically acceptable salt thereof;

(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.

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:

(a) reacting the reducing agent and the antibody in the presence of transition metal ions to selectively reduce the intrachain disulfide bonds of the antibody to hydroxyl groups;

(b) reacting the hydroxylated antibody obtained in step (a) with a drug linker intermediate or a pharmaceutically acceptable salt thereof;

(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.

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

The reoxidation step refers to adding an effective amount of an oxidizing agent (such as DHAA) to reoxidize the unreacted hydroxyl groups on the antibody after reduction.

In some embodiments, the transition metal ion in step (a) 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⁺ .

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.

In some embodiments, suitable zinc salts include, but are not limited to, ZnCl ₂ , Zn(NO ₃ ) ₂ , ZnSO ₄ , Zn(CH ₃ COO) ₂ , ZnI ₂ , ZnBr ₂ , zinc formate, and zinc tetrafluoroborate.

In some embodiments, in step (a), a suitable transition metal salt that is soluble and can release free Cd ²⁺ or Hg ²⁺ ions is added, including but not limited to: CdCl ₂ , Cd(NO ₃ ) ₂ , CdSO ₄ , Cd(CH ₃ COO) ₂ , CdI ₂ , CdBr ₂ , cadmium formate and cadmium tetrafluoroborate; HgCl ₂ , Hg(NO ₃ ) ₂ , HgSO ₄ , Hg(CH ₃ COO) ₂ , HgBr ₂ , mercury (II) formate, and mercury (II) tetrafluoroborate, etc.

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),

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.

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 0.13mM, about 0.14mM, about 0.15mM, about 0.20mM, about 0.25mM, about 0.30mM, about 0.35mM, about 0.40mM, about 0.45mM, about 0.50mM, or any value or range between any two values; in some embodiments, the final concentration of the antibody is selected from about 0.10mM to about 0.20mM. In some embodiments, the final concentration of the antibody is selected from about 0.12mM to about 0.14mM.

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 0.25mM, about 0.26mM, about 0.27mM, about 0.28mM, about 0.29mM, about 0.30mM, about 0.31mM, about 0.32mM, about 0.33mM, about 0.34mM, about 0.35mM, about 0.40mM, about 0.45mM, about 0.50mM, or any value or range between any two values; in some embodiments, the final concentration of the transition metal ion is selected from about 0.27mM to about 0.28mM.

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

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, such as 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 between any two values. 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.

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, such as 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 between any two values. In some embodiments, the final concentration equivalent ratio (mM/mM) of the antibody to the transition metal ion is about 1:2.

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.

In some embodiments, the buffer system used in step (a) depends on transition metal ions.

In some embodiments, the buffer system contains or does not contain a metal chelator. In some embodiments, the buffer does not contain a metal chelator.

In some embodiments, the pH of the buffer system used in step (a) is selected from about 4 to about 10, such as 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.

In some embodiments, step (a) is performed at about -10°C to about 40°C, such as 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 between any two values; in some embodiments, at about -5°C to about 37°C; in some embodiments, In some embodiments, the temperature is about 0°C to about 37°C; in some embodiments, the temperature is about 0°C to about 4°C; in some embodiments, the temperature is about 0°C to about 37°C; in some embodiments, the temperature is about 10°C to about 25°C; in some embodiments, the temperature is about -5°C to about 5°C; in some embodiments, the temperature is about 0°C to 30°C; in some embodiments, the temperature is about 0°C to 25°C.

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

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

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 a person 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 transition metal ions.

In some embodiments, the method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof disclosed herein further includes a step of adding a metal chelator between step (a) and step (b).

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

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, ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid dicalcium salt, diethylenetriaminepentaacetic acid or a mixture thereof.

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, such as about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, or any value or range between any two values. In some embodiments, the final concentration equivalent ratio (mM/mM) of the transition metal ion to the metal chelator is about 1:2.

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 styrene of the 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 the styrene group; in some embodiments, the drug linker intermediate or its pharmaceutically acceptable salt contains a maleimide group (such as an N-substituted maleimide group), bromine, iodine, fluorine, alkene, allene or sulfonyl group.

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

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:

in,

或

，該環A視需要被一個或多個取代基Q₁所取代； Ring A is

or

, the ring A is optionally substituted by one or more substituents Q ₁ ;

或

，該環B視需要被一個或多個取代基Q₁所取代； Ring B is

or

, the ring B is optionally substituted by one or more substituents Q ₁ ;

X ₁ is -(CR _5a R _5b )m- or a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group which is optionally substituted by one or more substituents Q ₁ , wherein the heteroaryl group contains at least one nitrogen atom;

R _5a and R _5b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, cyano and the following groups which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k and C ₁ -C ₆ alkoxy, or R _5a and R _5b together form a pendoxy group or a thioketone group;

、

、

、

和

，該環C視需要被一個或多個取代基Q₁所取代； Ring C selected from

,

,

,

and

, the ring C is optionally substituted by one or more substituents Q ₁ ;

或

，該環D視需要被一個或多個取代基Q₁所取代； Ring D is

or

, the ring D is optionally substituted by one or more substituents Q ₁ ;

_X2 is selected from -( _CR6aR6b )n-, -O-, -S-, _{-NR6c- ,} -CH2S-, _-CH2O- , _-NHCR6dR6e- and _a 6- to 10-membered _aryl group or a 5- to 10-membered heteroaryl group which is optionally substituted with one or more substituents _Q1 ;

R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, cyano and the following groups which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k and C ₁ -C ₆ alkoxy;

R _6c , R _6d and R _6e are each independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ halogenalkyl and C ₁ -C ₆ alkoxy;

、 _R2 is each independently selected from _{-CH2OH , -CH2SH , -CH2Cl ,} -SCH2Cl _, -SCH2F, -SCH2CF3, -OH, _-OCH2CN , -OCH2Cl _{, -OCH2F} , _-OCH3 , _-OCH2CH3 , _{-SCH2CN ,}

,

R _2a is hydrogen or C ₁ -C ₆ alkyl;

R _2b is C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy;

R _2c is selected from hydrogen, C ₁ -C ₆ alkyl, -CH ₂ OH and C ₁ -C ₆ alkoxy;

R _2d and R _2e are each independently hydrogen or C ₁ -C ₆ alkyl;

R ₃ are each independently hydrogen or a halogen;

m and n are each independently integers from 1 to 6;

The substituent groups _Q1 are each independently selected from halogen, hydroxyl, oxirane, deuterium, oxo _, thioketone, cyano, amine, carboxyl, _C1 - _C6 alkyl and C1- _C6 alkoxy groups;

R _i and R _j are each independently selected from a hydrogen atom, a hydroxyl group, a C ₁ -C ₆ alkyl group and a C ₁ -C ₆ alkoxy group;

R _k is independently selected from a hydrogen atom, a C ₁ -C ₆ alkyl group, a C ₁ -C ₆ halogenalkyl group, a C ₁ -C ₆ alkoxy group, a hydroxyl group and -NR _i R _j ;

R _1a are each independently selected from hydrogen, C ₁ -C ₆ alkyl and C ₁ -C ₆ alkoxy;

、

和 R _1b are each independently selected from hydrogen, PG-, HL ₁ -, PG-L ₁ -,

,

and

p is independently 1, 2, 3, 4, 5 or 6;

； _L1 is an amino acid unit, preferably -glycine-glutamic acid- or

;

X is a halogen;

PG is an amine protecting group; and

Provided that when _R5a is hydrogen or alkyl, _R5b is not hydrogen or alkyl.

In some specific embodiments, the drug linker intermediate is selected from:

，或其藥學上可接受的鹽。

, or a pharmaceutically acceptable salt thereof.

In some specific embodiments, the drug linker intermediate is selected from:

，或其藥學上可接受的鹽；一 些更具的實施方案中，X為鹵素，一些更具的實施方案中，X為氯或溴，一些更具的實施方案中，X選溴。

, or a pharmaceutically acceptable salt thereof; in some more preferred embodiments, X is a halogen, in some more preferred embodiments, X is chlorine or bromine, in some more preferred embodiments, X is bromine.

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).

(q為1-6的整數)的胺 基酸構成的肽殘基，示例性胺基酸單元包括但不限於纈胺酸-瓜胺酸(Val-Cit)、丙胺酸-苯丙胺酸(Ala-Phe)、苯丙胺酸-賴胺酸(Phe-Lys)、苯丙胺酸-高賴胺酸(Phe-Homolys)、N-甲基-纈胺酸-瓜胺酸(Me-Val-Cit)、丙胺酸-丙胺酸(Ala-Ala)、甘胺酸-谷胺酸(Gly-Glu)、谷胺酸-丙胺酸-丙胺酸(Glu-Ala-Ala)、甘胺酸-賴胺酸(Gly- Lys)、甘胺酸-纈胺酸-瓜胺酸(Glv-Val-Cit)和甘胺酸-甘胺酸-甘胺酸(Gly-Gly-Gly) In some embodiments, the linker comprises an amino acid unit _L1 , and the amino acid unit _L1 preferably comprises 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamate, aspartic acid, homolysine, N-methyl-valine,

The peptide residue is composed of amino acids (q is an integer of 1-6), 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-valeric acid-citrulline (Me-Val-Cit), alanine-alanine (Ala-Ala), glycine-glutamate (Gly-Glu), glutamate-alanine-alanine (Glu-Ala-Ala), glycine-lysine (Gly-Lys), glycine-valeric acid-citrulline (Glv-Val-Cit) and glycine-glycine-glycine (Gly-Gly-Gly)

In certain embodiments, the linker comprises a stretching unit, which is a chemical structure fragment covalently linked to the antibody via a carbon atom at one end and linked 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:

In some embodiments, the stretching unit is selected from:

、

和

，其中p各自獨立地為1、2、3、4、5 或6。

,

and

, where p is independently 1, 2, 3, 4, 5 or 6.

In some embodiments, the connector is selected from:

In some embodiments, the connector is selected from:

In some embodiments, the connector is selected from:

Depending on the desired drug and the selected linker, one of ordinary skill 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 by covalent attachment, such as a drug-maleimide intermediate (i.e., a maleimide-linked drug).

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, such as 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.

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

Examples of solvents that can be used to dissolve drug linker intermediates in the present disclosure include organic solvents, such as 50% acetone aqueous solution, 80% ethanol aqueous solution, 80% methanol aqueous solution, 80% isopropanol aqueous solution, 80% dimethyl sulfoxide aqueous solution, 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.

In some embodiments, step (b) is performed at about -10°C to about 40°C, such as 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 between any two values; 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.

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

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

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

A person with ordinary knowledge in the art can select an appropriate purification method to recover the obtained antibody-drug conjugate or its pharmaceutically acceptable salt. Many ADC purification methods are known in the art. For example, the obtained antibody-drug conjugate or its pharmaceutically acceptable salt can be purified by using a desalting column, size exclusion chromatography, ultrafiltration, dialysis, UF-DF, etc.

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%. , higher than about 69wt%, higher than about 70wt%, higher than about 71wt%, higher than about 72wt%, higher than about 73wt%, higher than about 74wt%, higher than about 75wt%; in some embodiments, based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the disclosed method contains D4 in an amount selected from about 55wt% to about 75wt%, about 60wt% to about 70wt%, about 55wt% to about 65wt%.

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

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the disclosed method 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 inter-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 disclosed method comprises D4a in an amount 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%, higher than about 76 wt%, higher than about 77 wt%, higher than about 78 wt%, higher than about 79 wt%, higher than about 80 wt%, higher than about 81 wt%, higher than about 82 wt%, higher than about 83 wt%, higher than about 84 wt%, higher than about 85 wt%, higher than about 86 wt%, higher than about 87 wt%, higher than about 88 wt%, higher than about 89 wt%, higher than about 90 wt%, higher than about 91 wt%, higher than about 92 wt%, higher than about 93 wt%, higher than about 94 wt%, higher than about 95 wt%, higher than about 96 wt%, higher than about 97 wt%, higher than about 98 wt%, higher than about 99 About 69wt%, higher than about 70wt%, higher than about 71wt%, higher than about 72wt%, higher than about 73wt%, higher than about 74wt%, higher than about 75wt%; In some embodiments, based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the disclosed method contains D4a in an amount selected from about 55wt% to about 75wt%, about 60wt% to about 70wt%, about 55wt% to about 65wt%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the disclosed method comprises D4b, wherein D4b 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 a heavy-heavy chain inter-chain bond. 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 40wt%, such as less than about 40wt%, less than about 30wt%, less than about 20wt%, less than about 15wt%, less than about 10wt%, less than about 5wt%; 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 5wt% to about 30wt%, about 5wt% to about 20wt%, about 5wt% to about 10wt%.

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 disclosed method contains less than about 20 wt% of D0+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 D0, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the disclosed method contains D0+D8 in an amount selected from about 3wt% to about 20wt%, about 5wt% to about 15wt%, and about 3wt% to about 10wt%.

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 D6 in an amount 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 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 D0, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the disclosed method contains D6 in an amount selected from about 5wt% to about 20wt%, about 5wt% to about 15wt%, and about 5wt% to about 10wt%.

In some embodiments, 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 coupling reaction.

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 the coupling reaction after purification and removal of small molecule process impurities (e.g., free toxins and organic solvents, etc.), wherein the purification step will not affect the different drug-loaded components of the prepared ADC.

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 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%,; In some embodiments, based on the sum of peak areas, the peak area percentage of D4 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the disclosed method is selected from about 55% to about 75%, about 60% to about 70%, about 55% to about 65%.

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

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the disclosed method 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 a styryl group 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 a pharmaceutically acceptable salt thereof prepared by the disclosed method 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 its pharmaceutically acceptable salt prepared by the disclosed method is selected from about 55% to about 75%, about 60% to about 70%, about 55% to about 65%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the disclosed method comprises D4b, wherein D4b 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 a heavy-heavy chain inter-chain bond. 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%, such as less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, 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%, about 5% to about 10%.

In some embodiments, based on the total peak area, the peak area percentage of D0+D8 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the disclosed method 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 total peak area, the peak area percentage of D0+D8 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the disclosed method is selected from about 3% to about 20%, about 5% to about 15%, about 3% to about 10%.

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 disclosed method 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 disclosed method is selected from about 5% to about 20%, about 5% to about 15%, about 5% to about 10%.

Based on the total peak area, the peak area percentage means: the antibody-drug conjugate (ADC) or its pharmaceutically acceptable salt prepared in the present disclosure is detected by the HIC-HPLC method, and the sum of the areas of each chromatographic peak after deducting the blank solution is calculated by comparing with the chromatogram of the blank solution, and the chromatogram is integrated to obtain the total peak area. The peak area of each component (such as D0, D2, D4, D6 or D8) is calculated by the area normalization method. The percentage of the peak area of the total peak area.

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

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

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%, 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 11 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, 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 its pharmaceutically acceptable salt prepared by the disclosed method is selected from about 55% to about 75%, about 60% to about 70%, about 55% to about 65%.

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 its pharmaceutically acceptable salt prepared by the disclosed method is greater than the content of D4 obtained using TCEP.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the disclosed method comprises D4a, wherein D4a is selected from an antibody-drug conjugate or its pharmaceutically acceptable salt having only 4 drug linkers, and the 4 drug linkers are bound to the heavy-light chain inter-group. 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 its pharmaceutically acceptable salt prepared by the disclosed method 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%, 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 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, 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 its pharmaceutically acceptable salt prepared by the disclosed method is selected from about 55% to about 75%, about 60% to about 70%, about 55% to about 65%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the disclosed method comprises D4b, wherein D4b 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 a heavy-heavy chain inter-chain bond. 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%, such as less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, 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%, about 5% to about 10%.

In some embodiments, based on the sum of the peak areas of D0, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the disclosed method 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%. 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 disclosed method is selected from about 3% to about 20%, about 5% to about 15%, about 3% to about 10%.

In some embodiments, based on the sum of the peak areas of D0, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the disclosed method contains 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%. 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 disclosed method is selected from about 5% to about 20%, about 5% to about 15%, about 5% to about 10%.

Based on the sum of the peak areas of D0, D2, D4, D6 and D8, the peak area percentage means: the antibody-drug conjugate (ADC) or its pharmaceutically acceptable salt prepared in the present disclosure 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 with the chromatogram of the blank solution, and the sum of the peak areas based on D0, D2, D4, D6 and D8 is obtained by integrating the chromatogram, and the peak area percentage of each component (such as D0, D2, D4, D6 or D8) is calculated by the area normalization method.

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

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

In some embodiments, the sum of the peak areas of D0, D2, D4, D6 and D8 of the antibody-drug conjugate or its pharmaceutically acceptable salt 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.

In some embodiments, the sum of the peak areas of D0, D2, D4, D6 and D8 of the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method disclosed herein refers to the sum of the peak areas of D0, D2, D4, D6 and D8 obtained after the conjugation reaction after purification and removal of 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.

In some embodiments, the average drug loading of the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the disclosed method is selected from about 3.0 to about 5.0, about 3.5 to about 4.5, such as 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.

In some embodiments, the antibody-drug conjugate (ADC) disclosed herein is selected from:

Ab-(LD) _k (I)

Among them, Ab is an antibody or its antigen-binding fragment, and L-D is a drug linker intermediate;

L is a linker that covalently connects 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),

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

in,

表示單鍵或雙鍵；

Indicates single or double keys;

R _1a is selected from hydrogen, alkyl and alkoxy, and the alkyl and alkoxy are each independently and optionally substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl and hydroxyalkyl;

Ring A is an aryl group or a heteroaryl group which is optionally substituted by one or more substituents Q ₁ ;

Ring B is an aryl group or a heteroaryl group which is optionally substituted by one or more substituents Q ₁ ;

X ₁ is -(CR _5a R _5b ) _m - and optionally substituted by one or more substituents Q ₁ , aryl or heteroaryl;

R and _R are each independently selected from hydrogen, _halogen , hydroxyl, halogen, deuterium, nitro, cyano and the following groups, which are optionally substituted with one or _more substituents Q, including alkyl, _-NRiRj , _-C (O) _Rk , -C(O) _ORk , -S(O) _Rk , -S(O)ORk, -S(O) ₍ O) _Rk , -S(O)(O) _ORk , -C(S) _Rk , alkoxy, alkylthio, alkenyl and alkynyl, or R and _R together form a pendoxy _group or a thioketo group;

Ring C and Ring D are each independently selected from aryl groups and heteroaryl groups which are optionally substituted by one or more substituents Q ₁ , and at least one of Ring C and Ring D is selected from fused aryl groups or fused heteroaryl groups which are optionally substituted by one or more substituents Q ₁ ;

_X2 is selected from -( _CR6aR6b ) _n- , aryl or heteroaryl optionally substituted by one or more substituents _Q1 , -O-, -S-, -S(O ₎ -, -S(O)(O)-, _{-NR6c- ,} -CH2S- _{, -CH2O-, -NHCR6dR6e- , -CR6f} = _CR6g- and -C≡C-, or _X2 is absent;

R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, alkyl, deuterium, nitro, cyano or the following groups which are optionally substituted by one or more substituents Q ₁ : 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 they are attached form a 3- to 10-membered cycloalkyl group, or R _6a and R _6b together form a pendoxy group or a thioketo group;

R _6c , R _6d , R _6e , R _6f and R _6g are each independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ halogenalkyl and C ₁ -C ₆ alkoxy;

_R1 is each independently selected from hydrogen, alkyl and alkoxy, wherein the alkyl and alkoxy are each independently optionally substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl and hydroxyalkyl;

_R2 is each independently selected from _{-CH2OH , -CH2SH , -CH2Cl ,} -SCH2Cl _, -SCH2F, -SCH2CF3, -OH, _-OCH2CN , -OCH2Cl _{, -OCH2F} , _-OCH3 , _-OCH2CH3 , _{-SCH2CN ,}

R _2a are each independently hydrogen or C ₁ -C ₆ alkyl;

R _2b are each independently C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy;

R _2c are each independently selected from hydrogen, C ₁ -C ₆ alkyl, -CH ₂ OH and C ₁ -C ₆ alkoxy;

R ₂ d and R _2e are each independently hydrogen or C ₁ -C ₆ alkyl;

R ₃ are each independently hydrogen or a halogen;

_R4 are each independently selected from hydrogen, halogen and hydroxyl;

m and n are each independently integers from 1 to 6;

The substituent groups _Q1 are each independently selected from _C1 - _C6 alkyl, halogen, deuterium, hydroxyl, hydroxyl _{, -NRiRj} , oxo, thioketo, -C(O) _Rk , -C(O) _ORk , -S(O) _Rk , -S(O) _ORk , -S(O)(O) _Rk , -S(O)(O) _ORk , -C(S) _Rk , nitro, cyano _, C1- _C6 alkoxy, _C1 - _C6 alkylthio, C2- _C6 alkenyl, _C2 - _{C6 alkynyl} , 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclic group, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused cycloaryl, and 5- to 12-membered fused heteroaryl;

R _i and R _j are each independently selected from a hydrogen atom, a hydroxyl group, a C ₁ -C ₆ alkyl group and a C ₁ -C ₆ alkoxy group;

_{Rk is} independently selected from a hydrogen atom, a _C1 - _C6 alkyl group, a _C1 - _C6 haloalkyl group, a _C1 - _C6 alkoxy group, a hydroxyl _group and _-NRiRj , wherein the alkyl group, the alkoxy group and the haloalkyl group are each independently and optionally substituted with one or more substituents selected from a _C1 - _C6 alkyl group, a halogen group, a hydroxyl group, a hydroxyl group, _-NRiRj , a pendoxy group, a thioketo group, a carboxyl group, a nitro group, a cyano group, a _C1 - _C6 alkoxy group, a _C1 - _C6 alkylthio group, a _C2 - _C6 alkenyl group, a _C2 - _C6 alkynyl group, a 3- to ₁₀ -membered cycloalkyl group, a 3- to 10-membered heterocyclo group, a 6- to 10-membered aryl group and a 5- to 10-membered heteroaryl group; and

Provided that when _R5a is hydrogen or alkyl, _R5b is not hydrogen or alkyl.

In certain embodiments, R _1a is each independently selected from hydrogen, C ₁ -C ₆ alkyl and C ₁ -C ₆ alkoxy, and the alkyl and alkoxy are each independently optionally substituted with one or more substituents selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, deuterium, amino, cyano and hydroxyl.

In certain embodiments, each R _1a is independently selected from hydrogen, C ₁ -C ₆ alkyl, and C ₁ -C ₆ alkoxy.

In certain embodiments, Ring A is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group which is optionally substituted by one or more substituents Q ₁ , and the heteroaryl group contains at least one nitrogen atom.

或

。 In certain embodiments, Ring A is optionally substituted with one or more substituents Q ₁

or

.

In certain embodiments, Ring B is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group which is optionally substituted by one or more substituents Q ₁ , wherein the heteroaryl group contains at least one nitrogen atom.

或

。 In certain embodiments, Ring B is optionally substituted with one or more substituents Q ₁

or

.

In certain embodiments, X ₁ is -(CR _5a R _5b ) _m - or a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group which is optionally substituted by one or more substituents Q ₁ , wherein the heteroaryl group contains at least one nitrogen atom.

In certain embodiments, R and _R are each independently selected from hydrogen, _halogen , hydroxyl, halogen, deuterium, nitro, cyano, and the following groups, optionally substituted with _one or more substituents Q, including _C1 - _{C6 alkyl} , _-NRiRj , -C(O) _Rk , -C(O) _ORk , -S(O) _Rk , -S(O) _ORk , -S(O)(O) _Rk , -S(O)(O) _ORk , -C(S) _Rk , _C1 - _C6 alkoxy, _C1 - _C6 alkylthio, _C2 - _C6 alkenyl, and _C2 - _C6 alkynyl, or R and R together form a _pendoxy group or a _thioketo group.

In certain embodiments, R and _R are each independently selected from hydrogen, _halogen , hydroxyl, halogen, deuterium, cyano, and the following groups, optionally substituted with _one or more substituents Q, including _C1 - _{C6 alkyl} , _-NRiRj , -C(O) _Rk , -C(O)ORk, -S( _{O)Rk ,} -S(O) _ORk , -S(O)(O) _Rk , -S(O)(O) _ORk , _C1 - _C6 alkoxy, _C1 - _C6 alkylthio, _C2 - _{C6 alkenyl} , and _C2 - _C6 alkynyl, or R and _R together form a pendoxy group or a thioketo group.

In certain embodiments, R _5a and R _5b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, cyano, and the following groups, which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k , -S(O)R _k , -S(O)(O)R _k , C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, and C ₂ -C ₆ alkynyl, or R _5a and R _5b together form a pendoxy group or a thioketo group.

In certain embodiments, R _5a and R _5b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, cyano, and the following groups, which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k and C ₁ -C ₆ alkoxy, or R _5a and R _5b together form a pendoxy group or a thioketone group.

In certain embodiments, Ring C and Ring D are each independently selected from a 6- to 10-membered aryl group, a 5- to 10-membered heteroaryl group, an 8- to 12-membered fused ring aryl group, or a 5- to 12-membered fused heteroaryl group, which is optionally substituted with one or more substituents Q ₁ , and the heteroaryl group or fused heteroaryl group contains at least one nitrogen atom.

In certain embodiments, Ring C and Ring D are each independently selected from the following groups which are optionally substituted with one or more substituents Q ₁ :

、

、

、

In certain embodiments, Ring C is selected from the group consisting of: optionally substituted with one or more substituents Q ₁ :

,

,

,

In certain embodiments, ring D is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group which is optionally substituted by one or more substituents Q ₁ , wherein the heteroaryl group contains at least one nitrogen atom.

或

。 In certain embodiments, ring D is optionally substituted with one or more substituents Q ₁

or

.

In certain embodiments, X ₂ is selected from -(CR _6a R _6b )n-, -O-, -S-, -NR _6c -, -CH ₂ S-, -CH ₂ O-, -NHCR _6d R _6e -, and a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q ₁ , wherein the heteroaryl group contains at least one nitrogen atom.

In certain embodiments, R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, alkyl, deuterium, cyano, and the following groups, which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ 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 ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, or R _6a , R _6b together with the carbon atom to which they are attached form a 3- to 10-membered cycloalkyl, or R _6a and R _6b together form a pendoxy or thioketo group.

In certain embodiments, R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, alkyl, deuterium, cyano and the following groups, which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k , -S(O)R _k , -S(O)(O)R _k , C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, or R _6a , R _6b together with the carbon atom to which they are attached form a 3- to 10-membered cycloalkyl, or R _6a and R _6b together form a pendoxy or thioketo group.

In certain embodiments, R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, cyano and the following groups, which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k and C ₁ -C ₆ alkoxy, or R _6a and R _6b together form a pendoxy group or a thioketone group.

In certain embodiments, R ₁ is each independently selected from hydrogen, C ₁ -C ₆ alkyl and C ₁ -C ₆ alkoxy, preferably hydrogen.

In certain embodiments, each R ₄ is independently hydrogen.

In certain embodiments, the substituent groups Q ₁ are each independently selected from halogen, hydroxyl, halogen, deuterium, oxo, thioketone, cyano, amine, carboxyl, C ₁ -C ₆ alkyl, and C ₁ -C ₆ alkoxy.

In certain embodiments, R _k is independently selected from a hydrogen atom, a C ₁ -C ₆ alkyl group, a C ₁ -C ₆ halogenalkyl group, a C ₁ -C ₆ alkoxy group, a hydroxyl group, and -NR _i R _j .

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

in,

R _1a are each independently selected from hydrogen, C ₁ -C ₆ alkyl and C ₁ -C ₆ alkoxy;

或

，該環A視需要被一個或多個取代基Q₁所取 代； Ring A is

or

, the ring A is optionally substituted by one or more substituents Q ₁ ;

或

，該環B視需要被一個或多個取代基Q₁所取代； Ring B is

or

, the ring B is optionally substituted by one or more substituents Q ₁ ;

X ₁ is -(CR _5a R _5b ) _m - or a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group which is optionally substituted by one or more substituents Q ₁ , wherein the heteroaryl group contains at least one nitrogen atom;

R _5a and R _5b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, cyano and the following groups which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k and C ₁ -C ₆ alkoxy, or R _5a and R _5b together form a pendoxy group or a thioketone group;

、

、

、

和

，該環C視需要被一個或多個取代基Q₁所取代； Ring C selected from

,

,

,

and

, the ring C is optionally substituted by one or more substituents Q ₁ ;

或

，該環D視需要被一個或多個取代基Q₁所取代； Ring D is

or

, the ring D is optionally substituted by one or more substituents Q ₁ ;

_X2 is selected from -( _CR6aR6b )n-, -O-, -S-, _{-NR6c- ,} -CH2S-, _-CH2O- , _-NHCR6dR6e- and _a 6- to 10-membered _aryl group or a 5- to 10-membered heteroaryl group which is optionally substituted with one or more substituents _Q1 ;

R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, cyano and the following groups which are optionally substituted with one or more substituents Q ₁ : C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)R _k , -C(O)OR _k and C ₁ -C ₆ alkoxy, or R _6a and R _6b together form a pendoxy group or a thioketone group;

R _6c , R _6d and R _6e are each independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ halogenalkyl and C ₁ -C ₆ alkoxy;

_R2 is each independently selected from _{-CH2OH , -CH2SH , -CH2Cl ,} -SCH2Cl _, -SCH2F, -SCH2CF3, -OH, _-OCH2CN , -OCH2Cl _{, -OCH2F} , _-OCH3 , _-OCH2CH3 , _{-SCH2CN ,}

R _2a are each independently hydrogen or C ₁ -C ₆ alkyl;

R _2b are each independently C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy;

R _2c are each independently selected from hydrogen, C ₁ -C ₆ alkyl, -CH ₂ OH and C ₁ -C ₆ alkoxy;

R _2d and R _2e are each independently hydrogen or C ₁ -C ₆ alkyl;

R ₃ are each independently hydrogen or a halogen;

m and n are each independently integers from 1 to 6;

The substituent groups _Q1 are each independently selected from halogen, hydroxyl, oxirane, deuterium, oxo _, thioketone, cyano, amine, carboxyl, _C1 - _C6 alkyl and C1- _C6 alkoxy groups;

R _i and R _j are each independently selected from a hydrogen atom, a hydroxyl group, a C ₁ -C ₆ alkyl group and a C ₁ -C ₆ alkoxy group;

R _k is independently selected from a hydrogen atom, a C ₁ -C ₆ alkyl group, a C ₁ -C ₆ halogenalkyl group, a C ₁ -C ₆ alkoxy group, a hydroxyl group, and -NR _i R _j ; and

Provided that when _R5a is hydrogen or alkyl, _R5b is not hydrogen or alkyl.

In certain embodiments, R _1a is hydrogen.

、

和

。 In certain embodiments, X ₁ is selected from -(CR _5a R _5b ) _m -, optionally substituted with one or more substituents Q ₁ :

,

and

.

In certain embodiments, R _5a and R _5b are both fluoro.

In certain embodiments, R _5a and R _5b together form a pendoxy group or a thioketone group, preferably a pendoxy group.

、

和

。 In certain embodiments, X ₂ is selected from -(CR _6a R _6b ) _n -, optionally substituted with one or more substituents Q ₁ :

,

and

.

In certain embodiments, R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, halogen, deuterium, cyano, C ₁ -C ₆ alkyl, -NR _i R _j , -C(O)OR _k and C ₁ -C ₆ alkoxy, or R _6a and R _6b together form a pendoxy group or a thioketone group.

In certain embodiments, _R2 is each independently selected from _-CH2OH , _-CH2SH , -OH and

In certain embodiments, R ₃ is hydrogen.

In certain embodiments, R ₃ is fluoro.

In some embodiments, k is any number between 1 and 10, and in some embodiments, k is any number between 2 and 5. k can be an integer or a decimal.

In some 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).

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

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:

Among them, k is selected from 1 to 10 and can be an integer or a decimal.

In some implementations, k is any number between 2 and 5. k can be an integer or a decimal.

In some 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 value or range between any two values).

The antibodies disclosed herein are not particularly limited. The choice of antibody depends on the disease or condition (e.g., cancer) to be treated by the antibody-drug conjugate (ADC). Antibodies can specifically bind to corresponding antigens (also called tumor-associated antigens (TAAs)), viral antigens, or microbial antigens expressed on cancer cells, have antibody-dependent cell-mediated phagocytosis (ADCP) activity, and have in vivo anti-tumor, anti-viral, or anti-microbial activity. The interchain S-S bonds in antibodies are the sites for connecting the drug-linker complex.

Antibodies suitable for use in the methods disclosed herein can be prepared 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.

In some embodiments, the antibodies disclosed herein are selected from single antibodies or multiple antibodies.

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.

In some embodiments, the isotype of the antibody disclosed herein is selected from IgG (IgG1, IgG2, IgG3 or IgG4). In some embodiments, the antibody is selected from IgG1 or IgG4 isotype. In some specific embodiments, the antibody is selected from IgG1 monoclonal antibody or IgG4 monoclonal antibody.

In some embodiments, the antibody disclosed herein is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUC1 antibody, anti-Lewis antibody Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP2 antibody, anti-CD79B antibody, anti-Mesothelin antibody, anti-TNF-α antibody, anti-folate receptor alpha antibody (anti-human folate receptor alpha (FRA) antibody), anti-Nectin-4 antibody, anti-AXL antibody, anti-CD19 antibody or its antigen-binding fragment.

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.

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.

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

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.

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 purulentumab, afelimomab, nerimumab, ozolazumab, puraculumab 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 purulentumab, afelimomab, nerimumab, ozolazumab, puraculumab or golimumab.

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 antigen-binding fragment thereof. In certain embodiments, the anti-TNFα antibody or antigen-binding fragment thereof is adalimumab.

In certain embodiments, the anti-TNFα antibody or antigen-binding fragment thereof comprises a sequence of adalimumab, infliximab, certolizumab purulent, afelimumab, nerimumab, ozolazumab, pruculumab, or golimumab, such as a complementarity determining region (CDR), a variable heavy chain domain (VH), and/or a variable light chain domain (VL).

The drugs used in the present disclosure are not particularly limited, as long as the drug molecule has the desired effect (e.g., cytotoxicity, anti-tumor, or labeling agent, etc.) 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 diagnostic agents, therapeutic agents, or labeling agents; in some embodiments, the drug includes but is not limited to cytotoxic agents, such as chemotherapeutic agents, immunotherapeutic agents, antiviral agents, or antimicrobial agents. In some embodiments, the drug can be selected from but not limited to Maytansinoids, Auristatins (such as MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), MMAF (monomethyl auristatin F), etc.), Calicheamicins, Doxorubicins, Duocarmycins and CC-1065, Camptothecins, Pyrrolobenzodiazepines and PBD Dimmers, etc. In some embodiments, the drug is selected from glucocorticoid receptor agonists.

In certain embodiments, the drug is defined as D in the antibody-drug conjugate (ADC) described in formula (I).

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:

(1) Add an appropriate amount of _ZnCl2 and the reducing agent diphenylphosphoacetic acid to the antibody solution to be reduced;

(2) adding a metal chelating agent to chelate Zn2 ⁺ ions;

(3) Add an appropriate amount of dissolved drug linker intermediate or its pharmaceutically acceptable salt and react for a certain period of time;

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

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.

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.

In some embodiments, the final concentration equivalent ratio (mM/mM) of the antibody to Zn ²⁺ ions in step (1) is about 1:2.

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

In some embodiments, step (1) is carried out in a buffer system, which is a histidine buffer solution with a pH of 6.0-7.0.

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

In some embodiments, the final concentration equivalent ratio (mM/mM) of Zn ²⁺ ions to metal chelating agent is about 1:2.

In some embodiments, the final concentration equivalent ratio (mM/mM) of the antibody and the drug linker intermediate or its pharmaceutically acceptable salt (based on the drug linker intermediate) in step (3) is about 1:6.

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

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

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.

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.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method disclosed herein 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 About 67wt%, higher than about 68wt%, higher than about 69wt%, higher than about 70wt%, higher than about 71wt%, higher than about 72wt%, higher than about 73wt%, higher than about 74wt%, higher than about 75wt%; In some embodiments, based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt contains D4 in an amount selected from about 55wt% to about 75wt%, about 60wt% to about 70wt%, about 55wt% to about 65wt%.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method described in the present disclosure contains a higher content of D4 than the content of D4 obtained using TCEP, 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 prepared by the method disclosed herein 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 a heavy-light chain inter-group; based on the total weight of D0, D2, D4, D6 and D8, the content of D4a contained in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is higher than about 50wt%, for example, higher than about 50wt%, higher than about 55wt%, higher than about 60wt%, higher than about 61wt%, higher than about 62wt%. %, higher than about 63wt%, higher than about 64wt%, higher than about 65wt%, higher than about 66wt%, higher than about 67wt%, higher than about 68wt%, higher than about 69wt%, higher than about 70wt%, higher than about 71wt%, higher than about 72wt%, higher than about 73wt%, higher than about 74wt%, higher than about 75wt%; in some embodiments, based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt contains D4a in an amount selected from about 55wt% to about 75wt%, about 60wt% to about 70wt%, about 55wt% to about 65wt%.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method disclosed herein comprises D4b, wherein D4b is selected from an antibody-drug conjugate or its pharmaceutically acceptable salt having only 4 drug linkers, and the 4 drug linkers are bound to a heavy-heavy chain inter-chain styrene group; based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt comprises D4b of The content is less than about 40wt%, such as less than about 40wt%, less than about 30wt%, less than about 20wt%, less than about 15wt%, less than about 10wt%, less than about 5wt%; 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 5wt% to about 30wt%, about 5wt% to about 20wt%, about 5wt% to about 10wt%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method disclosed herein comprises less than about 20 wt % of D0+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 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 wt %, less than about 19 wt %, less than about 19 wt %, less than about 11 wt %, less than about 11 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 18 wt %, less than about 19 ... About 11wt%, less than about 10wt%, less than about 9wt%, less than about 8wt%, less than about 7wt%, less than about 6wt%, less than about 5wt%, less than about 4wt%, less than about 3wt%; In some embodiments, based on the total weight of D0, D2, D4, D6 and D8, the content of D0+D8 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt is selected from about 3wt%-about 20wt%, about 5wt%-about 15wt%, about 3wt%-about 10wt%.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method disclosed herein contains less than about 20 wt % 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 15 wt %, less than about 16 wt %, less than about 17 wt %, less than about 18 ... About 12wt%, less than about 11wt%, less than about 10wt%, less than about 9wt%, less than about 8wt%, less than about 7wt%, less than about 6wt%, less than about 5wt%; In some embodiments, based on the total weight of D0, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt contains D6 in an amount selected from about 5wt% to about 20wt%, about 5wt% to about 15wt%, about 5wt% to about 10wt%.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt 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 coupling reaction.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt 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 D0, D2, D4, D6 and D8 obtained after the coupling reaction after purification and removal of small molecule process impurities (e.g., free toxins and organic solvents, etc.), wherein the purification step will not affect the different drug-loaded components of the prepared ADC.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods disclosed 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 10 ... 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 D4 selected from about 55% to about 75%, about 60% to about 70%, about 55% to about 65% based on the sum of peak areas.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method described in the present disclosure contains a peak area percentage of D4 greater than the peak area percentage of D4 obtained using TCEP based on the sum of peak areas.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method disclosed herein 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 a heavy-light chain inter-group; based on the sum of peak areas, 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 60%, or greater than about 70%. , 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 peak areas.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method described in 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 the four drug linkers are bound to a styryl group between the heavy-heavy chains; based on the sum of peak areas, the antibody-drug conjugate or its pharmaceutically acceptable salt The peak area percentage of D4b contained in the salt is less than about 40%, such as less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, 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 its pharmaceutically acceptable salt is selected from about 5% to about 30%, about 5% to about 20%, about 5% to about 10%.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method disclosed herein contains less than about 20% of the peak area percentage of D0+D8, 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% based on the total peak area; in some embodiments, the peak area percentage of D0+D8 contained in the antibody-drug conjugate or its pharmaceutically acceptable salt is selected from about 3%-about 20%, about 5%-about 15%, about 3%-about 10% based on the total peak area.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method disclosed herein contains less than about 20% of D6 by peak area, 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% based on the total peak area; in some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt contains D6 by peak area percentage selected from about 5%-about 20%, about 5%-about 15%, about 5%-about 10% based on the total peak area.

The peak area percentage based on the total peak area means: the antibody-drug conjugate (ADC) or its pharmaceutically acceptable salt prepared in the present disclosure is detected 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, and the total peak area is obtained by integrating the chromatogram, and the peak area percentage of each component (such as D0, D2, D4, D6 or D8) is calculated by the area normalization method.

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

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

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method disclosed 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 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, 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 its pharmaceutically acceptable salt is selected from about 55% to about 75%, about 60% to about 70%, about 55% to about 65%.

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

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method disclosed herein 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 a heavy-light chain inter-group; 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 60%, or greater than about 70%. , 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, 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 its pharmaceutically acceptable salt is selected from about 55% to about 75%, about 60% to about 70%, about 55% to about 65%.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method disclosed herein comprises D4b, wherein D4b 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 a styryl group between the heavy-heavy chains; based on the sum of the peak areas of D0, D2, D4, D6 and D8, the antibody-drug conjugate or its pharmaceutically acceptable salt is The peak area percentage of D4b contained in the salt is less than about 40%, such as less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, 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 its pharmaceutically acceptable salt is selected from about 5% to about 30%, about 5% to about 20%, about 5% to about 10%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure 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 15%, less than about 16%, less than about 17 ... 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 is selected from about 3%-about 20%, about 5%-about 15%, about 3%-about 10%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method disclosed herein comprises less than about 20% 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 16%, less than about 17%, less than about 18 ... 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 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 is selected from about 5%-about 20%, about 5%-about 15%, about 5%-about 10%.

In some embodiments, the sum of the peak areas of D0, D2, D4, D6 and D8 of the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method described in the present disclosure refers to the sum of the peak areas of unpurified D0, D2, D4, D6 and D8 obtained after the conjugation reaction.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method described in the present disclosure, based on the sum of the peak areas of D0, D2, D4, D6 and D8, refers to the sum of the peak areas of D0, D2, D4, D6 and D8 obtained after the coupling reaction, after purification and removal of small molecule process impurities (e.g., free toxins and organic solvents, etc.), wherein the purification step will not affect the different drug-loaded components of the prepared ADC.

The peak area percentage based on the sum of the peak areas of D0, D2, D4, D6 and D8 means: the antibody-drug conjugate (ADC) or its pharmaceutically acceptable salt prepared in the present disclosure 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 with the chromatogram of the blank solution, and the sum of the peak areas based on D0, D2, D4, D6 and D8 is obtained by integrating the chromatogram, and the peak area percentage of each component (such as D0, D2, D4, D6 or D8) is calculated by the area normalization method.

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

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

In some embodiments, the sum of the peak areas of D0, D2, D4, D6 and D8 of the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method described in the present disclosure refers to the sum of the peak areas of unpurified D0, D2, D4, D6 and D8 obtained after the conjugation reaction.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt prepared by the method described in the present disclosure, based on the sum of the peak areas of D0, D2, D4, D6 and D8, refers to the sum of the peak areas of D0, D2, D4, D6 and D8 obtained after the coupling reaction, after purification and removal of small molecule process impurities (e.g., free toxins and organic solvents, etc.), wherein the purification step will not affect the different drug-loaded components of the prepared ADC.

In some embodiments, the average drug loading of the antibody-drug conjugate or its pharmaceutically acceptable salt 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, such as 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 its pharmaceutically acceptable salt 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.

The antibody-drug conjugate or its pharmaceutically acceptable salt disclosed herein can be prepared into a reagent kit.

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.

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 drug for treating and/or preventing tumors, cancers, autoimmune diseases, or infectious diseases. In some embodiments, the cancer is a cancer associated with the expression of HER2, HER3, B7H3, CD19, CD30, CD33, Trop 2, 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, suppurative hidradenitis, uveitis, Behcet's disease, spondyloarthropathies, and psoriasis.

The present disclosure also provides a use of the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the preparation of a drug 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, neuroglioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

The active compound, such as an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, can be prepared into a form suitable for administration by any appropriate route, preferably in a unit dose, or in a form that a patient can self-administer in a single dose. The unit dose of the disclosed compound or composition can be expressed in the form of a tablet, capsule, cachet, bottled solution, powder, granules, tablet, suppository, reconstituted powder or liquid preparation.

The dosage of the antibody-drug conjugate or its pharmaceutically acceptable salt or compound or composition used in the treatment method 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 0.1~1000 mg.

The pharmaceutical composition disclosed herein may contain one or more excipients in addition to the active compound such as an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, and the excipients are selected from the following ingredients: fillers (diluents), binders, wetting agents, disintegrants or excipients, etc. 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.

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 enantiomer- or diastereomer-enriched mixtures, all of which are within the scope of the disclosure. Additional asymmetric carbon atoms may exist in substituents such as alkyl groups. Unless otherwise stated, all these isomers and their mixtures are included within the scope of this disclosure.

The compounds, drugs, intermediates, and conjugates disclosed herein containing asymmetric carbon atoms can be isolated in optically active pure forms or racemic forms. Optically active pure forms can be resolved from racemic mixtures or synthesized using chiral raw 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 disclosed herein 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 group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amine group) or an acidic functional group (such as a carboxyl group), diastereoisomers are formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art and the pure enantiomers are recovered. In addition, the separation of enantiomers and diastereomers is usually achieved by using chromatographic methods that use chiral stationary phases and are optionally combined with chemical derivatization methods (such as the formation of carbamates from amines).

Any isotopically labeled derivatives of the compounds, drugs, intermediates, conjugates or pharmaceutically acceptable salts thereof, or isomers thereof described in the present disclosure 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, etc. They can be replaced by isotopes such as 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.

Unless otherwise indicated, when a position is specifically designated as deuterium (D), the position is understood to have at least 1000 times greater abundance of deuterium (i.e., at least 10% deuterium incorporation) than the natural abundance of deuterium (which is 0.015%). Exemplary compounds having greater abundance of deuterium than the natural abundance of deuterium may be at least 1000 times greater abundance of deuterium, at least 2000 times greater abundance of deuterium, at least 3000 times greater abundance of deuterium (i.e., at least 45% deuterium incorporation), at least 4000 times greater abundance of deuterium, at least 5000 times greater abundance of deuterium, at least 6000 times greater abundance of deuterium, or more. The present disclosure also includes various deuterated forms of the compounds. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. A person skilled in the art can synthesize deuterated compounds by referring to relevant literature. Commercially available deuterated starting materials can be used in the preparation of deuterated compounds, or they can be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.

On the other hand, the present disclosure also provides a method for selectively reducing an antibody, comprising 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 the intrachain disulfide bonds of the antibody to alkyl groups.

In some embodiments, the transition metal ion in the method of selectively reducing the antibody 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⁺ .

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.

In some embodiments, suitable zinc salts include, but are not limited to, ZnCl ₂ , Zn(NO ₃ ) ₂ , ZnSO ₄ , Zn(CH ₃ COO) ₂ , ZnI ₂ , ZnBr ₂ , zinc formate, and zinc tetrafluoroborate.

In some embodiments, other suitable transition metal salts that are soluble in the reaction solution and can release free Cd ²⁺ or Hg ²⁺ ions include but are not limited to: CdCl ₂ , Cd(NO ₃ ) ₂ , CdSO ₄ , Cd(CH ₃ COO) ₂ , CdI ₂ , CdBr ₂ , cadmium formate and cadmium tetrafluoroborate; HgCl ₂ , Hg(NO ₃ ) ₂ , HgSO ₄ , Hg(CH ₃ COO) ₂ , HgBr ₂ , mercury(II) formate, and mercury(II) tetrafluoroborate, etc.

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.

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 transition metal ions.

In some embodiments, the buffer system contains or does not contain a metal chelator. In some embodiments, the buffer does not contain a metal chelator.

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, such as 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, and in some embodiments, selected from 6.0 to 7.0.

In some embodiments, the method of selectively reducing the antibody is performed at about -10°C to about 40°C, such as 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.

In some embodiments, the reaction time of the selective reduction of antibodies is selected from 1h to 24h, such as 2h, 4h, 8h, 12h, 16h; in some embodiments, the reaction time of the selective reduction of antibodies is 16h.

In some embodiments, the method for selectively reducing the antibody is selected from overnight incubation at 0-4°C or 2 hours at 25°C;

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 one of ordinary skill in the art. For example, the antibody to be conjugated is typically incubated overnight with the reducing agent in the presence of transition metal ions at 4°C.

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 0.14mM, about 0.15mM, about 0.20mM, about 0.25mM, about 0.30mM, about 0.35mM, about 0.40mM, about 0.45mM, about 0.50mM; in some embodiments, the final concentration of the antibody is selected from about 0.10mM to about 0.20mM; in some embodiments, the final concentration of the antibody is selected from about 0.12mM to about 0.14mM.

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 0.24mM, about 0.25mM, about 0.26mM, about 0.27mM, about 0.28mM, about 0.29mM, about 0.30mM, about 0.31mM, about 0.32mM, about 0.33mM, about 0.34mM, about 0.35mM, about 0.40mM, about 0.45mM, about 0.50mM; in some embodiments, the final concentration of transition metal ions is selected from about 0.27mM to about 0.28mM.

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

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.

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.

In some embodiments, the method for selectively reducing antibodies disclosed 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, ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid dicalcium salt, diethylenetriaminepentaacetic acid or a mixture thereof.

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, such as 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.

In some embodiments, the antibodies disclosed herein are selected from single antibodies or multiple antibodies.

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

In some embodiments, the isotype of the antibody disclosed herein is selected from IgG (IgG1, IgG2, IgG3 or IgG4). In some embodiments, the antibody is selected from IgG1 or IgG4 isotype. In some specific embodiments, the antibody is selected from IgG1 monoclonal antibody or IgG4 monoclonal antibody.

In some embodiments, the present disclosure provides a method for selectively reducing an antibody, comprising the following steps: (1a) adding an appropriate amount of ZnCl ₂ and a reducing agent diphenylphosphoacetic acid to an antibody solution to be reduced.

In some embodiments, the final concentration of the antibody in step (1a) 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.

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.

In some embodiments, the final concentration equivalent ratio (mM/mM) of the antibody to Zn ²⁺ ions in step (1a) is about 1:2.

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

In some embodiments, step (1a) is carried out in a buffer system, which is a histidine buffer solution with a pH of 6.0 to 7.0.

In some embodiments, the method for selectively reducing an antibody disclosed herein further comprises (2a) adding a metal chelator to chelate Zn ²⁺ ions.

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

In some embodiments, the final concentration equivalent ratio (mM/mM) of Zn ²⁺ ions to metal chelating agent is about 1:2.

In some embodiments, based on the total number of inter-chain disulfide bonds and intra-chain disulfide bonds of the antibody, the method for selectively reducing the antibody disclosed herein selectively reduces the ratio of the inter-chain disulfide bonds of the heavy-light chains to be higher than about 50wt%, such as higher than about 50wt%, higher than about 55wt%, higher than about 60wt%, higher than about 61wt%, higher than about 62wt%, higher than about 63wt%, higher than about 64wt%, higher than about 65wt%, higher than about 66wt %, higher than about 67wt%, higher than about 68wt%, higher than about 69wt%, higher than about 70wt%, higher than about 71wt%, higher than about 72wt%, higher than about 73wt%, higher than about 74wt%, higher than about 75wt%; in some embodiments, the ratio is selected from about 55wt% to about 75wt%, about 60wt% to about 70wt%, about 55wt% to about 65wt%.

This disclosure introduces the full text of WO2022166779A1.

Terminology

Unless otherwise defined, all technical and scientific terms used in this disclosure are consistent with the common understanding of those with ordinary knowledge in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described in this disclosure may be used to implement or test this disclosure, this disclosure describes preferred methods and materials. When describing and claiming protection for this disclosure, the following terms are used in accordance with the following definitions.

When a trade name is used in this disclosure, applicant intends to include the formulation of the trade name product, the generic drug product, and the active drug portion of the trade name product.

Unless otherwise stated, the terms used in the specification and application have the following meanings.

The term "antibody-drug conjugate" (ADC), also known as antibody-drug conjugate or antibody-drug conjugate, refers to an antibody connected to a drug via a linker. In the present disclosure, "antibody-drug conjugate" refers to an antibody or antigen-binding fragment connected to a biologically active toxic drug via a stable linker. In the present disclosure, antibody-drug conjugate, antibody-drug conjugate, and antibody-drug conjugate can be used interchangeably.

The term "linker", "linker", "linker unit", "linker unit" or "linker fragment" refers to a chemical structure fragment or bond that is linked to an antibody or antigen binding fragment at one end and to a drug at the other end. It can also be linked to other linkers and then to an antibody or drug.

The linker may comprise one or more linker components. Exemplary linker components include 6-maleimidohexanoyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with linker reagents: N-succinimidyl 4-(2-pyridylthioketone) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC, also known as MCC) and N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB). The linker may 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 those described in US2005-0238649A1. The linker can be a "cleavable linker" that facilitates release of the drug in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

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 to each antibody (Ab). In the embodiments disclosed herein, the drug loading can be exemplarily 3, 4, 5, or the average of any two values. The average amount of drug per ADC molecule after the coupling reaction can be identified by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays, monoclonal antibody size variant assays (CE-SDS), and HPLC characterization. For example, in one embodiment, HIC is used to determine the yield and isomer mixture of the obtained antibody-drug conjugate (e.g., for D4 conjugate). This technique is capable of separating antibodies loaded with various numbers of drug. The drug loading level can be determined based on, for example, the ratio of absorbance at 250nm and 280nm. For example, if the drug absorbs at 250nm and the antibody absorbs at 280nm. Therefore, the 250/280 ratio increases with drug loading. Using the bioconjugation method described herein, antibodies with an even number of drugs are generally observed to be conjugated to the antibody because reduction of the disulfide bonds produces an even number of free cysteine groups.

Typically, an antibody molecule belonging to the IgG1 or IgG4 subclass has 4 interchain S-S bonds, each formed by two -SH groups. The antibody molecule can undergo partial or complete reduction of one or more interchain S-S 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.

Therefore, 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 S-S bond between the heavy chain and the light chain, or can be coupled via a linker to a -SH group generated by reducing the S-S bond between the heavy chain and the light chain.

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 S-S bonds between the heavy chain and the light chain via a linker (the ADC is referred to as D4a), or the four drug molecules can be coupled to four -SH groups generated by reducing two S-S bonds between the heavy chain and the light chain via a linker (the ADC is referred to as D4b), or two drug molecules can be coupled to two -SH groups generated by reducing one S-S 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 S-S bond between the heavy chain and the light chain via a linker (the ADC is referred to as D4c).

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 S-S 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 S-S bond between the heavy chain and the light chain via a linker (the ADC is referred to as D6a), or four drug molecules can be coupled to four -SH groups generated by reducing two S-S 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 S-S bond between the heavy chain and the light chain via a linker (the ADC is referred to as D6b).

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

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. Typically, if the content of D4 in the mixture is high, the ADC is considered to have high homogeneity or high uniformity.

In this disclosure, the "homogeneity" or "uniformity" of an antibody-drug conjugate refers to having a high level of D4 in a mixture of the antibody-drug conjugate.

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

The term "interchain thiol" or "interchain thiol" is used to refer to the thiol groups obtained by reducing the interchain disulfides of an antibody.

The term "heavy-heavy chain thiol" is used to refer to the thiol group obtained by reducing the heavy-heavy chain disulfide of the antibody.

The term "heavy-light chain inter-thiol" is used to refer to the thiol obtained by reducing the heavy-light chain inter-disulfide of the antibody.

Unless otherwise specified, the use of the term "addition" does not limit the order, method, or how the added materials are combined. For example, "addition of A to B" may also describe "addition of B to A". In addition, "addition of A and B to C" may also describe other different combinations, such as "addition of A to B and C", "addition of A and C to B", "addition of B to A and C", "addition of B and C to A", and "addition of C to A and B".

The terms "thiol" and "thiol" are used interchangeably and refer to -SH.

。 The term "diphenylphosphino" refers to

.

The term "diphenylphosphinoacetic acid" is also known as "DPA" or "diphenylphosphinoacetic acid", which refers to

表示單鍵或雙鍵。 In the structure disclosed in the present invention,

Indicates a single key or two keys.

It is understood by those skilled in the art that, for example, when one of R _5a and R _5b is selected from a pendoxy group or a thiol group, the other one is absent.

The term "stretcher" refers to a chemical structural fragment that is covalently linked to an antibody via 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.

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, t-butyl, t-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 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, t-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, alkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, pendoxy, carboxyl or carboxylate.

The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of a parent alkane by removing two hydrogen atoms, and is a straight or branched chain group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (—CH ₂ —), 1,1-ethylene (—CH(CH ₃ )—), 1,2-ethylene (—CH ₂ CH ₂ )—, 1,1-propylene (—CH(CH ₂ CH ₃ )—), 1,2-propylene (—CH ₂ CH(CH ₃ )—), 1,3-propylene (—CH ₂ CH ₂ CH ₂ —), 1,4-butylene (—CH ₂ CH ₂ CH ₂ CH ₂ —), and the like. Alkylene groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment.

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.

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, etc.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic 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, etc.; polycyclic cycloalkyls include spirocyclic, fused-ring, and bridged-ring cycloalkyls. "Carbocycle" refers to the ring system in the cycloalkyl.

The term "spirocycloalkyl" refers to a polycyclic group in which a carbon atom (called a spiro atom) is shared between single rings of 5 to 20 members, which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 members, and more preferably, it is 7 to 10 members. According to the number of spiro atoms shared between rings, spirocycloalkyl is divided into monospirocycloalkyl, dispirocycloalkyl or polyspirocycloalkyl, preferably monospirocycloalkyl and dispirocycloalkyl. More preferably, it is 4-member/4-member, 4-member/5-member, 4-member/6-member, 5-member/5-member or 5-member/6-member monospirocycloalkyl. "Spirocarbocycle" refers to the ring system in spirocycloalkyl. Non-limiting examples of spirocycloalkyl include:

The term "fused cycloalkyl" refers to a full-carbon polycyclic group with 5 to 20 members, each ring in the system shares a pair of adjacent carbon atoms with other rings in the system, one or more of which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 members, and 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, and more preferably 5-member/5-member or 5-member/6-member bicyclic alkyl. "Fused carbocyclic" refers to the ring system in the fused cycloalkyl. Non-limiting examples of fused cycloalkyl include:

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. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. It is preferably 6-14 members, and more preferably 7-10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl include:

The cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring connected to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc. The cycloalkyl may be substituted or unsubstituted as desired, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, oxirane, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, pendoxy, carboxyl or carboxylate.

The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), but excluding the ring part of -OO-, -OS- or -SS-, and 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, thioketomorpholinyl, homopiperazinyl, etc., preferably piperidinyl and pyrrolidinyl. Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups. "Heterocyclic" refers to the ring system in the heterocyclic group.

The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group in which one atom (called a spiro atom) is shared between monocyclic rings of 5 to 20 members, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), 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. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. According to the number of spiro atoms shared between rings, the spiroheterocyclic group is divided into a single spiroheterocyclic group, a double spiroheterocyclic group or a multi-spiroheterocyclic group, preferably a single spiroheterocyclic group and a double spiroheterocyclic 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 monospiroheterocyclic group. "Spiroheterocyclic" refers to the ring system in the spiroheterocyclic group. Non-limiting examples of spiroheterocyclic groups include:

The term "fused heterocyclic group" refers to a polycyclic heterocyclic group having 5 to 20 members, 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, one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, the ring has 6 to 14 members, and more preferably, it has 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic groups. "Fused heterocyclic" refers to the ring system in the fused heterocyclic group. Non-limiting examples of fused heterocyclic groups include:

The term "bridged heterocyclic group" refers to a polycyclic heterocyclic group of 5 to 14 members, 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 heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6 to 14 members, and more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

The heterocyclic ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic ring, non-limiting examples of which include:

和

等。

and

wait.

The heterocyclic group may be substituted or unsubstituted as required. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, alkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, pendoxy, carboxyl or carboxylate.

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 with a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring can be fused to a heteroaryl, heterocyclic or cycloalkyl ring, where the ring attached to the parent structure is the aryl ring. "Aryl ring" refers to the ring system in an aryl group. Non-limiting examples of aryl include:

The aryl group may be 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, alkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate, preferably phenyl.

The term "fused aryl" can 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 groups, such as naphthalene, phenanthrene, etc., and also includes partially saturated fused aryl groups, such as benzo 3-8 member saturated monocyclic cycloalkyl, benzo 3-8 member partially saturated monocyclic cycloalkyl. "Fused aromatic ring" refers to the ring system in the fused aryl group. Specific examples of fused aryl groups include 2,3-dihydro-1H-indenyl, IH-indenyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl, etc.

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 is preferably 5 to 12 members, such as imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, pyrazolyl, pyrimidyl or thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring can be fused to an aryl, heterocyclo or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. "Heteroaryl ring" refers to the ring system in the heteroaryl. Non-limiting examples of heteroaryl include:

The heteroaryl group may be substituted or unsubstituted as desired. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, alkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

The term "fused heteroaryl" may be an unsaturated fused ring structure with aromaticity containing 5-14 ring atoms (including at least one hetero atom) formed by two or more ring structures sharing two adjacent atoms, and the carbon atoms, nitrogen atoms and sulfur atoms may be substituted with pendant oxy groups, preferably "5-12 membered fused heteroaryl", "7-12 membered fused heteroaryl", "9-12 membered fused heteroaryl" or "12 membered fused heteroaryl". "Aryl", etc., such as benzofuranyl, benzoisofuranyl, benzothiophenyl, indolyl, isoindolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolyl, acridinyl, phenanthridinyl, benzoxazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, phenolazine, pteridinyl, purinyl, naphthyridinyl, phenazine, phenothiazine, etc. "Fused aromatic ring" refers to the ring system in the fused aromatic group.

The fused heteroaryl group may be substituted or unsubstituted as required. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, alkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy groups may be substituted or unsubstituted as desired, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, oxalyl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

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 may be substituted or unsubstituted as desired, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, oxirane, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

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

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

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

The term "hydroxy" refers to the -OH group.

The term "oxo" refers to a =O group. For example, a carbon atom is connected to an oxygen atom via a double bond, forming a ketone or aldehyde group.

The term "thioketo" refers to a =S group. For example, a carbon atom is connected to a sulfur atom via a double bond to form a thiocarbonyl group -C(S)-.

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

The term "amine" refers to _-NH2 .

The term "cyano" refers to -CN.

The term "nitro" refers to _-NO2 .

The term "carboxyl" refers to -C(O)OH.

The term "aldehyde group" refers to -CHO.

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

The term "acyl halides" refers to compounds containing a -C(O)-halogen group.

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

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

The "amine protecting group" is a suitable group for protecting an amine group known in the art, see the amine protecting group in the literature ("Protective Groups in Organic Synthesis", 5 ^Th . Ed. TW Greene & PGM Wuts). Preferably, the amine protecting group can be a ( _C1-10 alkyl or aromatic) acyl group, such as methyl, acetyl, benzyl, etc.; can be a ( _C1-6 alkyl or _C6-10 aromatic) sulfonyl group; can also be a ( _C1-6 alkoxy or _C6-10 aromatic) carbonyl group, such as Boc or Cbz; can also be a substituted or unsubstituted alkyl group, such as trityl (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).

The term "transition metals" 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 the elemental or ionic state (or both). Sc and Y of group 3 are also often considered transition metals.

The terms "buffer" and "buffer system" refer to buffers that tolerate pH changes by virtue of their 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, glycine and other organic acid buffers.

"Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate and other buffers. The preferred histidine buffer is histidine-hydrochloride buffer. Histidine-hydrochloride buffer is prepared by histidine and hydrochloric acid or histidine and histidine hydrochloride.

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

"Phosphate buffer" is also called PBS buffer, which is a buffer containing phosphate ions. Examples of phosphate buffers include sodium dihydrogen phosphate-sodium dihydrogen phosphate, sodium dihydrogen phosphate-potassium dihydrogen phosphate, etc. The preferred phosphate buffer is sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer.

"Acetate buffer" is also called "acetate buffer", which is a buffer containing acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. The preferred acetate buffer is acetic acid-sodium acetate buffer.

The terms "about" and "approximately" refer to numerical values within the acceptable error range of a specific value determined by a person 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 including" 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%, between 0.5% and 1%. In this disclosure, each case in which the term "about" precedes a number or a numerical range also includes embodiments of the given number. Unless otherwise specified, when a specific value appears in this disclosure, "about" or "substantially including" should mean within the acceptable error range of the specific value.

The values disclosed are instrument measurement values or values calculated after instrument measurement. There is a certain degree of error. Generally speaking, plus or minus 10% is within the reasonable error range. Of course, the context in which the value is used needs to be considered. For example, for the content of total impurities, the error change after measurement does not exceed plus or minus 10%. It can 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%, preferably plus or minus 5%.

The term "optionally" or "as necessary" 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 or does not occur.

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

The term "pharmaceutical composition" refers to a mixture containing one or more compounds, conjugates or their physiologically/pharmaceutically acceptable salts or prodrugs described herein and 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, facilitate the absorption of active ingredients and thus exert biological activity. In this disclosure, "pharmaceutical composition" and "preparation" are not mutually exclusive.

The pharmaceutical composition may be in the form of a sterile injection suspension in water or oil for intramuscular and subcutaneous administration. The suspension may be prepared according to known techniques using appropriate dispersants or wetting agents and suspending agents as described above. Sterile injection preparations may also be sterile injection solutions or suspensions prepared in non-toxic parenterally acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. In addition, sterile fixed oils may be conveniently used as solvents or suspension media. For this purpose, any blended fixed oils may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may also be used to prepare injections.

The term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" refers to a salt of the ligand-drug conjugate of the present disclosure, or a salt of the compound described in the present disclosure, which is safe and effective when used in mammals and has the desired biological activity. The disclosed antibody-drug conjugate contains at least one amine group, and thus can form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrosulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrophosphate, dihydrogen phosphate, salicylate, hydrocitrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

The term "carrier" used in the drugs disclosed herein refers to a system that can change the way drugs enter the human body and their distribution in the body, control the release rate of drugs, and transport drugs to targeted organs. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure. Preferred examples include micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, and can be used as excellent drug carriers.

The term "excipient" refers to the additives in drug preparations other than the main drug, which can also be called excipients. For example, adhesives, fillers, disintegrants, and lubricants in tablets; the base part in semisolid preparations such as ointments and creams; preservatives, antioxidants, flavor enhancers, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, coloring agents, etc. in liquid preparations can all be called excipients.

The term "diluent" is also called filler, and its main purpose is to increase the weight and volume of tablets. The addition of diluents not only ensures a certain volume size, but also reduces the dosage deviation of the main ingredients and improves the compressibility of the drug. When the drug in the tablet contains oily components, an absorbent needs to be added to absorb the oily substance to keep it in a "dry" state, which is conducive to making tablets.

The terms "connected" and "bound" are used interchangeably. When referring to the connection between two molecules, it means that the two molecules are connected by a covalent bond or the two molecules are associated 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 via an intermediate group, compound or molecule (e.g., a linking group). "Linked" encompasses the linkage of amino acid residues via covalent bonding, including but not limited to amide bonding, disulfide bonding, methylene bonding (also known as methylene bridge bonding), thioether bonding, hydrogen bonding, and electrostatic bonding.

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

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 preventive uses, beneficial or desired results include eliminating or reducing the risk, reducing the severity or delaying the onset of a disorder, including the biochemical, histological and/or behavioral symptoms of the disorder, its complications and intermediate pathological phenotypes presented during the development of the disorder. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target gene, target mRNA or target protein-related diseases disclosed herein or improving one or more symptoms of the disease, reducing the dosage of other drugs required to treat the disease, enhancing the efficacy of another drug, and/or delaying the progression of the target gene, target mRNA or target protein-related disease disclosed herein in patients.

The term "antibody" refers to immunoglobulin. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The different amino acid compositions and arrangement sequences of the constant regions of the immunoglobulin heavy chains can classify immunoglobulins into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ, δ, γ, α, and ε chains. The same type of Ig can be divided into different subclasses based on the differences in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ or λ chains according to the differences in the constant regions. Each of the five Ig classes can have either a kappa chain or a lambda chain.

The sequences of about 110 amino acids near the N-terminus of the heavy and light chains of the full-length antibody vary greatly, which is the variable region (Fv region); the amino acid sequence near the C-terminus is relatively stable, which is the constant region. The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. The 3 hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions, and the order from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to 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 disclosed herein conform to the known IMGT rules.

In the disclosed embodiments, the antibody can form a connection bond with the linking unit through the impurity atoms on the antibody.

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

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, you must first establish a fusion tumor that secretes mouse-specific monoclonal antibodies, then select variable region genes from the mouse fusion tumor cells, and then select human antibody constant region genes as needed. The mouse variable region gene and the human constant region gene are connected to form a chimeric gene and inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into human antibody variable region frameworks, i.e., different types of human germline antibody framework sequences. It can overcome the heterologous reactions induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public databases including germline antibody gene sequences or published references. For example, the germline DNA sequences of human heavy chain 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), and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the variable region framework sequence of the human antibody can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies after further affinity maturation of CDR by phage display. Literature further describing methods for humanization using mouse antibodies includes, 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)].

The term "fully human antibody", "fully human antibody" or "completely human antibody", also known as "fully human monoclonal antibody", refers to an antibody whose variable and constant regions are all human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The relevant technologies for the preparation of human antibodies mainly include: human fusion tumor technology, EBV-transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

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 the antibody. Examples of binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab) ₂ fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge on the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; and (vi) isolated complementary determining regions (CDRs) or (vii) a combination of two or more isolated CDRs, which may be optionally linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker using recombinant methods, thereby enabling them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, e.g., 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 of ordinary skill in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen binding moieties may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

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

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

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

In addition, the Fab' can be produced by inserting a DNA encoding the Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or a eukaryotic organism to express the Fab'.

"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 is 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.

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

The term "single-chain antibody", "single-chain Fv" or "scFv" means 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 may 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 with 1-4 repeats are used (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.

The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of a CDR can be determined using any of a variety of well-known schemes, including the "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering rule (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering rule (Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, for the classical format, following the Kabat rule, 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 rule, 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 consist 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 26-35 (CDR1), 51-57 (CDR2), and 93- 102 (CDR3), and the CDR amino acid residues in VL are numbered approximately 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.

The term "antibody framework" refers to the part of the 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.

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

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. Typically, the antibody binds with an affinity (KD) of less than about ^10-7 M, such as less than about ^10-8 M, ^10-9 M or ^10-10 M or less.

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

In this disclosure, the CAS No. of mc-vc-pab-MMAE is: 646502-53-6, and has the following structure:

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

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

Figure 3 shows the HIC of adalimumab-MMAE-03 conjugate.

Figure 4 shows the HIC of adalimumab-MMAE-04 conjugate.

Figure 5 shows the HIC of adalimumab-MMAE-05 conjugate.

Figure 6 shows the HIC of adalimumab-MMAE-06 conjugate.

Figure 7 shows the HIC of adalimumab-MMAE-07 conjugate.

Figure 8 shows the HIC of adalimumab-MMAE-08 conjugate prepared without _ZnCl2 .

Figure 9 shows the HIC of Farletuzumab-MMAE-01 conjugate.

Figure 10 shows the HIC of Farletuzumab-MMAE-02 conjugate prepared without _ZnCl2 .

Figure 11 shows the HIC of Enoblituzumab-MMAE-01 conjugate.

Figure 12 shows the HIC of Enoblituzumab-MMAE-02 conjugate prepared without _ZnCl2 .

Figure 13 is a HIC of the adalimumab-4-B00 conjugate prepared using the disclosed process.

Figure 14 shows the HIC of adalimumab-4-B00 conjugate prepared using the traditional process.

The present disclosure is further described below in conjunction with the embodiments, but these embodiments do not limit the scope of the present disclosure. The experimental methods in the embodiments of the present disclosure that do not specify specific conditions are usually carried out according to conventional conditions, such as the Cold Spring Harbor Antibody Technology Experimental Manual and the Molecular Cloning Manual; or according to the conditions recommended by the raw material or product manufacturer. Reagents that do not specify the specific source are conventional reagents purchased on the market.

Implementation example

Example 1, HIC-HPLC method 1

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

1.1 Main equipment and mobile phase

High performance liquid chromatograph: Agilent 1260.

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

Mobile phase: Mobile phase A (MPA): 20 mM PB + 1.5 M (NH ₄ ) ₂ SO ₄ (pH 7.00).

Preparation example of mobile phase A: Weigh 1.22g sodium dihydrogen phosphate dihydrate (NaH ₂ PO ₄ ．2H ₂ O, MW: 156.01g/mol), 4.37g sodium dihydrogen phosphate dodecahydrate (Na ₂ HPO ₄ ．12H ₂ O, MW: 358.14g/mol), 198.21g ammonium sulfate ((NH ₄ ) ₂ SO ₄ , MW: 132.14g/mol), add 950ml purified water to dissolve, adjust the pH value to 7.00±0.05 with 5M NaOH solution, add purified water to dissolve to 1000ml, then filter with 0.22μm filter membrane, store at 2-8℃, valid for 1 month, filter before use.

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

Example of preparation of mobile phase B: Weigh 0.97g sodium dihydrogen phosphate dihydrate (NaH ₂ PO ₄ ．2H ₂ O, MW: 156.01g/mol), 3.50g sodium dihydrogen phosphate dodecahydrate (Na ₂ HPO ₄ ．12H ₂ O, MW: 358.14g/mol), add 700ml purified water to dissolve, adjust the pH value to 7.00±0.05 with 5M NaOH solution, add purified water to 750ml, add 250ml isopropanol, and then filter with 0.22μm filter membrane, store at 2-8℃, the validity period is 1 month, and filter before use.

1.2 Chromatographic methods and calculations

The chromatographic method is shown in Table 1.

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

Calculation formula: DAR=Σ(DARn peak area percentage (%)*n)/100

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

The total peak area refers to the total peak area of each component (D0, D2, D4, D6 and D8).

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

Example 2, HIC-HPLC method 2

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

2.1 Main equipment and mobile phase

High performance liquid chromatograph: Agilent 1260.

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

Mobile phase: Mobile phase A (MPA): 100 mM Citrate + 1.5 M (NH ₄ ) ₂ SO ₄ (pH 5.00).

Preparation example of mobile phase A: Weigh 8.62g of citric acid monohydrate (C ₆ H ₈ O ₇ ．H ₂ O, MW: 210.14g/mol), 17.35g of sodium citrate dihydrate (Na ₃ C ₆ H ₅ O ₇ ．2H ₂ O, MW: 294.10g/mol), 198.21g of ammonium sulfate ((NH ₄ ) ₂ SO ₄ , MW: 132.14g/mol), add 800ml of purified water to dissolve, adjust the pH value to 5.00±0.05 with 1M NaOH solution, add purified water to dissolve to 1000ml, then filter with 0.22μm filter membrane, store at 2-8℃, valid for 1 month, filter before use.

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

Preparation example of mobile phase B: weigh 6.90g of citric acid monohydrate (C ₆ H ₈ O ₇ ．H ₂ O, MW: 210.14g/mol), 13.88g of sodium citrate dihydrate (Na ₃ C ₆ H ₅ O ₇ ．2H ₂ O, MW: 294.10g/mol), 198.21g of ammonium sulfate ((NH ₄ ) ₂ SO ₄ , MW: 132.14g/mol), add 700ml of purified water to dissolve, adjust the pH value 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℃. The shelf life is 1 month. Filter before use.

2.2 Chromatographic methods and calculations

The chromatographic method is shown in Table 2.

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

Calculation formula: DAR=Σ(DARn peak area percentage (%)*n)/100

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

The total peak area refers to the total peak area of each component (D0, D2, D4, D6 and D8).

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

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

3.1 Referring to WO2020164561A1, adalimumab-MMAE samples were prepared: adalimumab-MMAE-01, adalimumab-MMAE-02, and adalimumab-MMAE-03. The reaction conditions are shown in Table 3, and the specific results are shown in Table 4 and Figures 1 to 3.

The specific steps are as follows:

(1) ZnCl ₂ (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 incubated at 0-4°C overnight;

(2) Add mc-vc-pab-MMAE (purchased from Lianning Biotechnology, 1.350 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) and react in a water bath at room temperature for 1 hour;

(3) According to Table 3, add or not add appropriate amount of N-acetylcysteine (NAC, 1.620mM, as quencher), EDTA solution (0.540mM, as metal chelator) and sodium ascorbate (DHAA, 1.080mM, as oxidant) solution to the system, and react at room temperature for 30 minutes;

(4) Use ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) to purify the reaction solution (remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC) and then use the HIC-HPLC method for characterization. The HIC-HPLC method refers to Example 1.

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 the process has a significant effect on the coupling reaction results. After increasing the coupling temperature, the selectivity becomes worse (the D4 ratio of the adalimumab-MMAE-01 sample is significantly reduced compared with the adalimumab-MMAE-01 sample), and an increase in the number of odd peaks can be seen. In addition, if the 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 hydroxyl group on the antibody, and the selectivity will be lost (the main component of the adalimumab-MMAE-03 sample changes from D4 to D6).

3.2 After replacing the reducing agent TCEP with diphenylphosphoacetic acid (DPA), the coupling experiment was performed again using the same process as in Section 3.1 to prepare adalimumab-MMAE samples: adalimumab-MMAE-04, adalimumab-MMAE-05, and adalimumab-MMAE-06. The reaction conditions are shown in Table 5, and the specific results are shown in Table 6 and Figures 4 to 6.

The specific steps are as follows:

(1) ZnCl ₂ (0.270 mM) and diphenylphosphoacetic acid (purchased from Yuhan Chemical, 0.390 mM) were added to a solution of adalimumab (0.135 mM) (histidine buffer, pH = 6.5) and incubated at 0-4°C overnight;

(2) Add mc-vc-pab-MMAE (purchased from Lianning Biotechnology, 1.350 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) and react in a water bath at room temperature for 1 hour;

(3) According to Table 5, add or not add an appropriate amount of N-acetylcysteine (NAC, 1.620mM, as a quencher) to the system, add EDTA solution (0.540mM, as a metal chelator) and sodium ascorbate (DHAA, 1.080mM, as an oxidant) solution, and react at room temperature for 30 minutes;

(4) Use ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) to purify the reaction solution (remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC) and then use the HIC-HPLC method for characterization. The HIC-HPLC method refers to Example 1.

The HIC results in Table 6 show that after the reducing agent is replaced 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.

From the results of Table 4 and Table 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 of the process in Example 3.1. It does not require the use of a reaction quenching reagent, and the coupling reaction is not limited by conditions such as temperature.

Example 4: Prepared adalimumab-MMAE conjugate and characterization of its drug distribution

On the basis of Example 3.1, the reoxidation step was further omitted to optimize the preparation method. Adalimumab-MMAE-07 was prepared. For specific results, see Table 7 and Figure 7.

The specific steps are as follows:

(1) ZnCl ₂ (0.270 mM) and diphenylphosphoacetic acid (purchased from Yuhan Chemical, 0.412 mM) were added to a solution of adalimumab (0.135 mM) (histidine buffer, pH = 6.5) and incubated at 0-4°C overnight;

(2) Add EDTA (0.540 mM) solution to chelate Zn2 ⁺ ions;

(3) Add mc-vc-pab-MMAE (purchased from Lianning Biotechnology, 0.810 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) and react in a water bath at room temperature for 1 hour;

(4) Ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) are used to purify the reaction solution (remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC).

Drug distribution characterization: HIC-HPLC was used to analyze the drug-antibody ratio (DAR) and drug distribution. The HIC-HPLC method is shown in Example 1.

Example 5: Prepared Farletuzumab-MMAE conjugate and characterization of its drug distribution

On the basis of Example 3.1, the reoxidation step was further omitted to optimize the preparation method. Farletuzumab-MMAE-01 was prepared. The specific results are shown in Table 8 and Figure 9. Among them, Farletuzumab is a humanized anti-human folate receptor alpha (FRA) antibody.

The specific steps are as follows:

(1) ZnCl ₂ (0.276 mM) and diphenylphosphinothioacetic 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 incubated at 0-4°C overnight;

(2) Add EDTA (0.552 mM) solution to chelate Zn2 ⁺ ions;

(3) Add mc-vc-pab-MMAE (purchased from Lianning Biotechnology, 0.828 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) and react in a water bath at room temperature for 1 hour;

(4) Use ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) to purify the reaction solution (remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC). Drug distribution characterization: Use HIC-HPLC to analyze the drug-antibody ratio (DAR) and drug distribution. The HIC-HPLC method refers to Example 1.

Example 6, prepared Enoblituzumab-MMAE conjugate and characterization of its drug distribution

On the basis of Example 3.1, the reoxidation step was further omitted to optimize the preparation method. Enoblituzumab-MMAE-01 was prepared. The specific results are shown in Table 9 and Figure 11. Among them, Enoblituzumab is an Anti-B7H3 antibody.

The specific steps are as follows:

(1) ZnCl ₂ (0.278 mM) and diphenylphosphinothioacetic 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 incubated at 0-4°C overnight;

(2) Add EDTA (0.556 mM) solution to chelate Zn2 ⁺ ions;

(3) Add mc-vc-pab-MMAE (purchased from Lianning Biotechnology, 0.834 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) and react in a water bath at room temperature for 1 hour;

(4) Use ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) to purify the reaction solution (remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC). Drug distribution characterization: Use HIC-HPLC to analyze the drug-antibody ratio (DAR) and drug distribution. The HIC-HPLC method refers to Example 1.

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.

Example 7: Investigating the effect of transition metal ions on the preparation method

The effect of not adding ZnCl ₂ on the preparation method was investigated. The preparation method of the sample with ZnCl ₂ added is referred to Example 4-6.

Without adding ZnCl ₂ , samples Adalimumab-MMAE-08 (without ZnCl 2), Farletuzumab-MMAE-02 (without ZnCl 2), and Enoblituzumab-MMAE-02 (without ZnCl 2) were prepared. For specific results, see Table 10 and Figures 8, 10, and 12. The only difference from Examples 4-6 is that ZnCl ₂ was not added.

Without adding _ZnCl2, the specific steps are as follows:

(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 leave it at 0-4°C overnight;

(2) Add 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), and react in a water bath at room temperature for 1 hour;

(3) Use ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) to purify the reaction solution (remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC). Drug distribution characterization: Use HIC-HPLC to analyze the drug-antibody ratio (DAR) and drug distribution. The HIC-HPLC method refers to Example 1.

The HIC results in Table 10 show that compared with using only the reducing agent DPA, the introduction of ZnCl ₂ can significantly improve the selectivity of the reduction reaction.

Example 8: Comparison of adalimumab-4-B00 conjugates prepared using the disclosed process and traditional process and their drug distribution

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.

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

(1) ZnCl ₂ (0.270 mM) and diphenylphosphoacetic acid (purchased from Yuhan Chemical, 0.412 mM) were added to a solution of adalimumab (0.135 mM) (histidine buffer, pH = 6.5) and incubated at 0-4°C overnight;

(2) Add EDTA (0.540 mM) solution to chelate Zn2 ⁺ ions;

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

(4) Add 4-B00 (0.81 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) and react in a water bath at room temperature for 3 hours;

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

(5) Ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) were used to purify the reaction solution (remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC). HIC-HPLC was used to analyze the drug-antibody ratio (DAR) and drug distribution. The HIC-HPLC method was referred to Example 2. The specific results are shown in Table 11 and Figure 13.

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

(1) Add tri(2-carboxyethyl)phosphine (TCEP, purchased from Aldrich Sigma, 0.297 mM) dissolved in water to a solution of adalimumab (0.135 mM) (histidine buffer, pH=6.5) and react at room temperature for 2 hours;

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

(3) Add 4-B00 (0.81 mM) dissolved in 100% DMSO (purchased from Aldrich Sigma) and react in a water bath at room temperature for 3 h;

(4) Ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) were used to purify the reaction solution (remove small molecule process impurities without affecting the different drug-loaded components of the prepared ADC). HIC-HPLC was used to analyze the drug-antibody ratio (DAR) and drug distribution. The HIC-HPLC method was referred to Example 2. The specific results are shown in Table 11 and Figure 14.

Table 11

The HIC results in Table 11 show that compared with the traditional 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.

Although the above invention has been described in detail with the aid of drawings and examples for the sake of clear understanding, the description and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (16)

A method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, comprising the following steps: (a) reacting the reducing agent and the antibody in the presence of transition metal ions to selectively reduce the intrachain disulfide bonds of the antibody to hydroxyl groups; (b) reacting the hydroxylated antibody obtained in step (a) with a drug linker intermediate or a pharmaceutically acceptable salt thereof; (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. The method as described in 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⁺ . The method as claimed in claim 1 or 2, wherein the reducing agent in step (a) is a reducing agent containing a diphenylphosphine 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. A method as described in 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. The method as claimed in 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, ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid dicalcium salt, diethylenetriaminepentaacetic acid or a mixture thereof. A method as described in any one of claims 1 to 5, wherein the drug linker intermediate or a pharmaceutically acceptable salt thereof contains a reactive group capable of reacting with a hydroxyl group. A method as described in any one of claims 1 to 6, wherein the average drug loading of the obtained antibody-drug conjugate or its pharmaceutically acceptable salt is 3.0 to 5.0; preferably greater than 3.0 and less than 5.0. A method as described in any one of claims 1 to 7, wherein the antibody is selected from a single antibody or multiple antibodies. A method as described in 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. A method as described in any one of claims 1 to 9, wherein the antibody is selected from IgG1 or IgG4 isotype. A method as described in any one of claims 1 to 10, wherein the drug is selected from a diagnostic agent, a therapeutic agent or a labeling agent. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, prepared by the method as described in any one of claims 1 to 11. A pharmaceutical composition comprising an effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 12, and a formulator, diluent or carrier as required. 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 or a pharmaceutically acceptable salt thereof as described in claim 12, or a pharmaceutical composition as described in claim 13; preferably, the condition or disease is selected from a tumor, 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, suppurative hidradenitis, uveitis, Behcet's disease, spondyloarthropathies, and psoriasis. A method for selectively reducing an antibody comprises step (a): reacting a reducing agent and an antibody in the presence of a transition metal ion to selectively reduce the intrachain disulfide bonds of the antibody to hydroxyl groups. A method for selectively reducing antibodies as described in claim 15, wherein step (a) is as defined in any one of claims 2 to 10.

Images