WO2023222108A1

WO2023222108A1 - Method for preparing antibody-drug conjugate

Info

Publication number: WO2023222108A1
Application number: PCT/CN2023/095226
Authority: WO
Inventors: 张�浩; 李雪晴; 马舒; 李磊
Original assignee: 上海迈晋生物医药科技有限公司
Priority date: 2022-05-20
Filing date: 2023-05-19
Publication date: 2023-11-23

Abstract

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

Description

Preparation method of antibody-drug conjugates

This disclosure claims priority from Chinese patent application 202210553520.X with a filing date of May 20, 2022. This disclosure cites the full text of the above-mentioned Chinese patent application.

Technical field

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

Background technique

Antibody drug conjugate (ADC) is obtained by connecting an antibody or antigen-binding fragment to a biologically active toxin through a stable chemical linker compound. It combines the powerful killing power of small molecule drugs with the specificity of antibodies and can It has the specificity of antibody binding to surface antigens of normal cells and tumor cells and the high efficiency of cytotoxicity, while avoiding the shortcomings of low antibody efficacy and excessive side effects of cytotoxicity. Antibody-drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells, thereby reducing adverse reactions during treatment (MullardA, (2013) Nature Reviews DrugDiscover y, 12:329–332; DiJoseph JF, Armellino DC , (2004) Blood, 103:1807-1814).

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

ADC drugs often connect antibodies and toxins through two chemical strategies. One is based on the coupling of lysine on the antibody, such as the marketed drugs Mylotarg, Kadcyla, and Besponsa, which all rely on the succinimide group of the linker. Bind to the lysine side chain amino group to connect the toxin to the monoclonal antibody. An antibody molecule contains 80 to 90 lysine, and conjugation may occur on nearly 40 different lysine residues, so even There are many joint sites and they are not fixed. The other is based on the conjugation of cysteine on the antibody. Compared with the intra-chain disulfide bonds that stabilize the structure of the antibody, the inter-chain disulfide bonds are easily partially reduced to free sulfhydryl groups, and then combined with the connecting group of the linker (such as the maleimide group). Both IgG1 and IgG4 antibodies contain 4 pairs of inter-chain 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, current cysteine technology still has shortcomings. For example, ADC samples with a mean DAR 4 have a normal distribution of ADC components, making the ADC drug non-uniform. Additionally, the average DAR4 ADC sample, while Containing a certain proportion of ADC components connected to 6 and 8 toxins makes ADC drugs easier to be eliminated 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 emerged to try to introduce cysteine into the antibody structure, thereby introducing sulfhydryl groups, thereby achieving site-specific coupling of antibodies and drug molecules. Union. For example, some literature points out that a stable and uniform ADC can be obtained by inserting cysteine after the 239th amino acid of the antibody heavy chain, introducing a free sulfhydryl group and coupling with the toxin (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 ADC drugs.

In addition, some scholars have chosen to carry out cysteine mutations at special sites on the antibody. The mutated antibody contains free sulfhydryl groups and can also meet the requirements of ADC uniformity. However, this method is a modification of the antibody structure. In order to find mutation sites that do not affect the antibody structure and function, a large number of site screenings are 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 sulfhydryl groups in the antibody hinge region, improving the probability of conjugation of the reduced sulfhydryl groups in the Fab region with toxins. This method significantly increases DAR 4 component ratio. However, in this method, excessive use of reducing agent in the reduction stage causes a large number of interchain disulfide bonds to open, and the toxin quenching and re-oxidation steps must be performed after the coupling is completed. The operation is complicated and process impurities are easily introduced. Based on this, the present disclosure uses process optimization methods to improve the uniformity of ADCs based on cysteine coupling.

The method provided by the present disclosure for preparing antibody-drug conjugates (ADC) or pharmaceutically acceptable salts thereof can prepare ADC with improved uniformity. The preparation method is simple and requires no quenching reaction to terminate the coupling reaction after the coupling reaction, and no reoxidation step is required to reoxidize the unreacted sulfhydryl groups of the antibody. At the same time, the coupling reaction is not limited by temperature and can be carried out at -10°C to 40°C. The coupling substrate has no effect on the selectivity of the coupling reaction. It can limit and increase the proportion of DAR 4 products (D4) in ADC, with optimized safety and efficacy.

Contents of the invention

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

(a) Reacting a reducing agent and an antibody in a buffer system in the presence of transition metal ions to selectively reduce the interchain disulfide bonds within the antibody to sulfhydryl groups;

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

In some embodiments, step (c) is also included or not included: step (b) obtains the antibody-drug conjugate or a pharmaceutically acceptable salt thereof without going through a quenching step and/or a re-oxidation step.

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

(a) 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 interchain disulfide bonds within the antibody to sulfhydryl groups;

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

(c) Step (b) obtains the antibody-drug conjugate or a pharmaceutically acceptable salt thereof without going through a quenching step and/or a re-oxidation step.

(a) Reacting a reducing agent and an antibody in the presence of transition metal ions to selectively reduce the interchain disulfide bonds within the antibody to sulfhydryl groups;

The quenching step refers to inactivating the reactivity of the unreacted drug linker intermediate to terminate the conjugation reaction step. The conjugation reaction is terminated, for example, by inactivating the reactivity of the unreacted drug linker intermediate with a sulfhydryl-containing reagent (eg, N-acetamide cysteine, cysteine).

The reoxidation step refers to adding an effective amount of oxidizing agent (such as DHAA) to reoxidize the unreacted thiol 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, ZnCl2 _, Zn( _NO3 ) ₂ , _ZnSO4 , Zn( _CH3COO ) ₂ , _ZnI2 , ZnBr2 _, zinc formate, and zinc tetrafluoroborate.

In some embodiments, appropriate transition metal salts that are soluble and can release free Cd ²⁺ or Hg ²⁺ ions are added in step (a), 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 formate ( II), and mercury(II) tetrafluoroborate, etc.

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

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

3-(Diphenylphosphino)benzenesulfonic acid (3-(Diphenylphosphino)benzenesulfonic acid),

4-(Diphenylphosphino)benzoic acid (4-(Diphenylphosphino)benzoic acid),

2-(Diphenylphosphino)ethylamine (2-(Diphenylphosphino)ethylamine),

3-(diphenylphosphino)propylamine (3-(diphenylphosphino)propylamine),

3-(Diphenylphosphino)propionic acid (3-(Diphenylphosphino)propionic acid),

2-(diisopropylphosphino)ethylamine (2-(diisopropylphosphino)ethylamine),

2-(diphenylphosphino)benzoic acid (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 0.01mM to about 0.50mM, such as about 0.01mM, about 0.02mM, about 0.03mM, about 0.04mM, about 0.05mM, about 0.06mM, about 0.07mM, about 0.08 mM, about 0.09mM, about 0.10mM, about 0.11mM, about 0.12mM, 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 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 0.01mM to about 0.50mM, such as about 0.01mM, about 0.02mM, about 0.03mM, about 0.04mM, about 0.05mM, about 0.06mM, about 0.07mM, About 0.08mM, about 0.09mM, about 0.10mM, about 0.15mM, about 0.20mM, about 0.21mM, about 0.22mM, about 0.23mM, about 0.24mM, about 0.25mM, about 0.26mM, about 0.27mM, about 0.28 mM, 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 between any two values. or range; in some embodiments, the final concentration of 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, from about 0.38mM to about 0.45mM.

In some embodiments, the final concentration equivalent ratio (mM/mM) of antibody to 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 between any two values or scope. In some embodiments, the final concentration equivalent ratio of antibody to reducing agent (mM/mM) is from about 1:2.5 to about 1:3.5.

In some embodiments, the final concentration equivalent ratio (mM/mM) of antibody to transition metal ion is from 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 antibody to 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 may or may not contain a metal chelating agent. In some embodiments, the buffer does not contain metal chelating agents.

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 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 - It is carried out at 5°C to about 37°C; in some embodiments, it is carried out at about 0°C to about 37°C; in some embodiments, it is carried out at about 0°C to about 4°C; in some embodiments, it is carried out at about 0°C to about 37°C. Conducted at about 37°C; in some embodiments, conducted at about 10°C to about 25°C; in some embodiments, conducted at about -5°C to about 5°C; in some embodiments, conducted at 0°C to 30°C ; In some embodiments, the temperature is from 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, step (a) The reaction time is 2h.

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

In some embodiments, the reaction conditions of step (a) depend on the specific antibody to be conjugated. Determination of the incubation period and temperature based on a particular antibody is within the ability of one of ordinary skill in the art. For example, the antibody to be conjugated is typically reacted with a reducing agent by incubation overnight at 4°C in the presence of transition metal ions.

In some embodiments, the method of preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof according to the present disclosure further includes adding a metal chelating agent between step (a) and step (b). A step of.

In some embodiments, the metal chelating agent will be filtered out in subsequent dialysis, ultrafiltration, or gel filtration.

In some embodiments, the metal chelating agent is used to chelate transition metal ions; in some embodiments, the metal chelating agent is selected from ethylenediaminetetraacetic acid (hereinafter also referred to as "EDTA"); in some embodiments, The metal chelating agent is selected from ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid dicalcium salt, diethylenetriaminepentacetic acid or mixtures 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 of transition metal ions to metal chelators (mM/mM) is about 1:2.

The drug linker intermediate of the present disclosure or its pharmaceutically acceptable salt is not particularly limited as long as it is a compound capable of reacting with the interchain sulfhydryl group of the antibody, such as a drug linker intermediate having an N-substituted maleimide group. . In some embodiments, the drug linker intermediate or a pharmaceutically acceptable salt thereof contains a reactive group capable of reacting with a thiol group; in some embodiments, the drug linker intermediate or a pharmaceutically acceptable salt thereof contains Maleimide (such as N-substituted maleimide), bromine, iodine, fluorine, alkene, alkene or sulfonyl.

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 compounds represented by formula (III-A') or (III-B') or pharmaceutically acceptable salts thereof:

in,

Ring A is The ring A is optionally substituted by one or more substituents _Q1 ;

Ring B is The ring B is optionally substituted by one or more substituents _Q1 ;

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

R _5a and R _5b are each independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by 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 oxo or thio;

Ring C is selected from The ring C is optionally substituted by one or more substituents _Q1 ;

Ring D is The ring D is optionally substituted by one or more substituents _Q1 ;

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 optionally one Or a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group substituted by multiple substituents Q ₁ ;

R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by 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 haloalkyl and C ₁ -C ₆ alkoxy;

R ₂ is each independently selected from -CH ₂ OH, -CH ₂ SH, -CH ₂ Cl, -SCH ₂ Cl, -SCH ₂ F, -SCH ₂ CF ₃ , -OH, -OCH ₂ CN, -OCH ₂ Cl. , -OCH ₂ F , -OCH ₃ , -OCH ₂ CH ₃ , -SCH ₂ CN,

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 is each independently hydrogen or halogen;

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

Each substituent group Q ₁ is independently selected from halogen, hydroxyl, mercapto, deuterium, oxo, thio, cyano, amino, carboxyl, C ₁ -C ₆ alkyl and C ₁ -C ₆ alkoxy;

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 hydrogen atom, C ₁ -C ₆ alkyl group, C ₁ -C ₆ haloalkyl group, C ₁ -C ₆ alkoxy group, hydroxyl group and -NR _i R _j ;

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

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

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

L ₁ is an amino acid unit, preferably -glycine-glutamic acid-or

X is halogen;

PG is an amino protecting group; and

The proviso is that when R _5a is hydrogen or alkyl, R _5b is not hydrogen or alkyl.

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

or a pharmaceutically acceptable salt thereof.

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

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

In some embodiments, the drug linker intermediate is a drug conjugated 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 a drug molecule and the other of which can covalently couple an antibody. In some embodiments, linkers include cleavable linkers and non-cleavable linkers. In some embodiments, the cleavable linker is selected from peptide linkers that are cleaved by intracellular proteases, such as lysins, and in some embodiments, body proteases or endosomal proteases.

In some embodiments, the linker comprises amino acid unit L ₁ , and said amino acid unit L ₁ preferably contains from 2 to 7 selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamine Acid, aspartic acid, homolysine, N-methyl-valine, Peptide residues composed of amino acids (q is an integer from 1 to 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-valine-citrulline (Me-Val-Cit) , alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala), glycine-lysine ( Gly-Lys), glycine-valine-citrulline (Glv-Val-Cit) and glycine-glycine-glycine (Gly-Gly-Gly) and

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

In certain embodiments, the stretching unit is selected from:

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

In certain embodiments, the linker is selected from:

In some embodiments, the linker is selected from:

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

In some embodiments, the amount of the drug linker intermediate or pharmaceutically acceptable salt thereof in step (b) is excess. In some embodiments, the final concentration equivalent ratio (mM/mM) of the antibody to the drug linker intermediate or a pharmaceutically acceptable salt thereof (based on the drug linker intermediate) is about 1:1 to about 1:20, for example about 1:1, John 1:2, John 1:3, John 1:4, John 1:5, John 1:6, John 1:7, John 1:8, John 1:9, John 1:10, John 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 a pharmaceutically acceptable salt thereof (based on the drug linker intermediate) is about 1:6.

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

Examples of solvents that can be used in the present disclosure to dissolve the drug linker intermediate include organic solvents such as 50% acetone aqueous solution, 80% ethanol aqueous solution, 80% methanol aqueous solution, 80% isopropyl alcohol aqueous solution, 80% dimethyl sulfoxide aqueous solution, dimethyl sulfoxide aqueous solution, Methyl 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 Conducted at 25°C; in some embodiments, conducted 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 1-2 hours.

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

Those skilled in the art can select appropriate purification methods 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 a pharmaceutically acceptable salt thereof 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 DO, D2, D4, D6 and D8, the antibody-drug conjugate prepared by the disclosed method or a pharmaceutically acceptable salt thereof contains a content of D4 greater than about 50 wt%, For example, above about 50 wt%, above about 55 wt%, above about 60 wt%, above about 61 wt%, above about 62 wt%, above about 63 wt%, above about 64 wt%, above about 65 wt%, high At about 66 wt%, above about 67 wt%, above about 68 wt%, above about 69 wt%, above about 70 wt%, above about 71 wt%, above about 72 wt%, above about 73 wt%, above about 74wt%, higher than about 75wt%; in some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate prepared by the disclosed method or its pharmaceutically acceptable salt contains The content of D4 is selected from about 55 wt% to about 75 wt%, about 60 wt% to about 70 wt%, and about 55 wt% to about 65 wt%.

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

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains D4a, and the D4a is selected from the group consisting of having and only 4 drug linkers, and the 4 drug linkers are combined with heavy- An antibody-drug conjugate with sulfhydryl groups between light chains or a pharmaceutically acceptable salt thereof. Based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate prepared by the disclosed method or a pharmaceutically acceptable salt thereof contains a content of D4a higher than about 50 wt%, for example, higher than about 50 wt. %, above about 55 wt%, above about 60 wt%, above about 61 wt%, above about 62 wt%, above about 63 wt%, above about 64 wt%, above about 65 wt%, above about 66 wt%, Above about 67 wt%, above about 68 wt%, above about 69 wt%, above about 70 wt%, Above about 71 wt%, above about 72 wt%, above about 73 wt%, above about 74 wt%, above about 75 wt%; in some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the The antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the disclosed method contains D4a in a content selected from about 55wt% to about 75wt%, about 60wt% to about 70wt%, and about 55wt% to about 65wt%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure includes D4b, and the D4b is selected from the group consisting of having and only 4 drug linkers, and the 4 drug linkers are combined with heavy- Antibody-drug conjugates with sulfhydryl groups between heavy chains or pharmaceutically acceptable salts thereof. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains D4b in an amount less than about 40 wt%, such as less than about 40 wt%, less than About 30 wt%, less than about 20 wt%, less than about 15 wt%, less than about 10 wt%, less than about 5 wt%; in some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody - The drug conjugate or a pharmaceutically acceptable salt thereof contains D4b in an amount selected from about 5 wt% to about 30 wt%, about 5 wt% to about 20 wt%, and about 5 wt% to about 10 wt%.

In some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the disclosed method contains a content of DO + D8 of less than about 20 wt. %, for example, 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%, low At about 3wt%. In some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the disclosed method contains a content of DO + D8 selected from about 3wt % to about 20 wt%, about 5 wt% to about 15 wt%, about 3 wt% to about 10 wt%.

In some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the disclosed method contains a content of D6 of less than about 20 wt%, For example, 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%, low At about 11 wt%, below about 10 wt%, below about 9 wt%, below about 8 wt%, below about 7 wt%, below about 6 wt%, below about 5 wt%. In some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the disclosed method contains a content of D6 selected from about 5 wt% to About 20 wt%, about 5 wt% to about 15 wt%, about 5 wt% to about 10 wt%.

In some embodiments, the total weight based on DO, D2, D4, D6 and D8 refers to the total weight of unpurified DO, D2, D4, D6 and D8 obtained after the coupling reaction.

In some embodiments, the total weight based on DO, D2, D4, D6 and D8 refers to DO, D2, D4 obtained after the coupling reaction and only purified to remove small molecule process impurities (for example: free toxins and organic solvents, etc.) , the total weight of D6 and D8, where the purification step will not affect the different drug-loading components of the prepared ADC.

In some embodiments, based on the sum of peak areas, the antibody-drug conjugate prepared by the disclosed method or a pharmaceutically acceptable salt thereof comprising 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%; 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 method of the present disclosure 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 antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains a peak area percentage of D4 that 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 method of the present disclosure contains D4a, and the D4a is selected from the group consisting of having and only 4 drug linkers, and the 4 drug linkers are combined with heavy- An antibody-drug conjugate with sulfhydryl groups between light chains or a pharmaceutically acceptable salt thereof. Based on the sum of peak areas, the antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains a peak area percentage of D4a 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%, or any value or range between any two values; in some embodiments, based on the sum of peak areas, the The antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the disclosed method contains a peak area percentage of D4a selected from about 55% to about 75%, about 60% to about 70%, and about 55% to about 65 %.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure includes D4b, and the D4b is selected from the group consisting of having and only 4 drug linkers, and the 4 drug linkers are combined with heavy- Antibody-drug conjugates with sulfhydryl groups between heavy chains or pharmaceutically acceptable salts thereof. The antibody-drug conjugate or pharmaceutically acceptable salt thereof comprises a peak area percentage of D4b of 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 antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains a peak area percentage of D4b 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 peak areas, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains a peak area percentage of DO+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%, less than about 3%. In some embodiments, based on the sum of peak areas, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains a peak area percentage of DO+D8 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 peak areas, the antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the method of the present disclosure 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%, less than about 5%. In some embodiments, based on the total peak area, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains a peak area percentage of D6 selected from about 5% to about 20%, about 5 % to about 15%, about 5% to about 10%.

Based on the sum of peak areas, the peak area percentage refers to: detecting the antibody-drug conjugate (ADC) prepared in the present disclosure or its pharmaceutically acceptable salt by HIC-HPLC method, and calculating the deduction by comparing with the spectrum of the blank solution Calculate the sum of the areas of each chromatographic peak after the blank solution. Integrate the chromatogram to obtain the sum of the peak areas. Use the area normalization method to calculate the peak area of each component (such as D0, D2, D4, D6 or D8) in the sum of the peak areas. percentage.

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 DO, D2, D4, D6 and D8, the antibody-drug conjugate prepared by the disclosed method or a pharmaceutically acceptable salt thereof contains a peak area percentage of D4 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%; in some embodiments, based on DO, D2, D4, The sum of the peak areas of D6 and D8. The antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains a peak area percentage of D4 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 DO, D2, D4, D6 and D8, the antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains a peak area percentage of D4 that is greater than that using TCEP Obtain the content of D4.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains D4a, and the D4a is selected from the group consisting of having and only 4 drug linkers, and the 4 drug linkers are combined with heavy- An antibody-drug conjugate with sulfhydryl groups between light chains or a pharmaceutically acceptable salt thereof. Based on the sum of the peak areas of DO, D2, D4, D6 and D8, the antibody-drug conjugate prepared by the disclosed method or a pharmaceutically acceptable salt thereof contains a peak area percentage of D4a 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%; in some embodiments, based on DO, D2, D4, The sum of the peak areas of D6 and D8, the antibody-drug conjugate prepared by the disclosed method or its pharmaceutically acceptable salt contains a peak area percentage of D4a 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 method of the present disclosure includes D4b, and the D4b is selected from the group consisting of having and only 4 drug linkers, and the 4 drug linkers are combined with heavy- Antibody-drug conjugates with sulfhydryl groups between heavy chains or pharmaceutically acceptable salts thereof. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains a peak area percentage of D4b of 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 DO, D2, D4, D6 and D8, the antibody-drug conjugate The substance or a pharmaceutically acceptable salt thereof contains D4b with a peak area percentage selected from about 5% to about 30%, about 5% to about 20%, and about 5% to about 10%.

In some embodiments, based on the sum of the peak areas of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains a peak area percentage of DO+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%, less than about 3%. In some embodiments, based on the sum of the peak areas of DO, D2, D4, D6 and D8, the peak area percentage of DO+D8 contained in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is selected. From about 3% to about 20%, from about 5% to about 15%, from about 3% to about 10%.

In some embodiments, based on the sum of the peak areas of DO, D2, D4, D6 and D8, the antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains a peak area percentage of D6 of less than about 20 %, for example, 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 sum of the peak areas of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure contains a peak area percentage of D6 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 refers to: the antibody-drug conjugate (ADC) prepared in the present disclosure or its pharmaceutically acceptable salt is detected by the HIC-HPLC method. Compare with the spectrum of the blank solution, calculate the sum of the areas of D0, D2, D4, D6, and D8 after deducting the blank solution, integrate the chromatogram, and obtain the sum of the peak areas based on D0, D2, D4, D6, and D8, using The area normalization method calculates the peak area of each component (such as D0, D2, D4, D6, or D8) as a percentage of the sum of the peak areas based on D0, D2, D4, D6, and D8.

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 based on DO, D2, D4, D6 and D8 of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method shown in the present disclosure refers to the value obtained after the coupling reaction. The sum of the peak areas of D0, D2, D4, D6 and D8 without purification.

In some embodiments, the sum of the peak areas based on DO, D2, D4, D6 and D8 of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method shown in the present disclosure refers to the value obtained after the coupling reaction. Only the sum of the peak areas of D0, D2, D4, D6 and D8 after purifying and removing small molecule process impurities (such as free toxins and organic solvents, etc.), where the purification step will not affect the different drug-loading components of the prepared ADC.

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

In some embodiments, the antibody-drug conjugates (ADCs) of the present disclosure are 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 covalently connecting 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 value between any two values),

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

in,

Represents a single or double bond;

R _1a is selected from hydrogen, alkyl and alkoxy, and each of the alkyl and alkoxy is independently selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl Substituted with one or more substituents in the hydroxyalkyl group;

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

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

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

R _5a and R _5b are each independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano and the following groups 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 _5a and R _5b together form oxo or thio;

Ring C and Ring D are each independently selected from aryl and heteroaryl groups optionally substituted by one or more substituents Q ₁ , and at least one of Ring C and Ring D is selected from the group consisting of optionally substituted by one or more substituents Q 1 A fused ring aryl group or a fused heteroaryl group substituted by substituent Q ₁ ;

X ₂ is selected from -(CR _6a R _6b ) _n -, aryl or heteroaryl optionally substituted by one or more substituents Q ₁ , -O-, -S-, -S(O)-, -S(O)(O)-, -NR _6c -, -CH ₂ S-, -CH ₂ O-, -NHCR _6d R _6e -, -CR _6f =CR _6g - and -C≡C-, or X ₂ does not exist;

R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano or the following groups 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 and the carbon atoms to which they are connected together form a 3- to 10-membered cycloalkane group, or R _6a and R _6b together form oxo or thio;

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 haloalkyl and C ₁ -C ₆ alkoxy;

R ₁ is each independently selected from hydrogen, alkyl and alkoxy, wherein each of said alkyl and alkoxy is independently selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, Substituted with one or more substituents among nitro, hydroxyl and hydroxyalkyl;

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

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

R _2c is 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 is each independently hydrogen or halogen;

R ₄ is each independently selected from hydrogen, halogen and hydroxyl;

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

Each substituent group Q ₁ is independently selected from C ₁ -C ₆ alkyl, halogen, deuterium, hydroxyl, mercapto, -NR _i R _j , oxo, thio, -C(O)R _k , -C(O )OR _k , -S(O)R _k , -S(O)OR _k , -S(O)(O)R _k , -S(O)(O)OR _k , -C(S)R _k , Nitro, cyano, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, 3 to 10-membered cycloalkyl, 3 to 10-membered heterocyclyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused-ring aryl, and 5- to 12-membered fused heteroaryl;

R _k is independently selected from hydrogen atoms, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy, hydroxyl and -NR _i R _j , wherein the alkyl, alkyl Oxygen and haloalkyl are each independently optionally selected from C ₁ -C ₆ alkyl, halogen, hydroxyl, mercapto, -NR _i R _j , oxo, thio, carboxyl, nitro, cyano, C ₁ - C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, 3 to 10 membered cycloalkyl, 3 to 10 membered heterocyclyl, 6 to 10 Substituted with one or more substituents of 5- to 10-membered aryl groups and 5- to 10-membered heteroaryl groups; and

In certain embodiments, R _1a is each independently selected from hydrogen, C ₁ -C ₆ alkyl, and C ₁ -C ₆ alkoxy, which alkyl and alkoxy are each independently selected from the group consisting of Substituted with one or more substituents 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 or 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q ₁ , the heteroaryl group containing at least one nitrogen atom.

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

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

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

_In _certain _embodiments _, _{_} The heteroaryl group contains at least one nitrogen atom.

In certain embodiments, R _5a and R _5b are each independently selected from hydrogen, halogen, hydroxyl, thiol, deuterium, nitro, cyano, and optionally substituted with one or more substituents Q ₁ Group: 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(S)R _k , C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, or R _5a and R _5b together form oxygen Generation or thio.

In certain embodiments, R _5a and R _5b are each independently selected from hydrogen, halogen, hydroxyl, thiol, deuterium, cyano, and the following groups 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 _5a and R _5b together form oxo or thio.

In certain embodiments, R _5a and R _5b are each independently selected from hydrogen, halogen, hydroxyl, thiol, deuterium, cyano, and the following groups 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 oxo or thio.

In certain embodiments, R _5a and R _5b are each independently selected from hydrogen, halogen, hydroxyl, thiol, deuterium, cyano, and the following groups 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 oxo or sulfur generation.

In certain embodiments, Ring C and Ring D are each independently selected from the group consisting of 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered aryl optionally substituted with one or more substituents _Q1 Condensed ring aryl or 5 to 12 membered fused heteroaryl, the heteroaryl or fused heteroaryl contains at least one nitrogen atom.

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

In certain embodiments, Ring C is selected from optionally substituted with one or more substituents _Q :

In certain embodiments, Ring D is a 6- to 10-membered ring optionally substituted with one or more substituents Q ₁ Aryl or 5 to 10 membered heteroaryl containing at least one nitrogen atom.

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

In certain embodiments, _X2 is selected _from -( _CR6aR6b )n-, -O-, -S-, _-NR6c- , _-CH2S- , _-CH2O- , _-NHCR6dR _6e - and a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted by one or more substituents Q ₁ , the heteroaryl group containing at least one nitrogen atom.

In certain embodiments, R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, thiol, deuterium, cyano, and 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 and R _6b together with the carbon atom to which they are connected form a 3- to 10-membered cycloalkyl group, or R _6a and R _6b together form an oxo or thio group.

In certain embodiments, R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, thiol, deuterium, cyano, and 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 and R _6b together with the carbon atoms to which they are connected form a 3- to 10-membered cycloalkyl group, or R _6a and R _6b together form oxo or thio.

In certain embodiments, R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, thiol, deuterium, cyano, and 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 oxo or sulfur generation.

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

In certain embodiments, each R ₄ is independently hydrogen.

In certain embodiments, each substituent group Q ₁ is independently selected from the group consisting of halogen, hydroxy, thiol, deuterium, oxo, thio, cyano, amino, carboxyl, C ₁ -C ₆ alkyl, and C ₁ -C ₆ alkoxy.

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

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

in,

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

R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted by 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 oxo or thio;

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

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

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

R ₃ is each independently hydrogen or halogen;

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

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

In certain embodiments, R _1a is hydrogen.

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

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

In certain embodiments, R _5a and R _5b together form oxo or thio, preferably oxo.

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

In certain embodiments, R _6a and R _6b are each independently selected from hydrogen, halogen, hydroxyl, thiol, 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 oxo or thio.

In certain embodiments, each R ₂ is independently selected from -CH ₂ OH, -CH ₂ SH, -OH, and

In certain embodiments, _R3 is hydrogen.

In certain embodiments, _R3 is fluoro.

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

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

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

Among them, Ab, D and k are as defined before, and p is each 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 certain embodiments, k is any number between 2-5. k can be an integer or a decimal.

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

The antibodies described in the present disclosure are not particularly limited. The choice of antibody depends on the disease or condition (eg, cancer) being treated by the antibody-drug conjugate (ADC). Antibodies can specifically bind to corresponding antigens expressed on cancer cells (also known as tumor-associated antigens (TAA)), viral antigens, or microbial antigens, have antibody-dependent cell-mediated phagocytosis (ADCP) activity, and have Antitumor, antiviral, or antimicrobial activity in vivo. The interchain S-S bond in the antibody is the site of attachment of the drug-linker complex.

Antibodies suitable for use in the methods of the present disclosure may 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 of the present disclosure are selected from monoclonal antibodies or polyclonal antibodies.

In some embodiments, the antibodies of the present disclosure are selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, or antigen-binding fragments thereof.

In some embodiments, the isotype of the antibodies of the present disclosure is selected from IgG (IgG1, IgG2, IgG3, or IgG4). In some embodiments, the antibody is selected from IgG1 or IgG4 isotypes. In some specific embodiments, the antibody is selected from the group consisting of antibodies, IgG1 monoclonal antibodies, or IgG4 monoclonal antibodies.

In some embodiments, the antibodies of the present disclosure are selected from the group consisting of anti-HER2 (ErbB2) antibodies, anti-EGFR antibodies, anti-B7-H3 antibodies, anti-c-Met antibodies, anti-HER3 (ErbB3) antibodies, anti-HER4 (ErbB4) antibodies, anti- CD20 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-MUCl antibody, anti-Lewis 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-human folate receptor alpha (FRA) antibody, anti-Nectin-4 antibody, anti-AXL antibody, anti-CD19 antibody or antigen-binding fragment thereof.

In some embodiments, the antibody is selected from the group consisting of Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Imatuzumab Anti-(Emibetuzumab), Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivazumab Anti-(Bivatuzumab), Lorvotuzumab (Lorvotuzumab), cBR96, adalimumab (adalimumab), Farletuzumab (Farletuzumab), Glematumamab and Enfortumab, or antigen-binding fragments thereof.

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

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

Anti-TNFα antibodies and antigen-binding fragments thereof include, for example, adalimumab, infliximab, certolizumab pegol, afitumomab, nerelimomab, ozolizumab Anti-(ozoralizumab), placulumab and golimumab or antigen-binding fragments thereof. Additional anti-TNFa 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 their antigen-binding fragments also include competitive inhibitors of adalimumab, infliximab, certolizumab, afitumomab, nerimumab, olzolazumab, and paclitaxel. Kulumab or golimumab binds to TNFα antibodies and their antigen-binding fragments. Anti-TNFα antibodies and their antigen-binding fragments also include combinations with adalimumab, infliximab, certolizumab, afitumomab, neretolizumab, olzolazumab, and prakuru Antibodies and antigen-binding fragments of monoclonal antibodies or golimumab that bind the same TNFα epitope.

In certain embodiments, an anti-TNFa antibody or antigen-binding fragment thereof competitively inhibits the interaction between adalimumab and Binding of TNFα. In certain embodiments, the anti-TNFa antibody or antigen-binding fragment thereof binds to the same TNFα epitope as adalimumab. In certain embodiments, the anti-TNFa antibody or antigen-binding fragment thereof is adalimumab or an antigen-binding fragment thereof. In certain embodiments, the anti-TNFa antibody or antigen-binding fragment thereof is adalimumab.

In certain embodiments, the anti-TNFa antibody or antigen-binding fragment thereof comprises adalimumab, infliximab, certolizumab, afitumomab, nerimumab, ozolizumab , prakulumab or golimumab sequences, such as complementarity determining regions (CDRs), variable heavy chain domains (VH) and/or variable light chain domains (VL).

The drug used in the present disclosure is not particularly limited as long as the drug molecule has a desired (eg, cytotoxic, anti-tumor, or labeling agent, etc.) effect and has at least one substituent group or partial structure that allows connection with the linker structure. In some embodiments, the drug is selected from the group consisting of 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 may be selected from, but is not limited to, maytansinoids, auristatins (e.g., MMAE (monomethyl auristatin E)), MMAD (monomethyl auristatin E), Monomethyl oristatin D), MMAF (monomethyl oristatin F), etc.), calicheamicins, doxorubicins, benzodipyrroles (duocarmycins and CC-1065 ), camptothecins, pyrrolobenzodiazepines and pyrrolobenzodiazepine dimers (PBD Dimmers), etc. In some embodiments, the drug is selected from glucocorticoid receptor agonists.

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

(1) Add an appropriate amount of ZnCl ₂ and the reducing agent diphenylphosphoacetic acid to the antibody solution that needs to be reduced;

(2) Add metal chelating agent to chelate Zn ²⁺ 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) Obtain an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof.

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

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 of the antibody to the reducing agent in step (1) is ( mM/mM) from 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 standing at 0-4°C overnight or reaction at 25°C for 2 hours.

In some embodiments, step (1) is reacted in a buffer system, the buffer system is histidine buffered liquid, pH is 6.0-7.0.

In some embodiments, the metal chelating agent in step (2) is selected from EDTA; in some embodiments, the metal chelating agent in step (2) is selected from ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium salt, ethylenediamine Dicalcium tetraacetate, diethylene triamine pentaacetic acid or mixtures 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 in step (3) to the drug linker intermediate or a pharmaceutically acceptable salt thereof (based on the drug linker intermediate) 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 the step of purifying the reaction solution. The purification is selected from using a desalting 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, prepared by the methods of the present disclosure.

The present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, which is obtained without a quenching step and/or a re-oxidation step.

In some embodiments, the antibody-drug conjugate prepared by the method of the present disclosure or a pharmaceutically acceptable salt thereof, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprising 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%, Above about 63 wt%, above about 64 wt%, above about 65 wt%, above about 66 wt%, above about 67 wt%, above about 68 wt%, above about 69 wt%, above about 70 wt%, above About 71 wt%, greater than about 72 wt%, greater than about 73 wt%, greater than about 74 wt%, greater than about 75 wt%; in some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody -The drug conjugate or a pharmaceutically acceptable salt thereof contains D4 in an amount selected from about 55 wt% to about 75 wt%, about 60 wt% to about 70 wt%, and about 55 wt% to about 65 wt%.

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

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods of the present disclosure, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises D4a, the D4a Selected from antibody-drug conjugates or pharmaceutically acceptable salts thereof having only 4 drug linkers, and the 4 drug linkers are combined with sulfhydryl groups between heavy and light chains; based on D0, D2, D4, D6 and D8 The total weight of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof includes D4a 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%, Above about 61 wt%, above about 62 wt%, above about 63 wt%, above about 64 wt%, above about 65 wt%, above about 66 wt%, above about 67 wt%, above about 68 wt%, above 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 DO, D2, The total weight of D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains D4a The content is selected from about 55wt% to about 75wt%, about 60wt% to about 70wt%, and about 55wt% to about 65wt%.

In some embodiments, the antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, prepared by the methods of the present disclosure, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises D4b, the D4b Selected from antibody-drug conjugates or pharmaceutically acceptable salts thereof having only 4 drug linkers, and the 4 drug linkers are combined with sulfhydryl groups between heavy and heavy chains; based on D0, D2, D4, D6 and D8 The total weight of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof includes D4b in an amount less than about 40 wt%, such as less than about 40 wt%, less than about 30 wt%, less than about 20 wt%, Less than about 15 wt%, less than about 10 wt%, less than about 5 wt%; in some embodiments, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or pharmaceutically acceptable Acceptable salts contain D4b in an amount selected from about 5 wt% to about 30 wt%, about 5 wt% to about 20 wt%, and about 5 wt% to about 10 wt%.

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

In some embodiments, the antibody-drug conjugate prepared by the method of the present disclosure or a pharmaceutically acceptable salt thereof, based on the total weight of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprising D6 in an amount 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%, Below about 14 wt%, below about 13 wt%, below about 12 wt%, below about 11 wt%, below about 10 wt%, below about 9 wt%, below about 8 wt%, below about 7 wt%, below About 6 wt%, less than about 5 wt%; in some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains an amount of D6 based on the total weight of DO, D2, D4, D6 and D8 Selected from about 5wt% to about 20wt%, about 5wt% to about 15wt%, and about 5wt% to about 10wt%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure, based on the total weight of DO, D2, D4, D6 and D8 refers to the unconjugated antibody obtained after the coupling reaction. Total weight of purified DO, D2, D4, D6 and D8.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure, based on the total weight of DO, D2, D4, D6 and D8, refers to only the amount obtained after the coupling reaction. The total weight of D0, D2, D4, D6, and D8 after purification to remove small molecule process impurities (such as free toxins and organic solvents, etc.). The purification step will not affect the different drug-loading components of the prepared ADC.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure, based on the sum of peak areas, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises of D4 has a peak area percentage 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, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof includes a peak area percentage of D4 selected from about 55% to about 75%, about 60% to about 70, based on the sum of peak areas. %, about 55% to about 65%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure, based on the sum of peak areas, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises The peak area percentage of D4 is greater than that of D4 obtained using TCEP.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the methods of the present disclosure, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises D4a, the D4a Selected from antibody-drug conjugates having and only 4 drug linkers, and the 4 drug linkers are combined with sulfhydryl groups between heavy and light chains, or pharmaceutically acceptable salts thereof; based on the sum of peak areas, the antibody-drug conjugate The conjugate or a pharmaceutically acceptable salt thereof contains D4a with a peak area percentage 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 antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains a peak area percentage of D4a 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 methods of the present disclosure, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises D4b, the D4b Selected from antibody-drug conjugates having and only 4 drug linkers, and the 4 drug linkers are combined with sulfhydryl groups between heavy and heavy chains, or pharmaceutically acceptable salts thereof; based on the sum of peak areas, the antibody-drug conjugate The conjugate or a pharmaceutically acceptable salt thereof contains D4b with a peak area percentage of 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, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains a peak area percentage of D4b selected from about 5% to about 30%, about 5%, based on the sum of peak areas. 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 of the present disclosure, based on the sum of peak areas, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises The peak area percentage of DO+D8 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%; some In embodiments, based on the sum of peak areas, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains a peak area percentage of DO+D8 selected from about 3% to about 20%, about 5% to about 15 %, about 3%-about 10%.

In some embodiments, antibody-drug conjugates prepared by methods described in the present disclosure or pharmaceutically acceptable Acceptable salts, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprise 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%, less than about 5%; in some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains a peak area percentage of D6 selected from about 5 based on the sum of peak areas. %-about 20%, about 5%-about 15%, about 5%-about 10%.

The peak area percentage based on the sum of peak areas refers to: detecting the antibody-drug conjugate (ADC) prepared in the present disclosure or its pharmaceutically acceptable salt by HIC-HPLC method, and calculating the deduction by comparing it with the spectrum of the blank solution Calculate the sum of the areas of each chromatographic peak after the blank solution. Integrate the chromatogram to obtain the sum of the peak areas. Use the area normalization method to calculate the peak area of each component (such as D0, D2, D4, D6 or D8) in the sum of the peak areas. percentage.

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 of the present disclosure, based on the sum of the peak areas of DO, D2, D4, D6 and D8, the antibody-drug conjugate The substance or a pharmaceutically acceptable salt thereof contains D4 with a peak area percentage 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 the peak areas of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises The peak area percentage of D4 is selected from about 55% to about 75%, about 60% to about 70%, and about 55% to about 65%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure, based on the sum of the peak areas of DO, D2, D4, D6 and D8, the antibody-drug conjugate The substance or a pharmaceutically acceptable salt thereof 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 methods of the present disclosure, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises D4a, the D4a Selected from antibody-drug conjugates or pharmaceutically acceptable salts thereof having only 4 drug linkers, and the 4 drug linkers are combined with sulfhydryl groups between heavy and light chains; based on D0, D2, D4, D6 and D8 The sum of the peak areas of D4a, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains a peak area percentage of D4a 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%; in some embodiments, based on the sum of the peak areas of DO, D2, D4, D6 and D8, the antibody-drug couple The conjugate or a pharmaceutically acceptable salt thereof contains D4a with a peak area percentage 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 methods of the present disclosure, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises D4b, the D4b Selected from antibody-drug conjugates or pharmaceutically acceptable salts thereof having only 4 drug linkers, and the 4 drug linkers are combined with sulfhydryl groups between heavy and heavy chains; based on D0, D2, D4, D6 and D8 The sum of the peak areas of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof includes a peak area percentage of D4b of 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 DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof D4b is included in a peak area percentage selected from about 5% to about 30%, about 5% to about 20%, and about 5% to about 10%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure, based on the sum of the peak areas of DO, D2, D4, D6 and D8, the antibody-drug conjugate The substance or a pharmaceutically acceptable salt thereof contains a peak area percentage of DO+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%, less than about 3%; in some embodiments, based on the sum of the peak areas of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises DO+D8 The peak area percentage is selected from about 3% to about 20%, about 5% to about 15%, and about 3% to about 10%.

In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method of the present disclosure, based on the sum of the peak areas of DO, D2, D4, D6 and D8, the antibody-drug conjugate The substance or a pharmaceutically acceptable salt thereof contains D6 with a peak area percentage 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%; some embodiments , based on the sum of the peak areas of DO, D2, D4, D6 and D8, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof contains a peak area percentage of D6 selected from about 5% to about 20%, About 5% to about 15%, about 5% to about 10%.

In some embodiments, the sum of the peak areas based on DO, D2, D4, D6 and D8 of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure refers to the value obtained after the coupling reaction. The sum of the peak areas of D0, D2, D4, D6 and D8 without purification.

In some embodiments, the sum of the peak areas based on DO, D2, D4, D6 and D8 of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof prepared by the method described in the present disclosure refers to the value obtained after the coupling reaction. Only the sum of the peak areas of D0, D2, D4, D6 and D8 after purifying and removing small molecule process impurities (such as free toxins and organic solvents, etc.), where the purification step will not affect the different drug-loading components of the prepared ADC.

The peak area percentage based on the sum of the peak areas of D0, D2, D4, D6 and D8 refers to: the antibody-drug conjugate (ADC) prepared in the present disclosure or its pharmaceutically acceptable salt is detected by the HIC-HPLC method. and empty Compare the spectra of the white solution, calculate the sum of the areas of D0, D2, D4, D6, and D8 after deducting the blank solution, integrate the chromatogram, and obtain the sum of the peak areas based on D0, D2, D4, D6, and D8, using The area normalization method calculates the peak area of each component (such as D0, D2, D4, D6, or D8) as a percentage of the sum of the peak areas based on D0, D2, D4, D6, and D8.

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 average drug loading of the antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the method of the present disclosure is selected from about 3.0 to about 5.0, about 3.5 to about 4.5, 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 capacity of the antibody-drug conjugate or pharmaceutically acceptable salt thereof prepared by the disclosed method is greater than 3.0 and less than 5.0; in some embodiments, the average drug loading capacity 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 aforementioned antibody-drug conjugates or pharmaceutically acceptable salts thereof of the present disclosure can be prepared into kits.

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 the use of the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the preparation of a medicament for the treatment and/or prevention of tumors, cancer, autoimmune diseases, or infectious diseases. . In some embodiments, the cancer is a cancer associated with expression of HER2, HER3, B7H3, CD19, CD30, CD33, Trop2, CD79b, Nectin-4, TNF-alpha, folate receptor alpha, or EGFR. In some embodiments, the infectious disease is a viral or microbial infection. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcers colitis, hidradenitis suppurativa, uveitis, Behcet's disease, spondyloarthropathy, and psoriasis.

The present disclosure also provides the use of the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the preparation of a medicament for the treatment and/or prevention of cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethra cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, Melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, Pancreatic cancer and lymphoma.

The active compounds, such as antibody-drug conjugates or pharmaceutically acceptable salts thereof, may be formulated in a form suitable for administration by any appropriate route, preferably in unit dosage form, or in a form which may be administered to the patient in a single dose. Modes of self-administration. The unit dosage form of a compound or composition of the present disclosure may be expressed as a tablet, capsule, cachet, bottled solution, powder, granule, lozenge, suppository, reconstituted powder or liquid preparation.

The dosage of the antibody-drug conjugate or pharmaceutically acceptable salt or compound or composition used in the treatment methods of the present disclosure will generally vary with the severity of the disease, the body weight of the patient, and the relative efficacy of the compound. However, as a general guide, suitable unit doses may range from 0.1 to 1000 mg.

In addition to active compounds such as antibody-drug conjugates or pharmaceutically acceptable salts thereof, the pharmaceutical compositions of the present disclosure may contain one or more auxiliary materials, and the auxiliary materials are selected from the following ingredients: fillers (diluents), Binders, wetting agents, disintegrating agents or excipients, etc. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of active compound such as an antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

On the other hand, without specifying the configuration, the compounds, drugs, intermediates, and conjugates of the present disclosure may exist in specific isomeric forms, such as tautomers, rotamers, and geometric isomers. , diastereomers, racemates and enantiomers. Without specifying a configuration, this disclosure contemplates all such compounds, conjugates, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and their racemic and other mixtures, such as enantiomeric or non-enantiomeric Enantiomerically enriched mixtures, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. Unless otherwise stated, all such isomers, as well as mixtures thereof, are included within the scope of this disclosure.

The compounds, drugs, intermediates, and conjugates containing asymmetric carbon atoms of the present disclosure can be isolated in optically active pure form or racemic form. Optically active pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents. The optically active (R)- and (S)-isomers as well as the D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one wants to obtain an enantiomer of a compound, drug, intermediate, or conjugate of the present disclosure, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, in which 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 amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with a suitable optically active acid or base, and then the salt is formed by conventional methods known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally combined with chemical derivatization methods (e.g., generation of amino groups from amines). formate).

Any isotopically labeled derivatives of the compounds, drugs, intermediates, conjugates, pharmaceutically acceptable salts thereof, or isomers described in this disclosure are covered by this disclosure. Atoms that can be labeled with isotopes include, but are not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, iodine, etc. They can be replaced by the isotopes 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 respectively.

Unless otherwise stated, when a position is specifically designated as deuterium (D), that position is understood to have an abundance of deuterium that is at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10 % deuterium incorporation). Examples of compounds having a natural abundance greater than deuterium may be at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium (i.e., at least 45% deuterium incorporation) , at least 4000 times the abundance of deuterium, at least 5000 times the abundance of deuterium, at least 6000 times the abundance of deuterium, or a higher abundance of deuterium. The present disclosure also includes various deuterated forms of the compounds. Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can refer to relevant literature to synthesize deuterated forms of compounds. Commercially available deuterated starting materials may be used in the preparation of deuterated forms of the compounds, or they may be synthesized using conventional techniques using deuterated reagents including, but not limited to, deuterated borane, trideuterated borane in tetrahydrofuran. , 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 antibodies, which includes 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 intra-antibody interchain ions. The step in which the sulfur bond is a sulfhydryl group.

In some embodiments, the transition metal ion in the method of selectively reducing an 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 other 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 of selectively reducing antibodies is a reducing agent containing 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)ethyl Amine, 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 of 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 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 an 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, it is carried out at about 0°C to about 4°C; in some embodiments, it is carried out at about 0°C to about 37°C; in some embodiments, it is carried out at about 10°C to about 25°C; in some embodiments , carried out at about -5°C to about 5°C.

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

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

In some embodiments, reaction conditions depend on the specific antibody to be reduced. Determination of the incubation period and temperature based on a particular antibody is within the ability of one of ordinary skill in the art. For example, the antibody to be conjugated is typically reacted with a reducing agent by incubation overnight at 4°C in the presence of transition metal ions.

In some embodiments, the final concentration of the antibody is selected from about 0.01mM to about 0.50mM, such as about 0.01mM, about 0.02mM, about 0.03mM, about 0.04mM, about 0.05mM, about 0.06mM, about 0.07mM, about 0.08 mM, about 0.09mM, about 0.10mM, about 0.11mM, about 0.12mM, 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; 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 0.01mM to about 0.50mM, such as about 0.01mM, about 0.02mM, about 0.03mM, about 0.04mM, about 0.05mM, about 0.06mM, about 0.07mM, About 0.08mM, about 0.09mM, about 0.10mM, about 0.15mM, about 0.20mM, about 0.21mM, about 0.22mM, about 0.23mM, about 0.24mM, about 0.25mM, about 0.26mM, about 0.27mM, about 0.28 mM, 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 transition metal ion The final concentration 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 reducing agent is The final concentration 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 antibody to reducing agent is about 3:1 to about 1:10, such as about 3:1, about 2:1, about 1:1, about 1:2 , John 1:3, John 1:4, John 1:5, John 1:6, John 1:7, John 1:8, John 1:9, John 1:10. In some embodiments, the final concentration equivalent ratio of antibody to reducing agent (mM/mM) is from about 1:2.5 to about 1:3.5.

In some embodiments, the final concentration equivalent ratio (mM/mM) of antibody to transition metal ion is from 5:1 to about 1:5, such as about 5:1, about 4:1, about 3:1, about 2:1 , John 1:1, John 1:2, John 1:3, John 1:4, John 1:5. In some embodiments, the final concentration equivalent ratio (mM/mM) of antibody to transition metal ion is about 1:2.

In some embodiments, the method of selectively reducing antibodies described in the present disclosure further includes the step of adding a metal chelating agent. The metal chelating agent is used to chelate transition metal ions. In some embodiments, the metal chelating agent is selected from EDTA; in some embodiments, the metal chelating agent is selected from ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid dicalcium salt, diethylene Triaminepentaacetic acid or mixtures 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, John 1:4, John 1:5. In some embodiments, the final concentration equivalent ratio (mM/mM) of transition metal ion to metal chelator is about 1:2.

In some embodiments, the antibodies of the present disclosure are selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies or antigen-binding fragments thereof.

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

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

In some embodiments, the final concentration equivalent ratio (mM/mM) of antibody to reducing agent in step (1a) is from 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 standing at 0-4°C overnight or reaction at 25°C for 2 hours.

In some embodiments, step (1a) is reacted in a buffer system, which is a histidine buffer, pH 6.0 to 7.0.

In some embodiments, the method of selectively reducing antibodies of the present disclosure further includes (2a) adding a metal chelating agent to chelate Zn ²⁺ ions.

In some embodiments, the metal chelating agent of step (2a) is selected from EDTA. In some embodiments, the step The metal chelating agent in step (2a) is selected from ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid dicalcium salt, diethylenetriaminepentacetic acid or mixtures thereof.

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

This disclosure incorporates the entire text of WO2022166779A1.

Terminology

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

When a trade name is used in this disclosure, Applicants intend to include the formulations of the trade name product, the generic and active pharmaceutical portions of the trade name product.

Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

The term "antibody drug conjugate" (antibody drug conjugate, ADC), also known as antibody-drug conjugate or antibody-drug conjugate, refers to the fact that the antibody is connected to the drug through a linking unit. In the present disclosure, "antibody-drug conjugate" refers to an antibody or antigen-binding fragment connected to a biologically active toxic drug through a stable linking unit. Antibody-drug conjugates are used interchangeably with antibody-drug conjugates and antibody-drug conjugates in this disclosure.

The terms "linker", "linker", "linker unit", "linker unit" or "linker fragment" refer to a chemical structural fragment or bond that is connected to an antibody or antigen-binding fragment at one end and to a drug at the other end. It can also be used Connect other connectors before connecting to antibodies or drugs.

A joint may contain one or more joint components. Exemplary linker building blocks include 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenyl Alanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with a linker reagent: N-succinimidyl 4-(2-pyridylthio)valerate ( SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate (SMCC, also known as MCC) and N-succinimidyl (4-iodo- Acetyl)aminobenzoate (SIAB). Linkers may include stretch units, spacer units, amino acid units, and extension units. 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 the cell. For example, acid labile linkers (e.g., hydrazone), protease sensitive (e.g., peptidase sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used Connector (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 carried per antibody-drug conjugate molecule in the population of antibody-drug conjugates, or Expressed as the ratio of drug amount to antibody amount. Drug loading can range from 1 to 20 linkages per antibody (Ab). In embodiments of the present disclosure, the drug loading amount may be, for example, 3, 4, 5, or the average of any two values. The average number of drugs per ADC molecule after the coupling reaction can be characterized using conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA testing, MAb size variant determination (CE-SDS), and HPLC. For example, in one embodiment, HIC is used to determine the yield and isomeric mixture obtained for an antibody-drug conjugate (eg, for a D4 conjugate). This technology is capable of isolating antibodies loaded with various numbers of drugs. Drug loading levels can be determined based on, for example, the ratio of absorbance at 250 nm and 280 nm. For example, if a drug absorbs at 250nm and an antibody absorbs at 280nm. Therefore, the 250/280 ratio increases with drug loading. Using the bioconjugation methods described herein, typically antibodies with an even number of drugs are observed to be conjugated to the antibody because reduction of disulfide bonds yields an even number of free cysteine sulfhydryl groups.

Typically, an antibody molecule belonging to the IgG1 or IgG4 subclasses has four interchain S-S bonds, each formed by two -SH groups. The antibody molecule may 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, thus conjugating to a single antibody molecule The number of drugs is 2, 4, 6 or 8. Depending on the number of drugs conjugated to a single antibody molecule, different conjugates containing different numbers of drug molecules are named DO, 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, where the two drug molecules can be coupled via a linker to a -SH group generated by reduction of the S-S bond between the heavy and light chains, or Coupling can be via a linker with a -SH group generated by reduction of the heavy chain and the S-S bond between the heavy chain.

D4 refers to an ADC in which four drug molecules are coupled to a single antibody molecule, where the four drug molecules can be coupled via linkers to four -SH groups generated by reducing the two S-S bonds between the heavy and light chains. (this ADC is called D4a), or four drug molecules can be coupled via linkers to four -SH groups created by reduction of the heavy chain and the two S-S bonds between the heavy chains (this ADC is called D4b), or Two drug molecules can be coupled via a linker to two -SH groups created by reducing one S-S bond between the heavy chain and the light chain and the other two drug molecules can be coupled via the linker with the two -SH groups created by reducing the heavy chain and the heavy chain. One S-S bond creates the coupling of two -SH groups (this ADC is called D4c).

D6 refers to an ADC in which six drug molecules are coupled to a single antibody molecule, where four drug molecules can be coupled via linkers to four -SH groups generated by reducing the two SS bonds between the heavy and light chains. And two drug molecules can be coupled via a linker with two -SH groups generated by reducing the heavy chain and one SS bond between the heavy chains (this ADC is called D6a), or four drug molecules can be coupled via a linker with The four -SH groups generated by reduction of two SS bonds between heavy and heavy chains are coupled and two drug molecules can be coupled via a linker with the two -SH groups generated by reduction of one SS bond between heavy and light chains. SH group coupling (this ADC is called D6b).

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

Typically, the heterogeneous mixture of ADC molecules produced by conventional conjugation methods or the methods of the present disclosure is a mixture of DO, D2, D4, D6, and D8. Thus, "homogeneity" or "uniformity" of an antibody-drug conjugate is used to describe a specific type of antibody-drug conjugate (i.e., one selected from the group consisting of DO, D2, D4, D6, and D8 conjugates). (a type of antibody) advantageous properties in a given mixture of antibody-drug conjugates. Generally, an ADC is considered to have high homogeneity or high uniformity if the content of D4 in the mixture is high.

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

The term "interchain disulfide" is used to mean 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. Sulfides (heavy-light interchain disulfides).

The term "interchain thiol" or "interchain thiol" is used to mean a thiol group obtained by reducing the interchain disulfide of an antibody.

The term "heavy-heavy chain thiol" is used to mean a sulfhydryl group obtained by reducing the heavy-heavy chain disulfide of an antibody.

The term "heavy-light interchain thiol" is used to mean a thiol group obtained by reducing the heavy-light interchain disulfide of an antibody.

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

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

The term "diphenylphosphino" refers to

The term "diphenylphosphineacetic acid", also known as "DPA" and "diphenylphosphineacetic acid", refers to

In the structure described in this disclosure, Represents a single or double bond.

It will be understood by those skilled in the art that, for example, when one of R _5a and R _5b is selected from oxo or thio, the other is not present.

The term "stretch unit" refers to one end covalently linked to the antibody through a carbon atom and the other end to an amino acid unit, Chemical structural fragments linked to disulfide moieties, sulfonamide moieties, or non-peptide chemical moieties.

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

The term "alkylene" refers to a saturated linear or branched aliphatic hydrocarbon radical having 2 residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which is Linear or branched chain groups containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, more preferably alkylene groups containing 1 to 6 carbon atoms. Non-limiting examples of alkylene 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 -), etc. The alkylene group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment.

The term "alkenylene" refers to linear alkenes containing from 2 to 8 carbon atoms, preferably from 2 to 6 carbon atoms, more preferably from 2 to 4 carbon atoms and having at least one double bond at any position Groups include, for example, vinylene, allylene, propenylene, butenylene, prenylene, butadienylene, pentenylene, pentylene Alkenyl, hexenyl, hexadienyl, etc.

The term "alkynylene" includes linear alkynes having from 2 to 8 carbon atoms, preferably from 2 to 6 carbon atoms, more preferably from 2 to 4 carbon atoms and having at least one triple bond at any position Groups include, for example, ethynylene, propynylene, butynylene, pennylene, hexynylene, and the like.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. The cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene base, cyclooctyl, etc.; polycyclic cycloalkyl includes spiro ring, fused ring and bridged ring cycloalkyl. "Carbocycle" refers to the ring system in a cycloalkyl group.

The term "spirocycloalkyl" refers to a polycyclic group with 5 to 20 membered monocyclic rings sharing one carbon atom (called a spiro atom). It may contain one or more double bonds, but no ring is fully conjugated. pi electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of shared spiro atoms between the rings, the spirocycloalkyl group is divided into a single spirocycloalkyl group, a double spirocycloalkyl group or a polyspirocycloalkyl group, and is preferably a single spirocycloalkyl group and a double spirocycloalkyl 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 monospirocyclic alkyl group. "Spirocarbocycle" refers to the ring system in a spirocycloalkyl group. Non-limiting examples of spirocycloalkyl groups include:

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

The term "bridged cycloalkyl" refers to an all-carbon polycyclic group of 5 to 20 members, with any two rings sharing two carbon atoms that are not directly connected. It may contain one or more double bonds, but no ring has a complete Conjugated pi electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl groups include:

The cycloalkyl ring can be fused to an aryl, heteroaryl or heterocycloalkyl ring, where the ring connected to the parent structure is a cycloalkyl group, non-limiting examples include indanyl, tetralin base, benzocycloheptyl, etc. Cycloalkyl may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkyl, Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio group, oxo group, carboxyl group or carboxylate group.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O) _m (where m is an integer from 0 to 2), excluding the ring portion 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 heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuryl, dihydropyrazolyl, dihydropyrrolyl, piperidine group, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., with piperidinyl and pyrrolidinyl being preferred. Polycyclic heterocyclyl groups include spirocyclic, fused cyclic and bridged cyclic heterocyclyl groups. "Heterocycle" refers to the ring system in a heterocyclyl group.

The term "spiroheterocyclyl" refers to a polycyclic heterocyclic group with 5 to 20 membered monocyclic rings sharing one atom (called a spiro atom), in which one or more ring atoms are selected from nitrogen, oxygen or S(O ) _m (where m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a fully conjugated pi-electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of shared spiro atoms between the rings, the spiroheterocyclyl group is divided into a single spiroheterocyclyl group, a double spiroheterocyclyl group or a polyspiroheterocyclyl group, and is preferably a single spiroheterocyclyl group and a double spiroheterocyclyl 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 single spiroheterocyclic group. "Spiroheterocycle" refers to a ring system in a spiroheterocyclyl group. Non-limiting examples of spiroheterocyclyl include:

The term "fused heterocyclyl" refers to a polycyclic heterocyclic group with 5 to 20 members, each ring in the system shares an adjacent pair of atoms with other rings in the system, and one or more rings may contain one or more Double bonds, but no ring has a fully conjugated pi electron system, one or more of the ring atoms is a heteroatom selected from nitrogen, oxygen, or S(O) _m (where m is an integer 0 to 2), and the remaining rings The atom is carbon. Preferably it is 6 to 14 yuan, more preferably 7 yuan to 10 yuan. 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. "Condensed heterocycle" refers to the ring system in a fused heterocyclyl group. Non-limiting examples of fused heterocyclyl groups include:

The term "bridged heterocyclyl" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two atoms that are not directly connected. It may contain one or more double bonds, but no ring has a completely shared bond. A yoke of pi-electron systems in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _m (where m is an integer from 0 to 2) and the remaining ring atoms are carbon. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclyl groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclyl groups include:

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

wait.

Heterocyclyl may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkyl, Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio group, oxo group, carboxyl group or carboxylate group.

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

The aryl group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle Alkylthio, carboxyl or carboxylate group, preferably phenyl.

The term "fused ring aryl" can be an unsaturated aromatic fused ring structure containing 8-14 ring atoms formed by two or more ring structures connected by sharing two adjacent atoms. The number of atoms is preferably 8-12. For example, it includes all unsaturated fused ring aryl groups, such as naphthalene, phenanthrene, etc., and also includes partially saturated fused ring aryl groups, such as benzo 3-8 membered saturated monocyclic cycloalkyl, benzo 3-8 membered partially saturated monocyclic ring. alkyl. "Condensed aromatic ring" refers to the ring system in a fused aromatic ring. Specific examples of condensed ring 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, where the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 12 yuan, such as imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazine group, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, where the ring attached to the parent structure is the heteroaryl ring. "Heteroaryl ring" refers to the ring system in a heteroaryl group. Non-limiting examples of heteroaryl groups include:

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

The term "fused heteroaryl" can be an unsaturated group containing 5-14 ring atoms (including at least one heteroatom) formed by two or more cyclic structures connected to each other by sharing two adjacent atoms. Fang The aromatic condensed ring structure, including carbon atoms, nitrogen atoms and sulfur atoms, can be oxygenated, preferably "5-12-membered condensed heteroaryl", "7-12-membered condensed heteroaryl", "9-12-membered condensed heteroaryl""Condensedheteroaryl", etc., such as benzofuryl, benzisofuryl, benzothienyl, indolyl, isoindole, benzoxazolyl, benzimidazolyl, indazolyl, benzotrizoyl Azolyl, quinolinyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinolyl Zozolinyl, quinoxalinyl, phenolazine, pteridinyl, purinyl, naphthyridinyl, phenazine, phenothiazine, etc. "Condensed heteroaromatic ring" refers to the ring system in a fused heteroaryl group.

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

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

The term "alkylthio" refers to -S-(alkyl) and -S-(unsubstituted cycloalkyl), where alkyl is as defined above. Non-limiting examples of alkylthio groups include: methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio. The alkylthio group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkyl Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , substituted by one or more substituents in the heterocycloalkylthio group.

The term "hydroxyalkyl" refers to an alkyl group substituted by hydroxyl, wherein alkyl is as defined above.

The term "haloalkyl" refers to an alkyl group substituted by 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 the =O group. For example, a carbon atom is connected to an oxygen atom through a double bond, in which a ketone or aldehyde group is formed.

The term "thio" refers to the =S group. For example, a carbon atom is double bonded to a sulfur atom to form thiocarbonyl -C(S)-.

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

The term "amino" refers to _-NH2 .

The term "cyano" refers to -CN.

The term "nitro" refers to _-NO2 .

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

The term "aldehyde group" refers to -CHO.

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

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

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

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

"Amino protecting group" is a suitable group for amino protection known in the art, see the amino protecting group in the literature ("Protective Groups in Organic Synthesis", 5 ^Th . Ed. TW Greene & P. GMWuts), preferably , the amino protecting group can be (C _1-10 alkyl or aryl) acyl group, such as: formyl, acetyl, benzoyl, etc.; can be (C _1-6 alkyl or C _6-10 aryl) base) sulfonyl group; it can also be (C _1-6 alkoxy or C _6-10 aryloxy) carbonyl, such as: Boc or Cbz; it can also be substituted or unsubstituted alkyl, such as: triphenylmethyl base (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).

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

The terms "buffer" and "buffer system" refer to a buffer that is resistant to changes in pH through the action of its acid-base conjugated components. Examples of buffers that control the pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate , glycylglycine 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 Acid-HCl buffer. Histidine-HCl buffer is prepared from histidine and hydrochloric acid or histidine and histidine hydrochloride.

"Succinate buffer" is a buffer that includes succinate ions. Examples of succinate buffer solutions include sodium succinate-succinate, histidine succinate, potassium succinate-succinate, calcium succinate-succinate, and the like. The preferred succinate buffer is succinate-sodium succinate buffer.

"Phosphate buffer", also known as PBS buffer, is a buffer containing phosphate ions. Examples of the phosphate buffer include disodium phosphate-sodium dihydrogen phosphate, disodium phosphate-potassium dihydrogen phosphate, and the like. A preferred phosphate buffer is disodium phosphate-sodium phosphate dibasic acid buffer.

"Acetate buffer", also known as "acetate buffer", is a buffer containing acetate ions. Examples of acetate buffers include acetate-sodium acetate, histidine acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, and the like. The preferred acetate buffer is acetic acid-sodium acetate buffer.

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

The published values are instrument measured values or calculated values after instrument measurements. There is a certain degree of error. Generally speaking, plus or minus 10% is within the reasonable error range. Of course, it is necessary to consider the context in which this value is used. For example, the content of total impurities. This value means that 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 7%, plus or minus 10%. 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 terms "optionally" or "optionally" are 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 does or does not occur.

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

The term "pharmaceutical composition" means a mixture containing one or more compounds, conjugates or physiologically/pharmaceutically acceptable salts or prodrugs thereof as described herein and other chemical components, as well as other components such as physiologically/ Pharmaceutically acceptable carriers and excipients. The purpose of pharmaceutical compositions is to facilitate administration to living organisms and facilitate the absorption of active ingredients to exert biological activity. In this disclosure, "pharmaceutical composition" and "preparation" are not mutually exclusive.

Pharmaceutical compositions may be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. The suspension may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents such as those mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a nontoxic parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. In addition, sterile fixed oil can be conveniently used as the solvent or suspending medium. For this purpose any blended fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables.

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of a ligand-drug conjugate of the present disclosure, or a salt of a compound described in the present disclosure, when such salt is administered in a mammal It is safe and effective, and has due biological activity. The antibody-drug conjugate of the present disclosure contains at least one amino group, so it can form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, Hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartaric acid Salt, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.

The term "carrier" is used for the drugs of this disclosure and refers to a substance that changes the way the drug enters and remains in the body. A system that distributes, controls the release rate of drugs, and delivers 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 entrap drug molecules and have good permeability to the membrane, making them excellent drug carriers.

The term "excipient" is an addendum in a pharmaceutical preparation other than the main drug, and may also be called an auxiliary material. Such as binders, fillers, disintegrants, and lubricants in tablets; matrix parts in semi-solid ointments and creams; preservatives, antioxidants, flavorings, aromatics, and auxiliaries in liquid preparations. Solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.

The term "diluent" is also known as filler, and its main purpose is to increase the weight and volume of the tablet. The addition of diluent not only ensures a certain volume, but also reduces the dosage deviation of the main ingredients and improves the compression moldability of the drug. When the tablet medicine contains oily components, an absorbent needs to be added to absorb the oily substances and keep it in a "dry" state to facilitate the preparation of tablets.

The terms "connected" and "bound" are used interchangeably. When referring to a connection between two molecules, they mean that the two molecules are connected by a covalent bond or that the two molecules are connected by a non-covalent bond (e.g., hydrogen bonding or ionic bonding). ) association. Connections include direct connections and indirect connections, direct combinations and indirect combinations. The terms "directly linked" or "directly bound" mean that a first compound or group is linked to a second compound or group without any intervening atoms or groups of atoms. The terms "indirectly linked" and "indirectly bound" mean that a first compound or group and a second compound or group are connected through an intermediate group, compound or molecule (eg, a linking group). "Connection" covers the connection of amino acid residues through covalent bonding, including but not limited to amide bonding, disulfide bonding; methylene bonding (also known as methylene bridge connection), 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 obtain any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include elimination or reduction of risk, reduction of severity, or delay of onset of a condition, including biochemical, histological, and biological aspects of the condition, its complications, and intermediate pathological phenotypes present during the development of the condition. academic and/or behavioral symptoms. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of, or ameliorating one or more symptoms of, various target gene, target mRNA, or target protein-related disorders of the present disclosure, reducing the treated disorder The dosage of the other agent required to enhance the efficacy of the other agent and/or delay the progression of a disorder associated with the target gene, target mRNA or target protein of the present disclosure in a patient.

The term "antibody" refers to an immunoglobulin. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by inter-chain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different. Immunoglobulins can be divided into five categories, or called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE. Their corresponding The heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain respectively. The same type of Ig can be divided into different types based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. Different subclasses, such as IgG, can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have either a kappa chain or a lambda chain.

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

In embodiments of the present disclosure, the antibody can form a linkage bond with the linking unit through a heteroatom on the antibody.

In this disclosure, the term "murine antibody" refers to antibodies prepared in mice according to the knowledge and skill in the art. They are prepared by injecting test subjects with a specific antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional properties.

The term "chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by murine antibodies. To establish a chimeric antibody, you must first establish a hybridoma that secretes mouse-derived specific monoclonal antibodies, then clone the variable region gene from the mouse hybridoma cells, and then clone the constant region gene of the human antibody as needed, and combine the mouse variable region gene with It is connected to the human constant region gene to form a chimeric gene and then 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 transplanting mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline antibodies Antibodies generated within framework sequences. It can overcome the heterologous reaction induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes are available in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase ), and in Kabat, EA et al. Found in Man, 1991 Sequences of Proteins of Immunological Interest, 5th ed. In order to avoid a decrease in activity caused by a decrease in immunogenicity, a minimum of reverse mutation or back mutation can be performed on the human antibody variable region framework sequence to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies that further undergo affinity maturation of CDRs by phage display. Further literature describing methods involving the use of humanized mouse antibodies includes, for example, the methods of Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and 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 "fully human antibody" is also called "fully human monoclonal antibody". The variable region and constant region of the antibody are both human, and the immunogen is removed. Sex and Toxic Side Effects use. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. Relevant technologies for the preparation of human antibodies mainly include: human hybridoma 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.

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of full-length antibodies can be utilized for the antigen-binding function of the antibody. Examples of binding fragments encompassed by "antigen-binding fragments" include (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab') ₂ fragments, which include fragments that pass through the hinge region A bivalent fragment of two Fab fragments connected by a disulfide bridge; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) ) a single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) optionally by A synthetic linker is a combination of two or more separate CDRs joined together. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by synthetic linkers using recombinant methods, allowing the production of a single protein in which the VL and VH regions pair up to form a monovalent molecule. chain (referred to as 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 within the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity among fragments obtained by treating an IgG antibody molecule with the protease papain (cleaving the 224th amino acid residue of the H chain), in which the N-terminal side of the H chain About half and the entire L chain are held together by disulfide bonds.

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

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

Furthermore, Fab' can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into the prokaryotic or 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 are combined with the second and third constant regions of the second heavy chain. Binding via disulfide bonds. Fc part of antibody 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 includes the portion of the heavy chain molecule connecting the CH1 and CH2 domains. The hinge region includes approximately 25 amino acid residues and is flexible, thereby allowing independent movement of the two N-terminal antigen binding regions.

The term "single chain antibody", "single chain Fv" or "scFv" means an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) linked by a linker of molecules. 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 using variants with 1 to 4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) . Other linkers useful in the present disclosure are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immuno 1.31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, described by 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 within 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 EA 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 of the light chain variable domain (CDR L1, CDR L2, CDR L3 or L1, L2, L3), and to the heavy chain variable domain CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3). Typically, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). Amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known protocols, including the "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al. (1997) JMB 273:927-948) and the ImMunoGenTics (IMGT) numbering convention (Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)) et al. For example, for the classic format, following Kabat's rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); light The CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Following the Chothia rules, the CDR amino acid residue numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residue numbers in VL are 26-32 (LCDR1), 50- 52(LCDR2) and 91-96(LCDR3). Defined by combining the CDRs of both Kabat and Chothia, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24- in human VL Composed of 34(LCDR1), 50-56(LCDR2) and 89-97(LCDR3). Following the IMGT rules, the CDR amino acid residue numbers in VH are roughly 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residue numbers in VL are roughly 27-32 (CDR1 ), 50-52(CDR2) and 89-97(CDR3). Following the IMGT rules, the CDR regions of the antibody can be determined using the program IMGT/DomainGap Align.

The term "antibody framework" refers to a portion of a variable domain, VL or VH, that serves as a scaffold for the antigen-binding loop (CDR) 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. Epitopes generally include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996).

The terms "specifically binds", "selectively binds", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, antibodies bind with an affinity (KD) of about less than 10 ^"7 M, such as about less than 10 ^"8 M, 10 ^"9 M, or 10 ^"10 M or less.

Antibodies or antigen-binding fragments of the present disclosure may be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect host cells. As a more recommended prior art mammalian expression system results in glycosylation of the antibody, particularly at the N-terminal site of the Fc region. Positive clones were expanded and cultured in bioreactor media to produce antibodies. Culture media secreting antibodies can be purified using conventional techniques. For example, use A or G Sepharose FF columns for purification. Wash away non-specifically bound components. Then use the pH gradient method to elute the bound antibody, and use SDS-PAGE to detect the antigen-binding fragments and collect them. Antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be frozen immediately, 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:

Description of the drawings

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 adding _ZnCl2 .

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

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

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

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

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

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

Detailed ways

The present disclosure is further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure. Experimental methods without specifying specific conditions in the disclosed examples usually follow conventional conditions, such as Cold Spring Harbor's Antibody Technology Experiment Manual, Molecular Cloning Manual; or conditions recommended by raw material or product manufacturers. Reagents whose specific sources are not indicated are conventional reagents purchased in the market.

Example

Example 1, HIC-HPLC method 1

Determine the yield and isomer mixture of the antibody-drug conjugates obtained (e.g., for D4 conjugate) 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): 20mM PB+1.5M (NH ₄ ) ₂ SO ₄ (pH 7.00).

Preparation example of mobile phase A: Weigh 1.22g sodium hydrogen phosphate dihydrate (NaH ₂ PO ₄ ·2H ₂ O, MW: 156.01g/mol), 4.37g sodium hydrogen 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

Preparation example of mobile phase B: weigh 0.97g sodium hydrogen phosphate dihydrate (NaH ₂ PO ₄ ·2H ₂ O, MW: 156.01g/mol), 3.50g sodium hydrogen 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, 2 Store at -8℃, valid for 1 month, filter before use.

1.2 Chromatographic methods and calculations

The chromatographic method is shown in Table 1.

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

Calculation formula: DAR=∑(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 sum of the peak areas refers to the sum of the peak areas of each component (D0, D2, D4, D6 and D8).

Peak area percentage refers to: detecting the antibody-drug conjugate (ADC) prepared in the present disclosure or its pharmaceutically acceptable salt by HIC-HPLC method, comparing with the spectrum of the blank solution, and calculating each chromatogram after subtracting the blank solution. The sum of the peak areas, integrate the chromatogram to obtain the sum of the peak areas, and use the area normalization method to calculate the percentage of the peak area of each component (such as D0, D2, D4, D6 or D8) to the total peak area.

Table 1. Chromatographic method 1

Example 2, HIC-HPLC method 2

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

Preparation example of mobile phase A: Weigh 8.62g citric acid monohydrate (C ₆ H ₈ O ₇ ·H ₂ O, MW: 210.14g/mol), 17.35g sodium citrate dihydrate (Na ₃ C ₆ H ₅ O ₇ ·2H ₂ O, MW: 294.10g/mol), 198.21g ammonium sulfate ((NH ₄ ) ₂ SO ₄ , MW: 132.14g/mol), add 800ml purified water to dissolve, and adjust the pH value to 5.00± with 1M NaOH solution 0.05, 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 citric acid monohydrate (C ₆ H ₈ O ₇ ·H ₂ O, MW: 210.14g/mol), 13.88g sodium citrate dihydrate (Na ₃ C ₆ H ₅ O ₇ ·2H ₂ O, MW: 294.10g/mol), 198.21g ammonium sulfate ((NH ₄ ) ₂ SO ₄ , MW: 132.14g/mol), add 700ml purified water to dissolve, adjust the pH value to 5.00±0.05 with 1M NaOH solution, add purified water to 800ml, add 200ml isopropanol, and then use 0.22 Filter with μm membrane, store at 2-8℃, valid for 1 month, need to be filtered before use.

2.2 Chromatographic methods and calculations

The chromatographic method is shown in Table 2.

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

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

Table 2. Chromatographic method 2

Example 3. Investigation of the influence of quenching agent on the preparation method

3.1 Refer to WO2020164561A1 to prepare adalimumab-MMAE samples: adalimumab-MMAE-01, adalimumab-MMAE-02, and adalimumab-MMAE-03. See Table 3 for reaction conditions, and see Table 4 and Figures 1-3 for specific results.

Specific steps are as follows:

(1) Add ZnCl ₂ (0.270mM) and TCEP (purchased from Aldrich Sigma, 0.540mM) to the solution of adalimumab (Shanghai Maijin Biomedical Technology Co., Ltd., 0.135mM) (PB buffer, pH=6.5) medium, let stand overnight at 0-4°C;

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

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

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

Table 3. Reaction conditions

Note: “√” means that this reagent was added to the reaction, and “×” means that this reagent was not added to the reaction.

Table 4. Result characterization

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 impact on the coupling reaction results. When the coupling temperature is increased, the selectivity becomes worse. (The D4 ratio of the adalimumab-MMAE-01 sample is significantly lower than that of the adalimumab-MMAE-01 sample), and an obvious increase in odd peaks can be seen; in addition, if a quencher is not used to react with the remaining toxins, After adding the EDTA solution, unreacted toxins and unreacted sulfhydryl groups on the antibody will continue to react, losing selectivity (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), perform the coupling experiment again using the same process as in Section 3.1 to prepare adalimumab-MMAE samples: adalimumab-MMAE-04, adalimumab Dalimumab-MMAE-05, adalimumab-MMAE-06. See Table 5 for reaction conditions, and see Table 6 and Figure 4-6 for specific results.

Specific steps are as follows:

(1) Add ZnCl ₂ (0.270mM) and diphenylphosphoacetic acid (purchased from Yuhan Chemical Industry, 0.390mM) into the solution of adalimumab (0.135mM) (histidine buffer, pH=6.5) , let stand overnight at 0-4℃;

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

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

Table 5. Process parameters

Table 6. Result characterization

The HIC results in Table 6 show that after replacing the reducing agent with diphenylphosphoacetic acid, the proportion of the D4 part is maintained, and the coupling reaction temperature has no effect on the reaction selectivity, and the reaction will not be significantly affected without quenching. Process selectivity.

Based on the results in 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 Section 3.1 of Example, and does not require the use of reaction quenching reagents, and the coupling reaction is also There are no restrictions on conditions such as temperature.

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

On the basis of Example 3.1, the re-oxidation step was further omitted to optimize the preparation method. Adalimumab-MMAE-07 was prepared. See Table 7 and Figure 7 for specific results.

Specific steps are as follows:

(1) Add ZnCl ₂ (0.270mM) and diphenylphosphoacetic acid (purchased from Yuhan Chemical Industry, 0.412mM) into the solution of adalimumab (0.135mM) (histidine buffer, pH=6.5) , let stand overnight at 0-4℃;

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

(3) Add mc-vc-pab-MMAE (purchased from Lianning Biotech, 0.810mM) 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) purify the reaction solution (removing small molecule process impurities without affecting the different drug-loading components of the prepared ADC).

Drug distribution characterization: Use HIC-HPLC to analyze the drug-antibody ratio (DAR) and drug distribution. Refer to Example 1 for the HIC-HPLC method.

Table 7. Result characterization

Example 5. Characterization of the prepared Farletuzumab-MMAE conjugate and its drug distribution

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

Specific steps are as follows:

(1) Add ZnCl ₂ (0.276mM) and diphenylphosphoacetic acid (purchased from Yuhan Chemical Industry, 0.469mM) into Farletuzumab (Shanghai Maijin Biomedical Technology Co., Ltd., 0.138mM) solution (histidine buffer, pH=6.5), let stand overnight at 0-4°C;

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

(3) Add mc-vc-pab-MMAE (purchased from Aldrich Sigma) dissolved in 100% DMSO (purchased from Aldrich Sigma) Zilian Biotechnology, 0.828mM), 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-loading components of the prepared ADC). Drug distribution characterization: Drug-to-antibody ratio (DAR) and drug distribution were analyzed using HIC-HPLC. Refer to Example 1 for HIC-HPLC method.

Table 8. Result characterization

Example 6. Characterization of the prepared Enoblituzumab-MMAE conjugate and its drug distribution

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

Specific steps are as follows:

(1) Add ZnCl ₂ (0.278mM) and diphenylphosphoacetic acid (purchased from Yuhan Chemical Industry, 0.390mM) to the solution of Enoblituzumab (Shanghai Maijin Biomedical Technology Co., Ltd., 0.139mM) (histidine buffer , pH=6.5), let stand at 0-4°C overnight;

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

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

Table 9. Result characterization

According to the results in Table 7-9, adding a metal chelating agent after the reduction reaction is completed can further omit the re-oxidation step and has almost no impact on the selectivity of the reaction.

Example 7. Investigation of the influence of transition metal ions on the preparation method

Investigate the impact of not adding ZnCl ₂ on the preparation method. Among them, the preparation method of the sample adding _ZnCl2 Reference Examples 4-6.

The samples Adalimumab-MMAE-08 (without ZnCl2), Farletuzum-MMAE-02 (without ZnCl2), and Enoblituzumab-MMAE-02 (without ZnCl2) were prepared without adding ZnCl2. The specific results are shown in Table 10 and Figures 8 _, 10, 12. The only difference from Example 4-6 is that ZnCl ₂ is not added.

The specific steps without adding ZnCl ₂ are as follows:

(1) Add an appropriate amount of diphenylphosphoacetic acid (purchased from Yuhan Chemical Industry, the concentration is the same as in Example 4-6) to the antibody solution that needs to be reduced (the antibody type and concentration are the same as in Example 4-6), Let stand at 0-4℃ overnight;

(2) Add mc-vc-pab-MMAE (purchased from Lianning Biotech) dissolved in 100% DMSO (purchased from Aldrich Sigma), and the concentration is the same as in Example 4-6, 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-loading components of the prepared ADC). Drug distribution characterization: Drug-to-antibody ratio (DAR) and drug distribution were analyzed using HIC-HPLC. Refer to Example 1 for HIC-HPLC method.

Table 10. Result characterization

Table 10 HIC results show that compared with only using 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 processes and their drug distribution

The adalimumab-4-B00 conjugate was prepared using the disclosed process and the traditional process (using TCEP as the reducing agent), where Adam represents adalimumab and n represents DAR.

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

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

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

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

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

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

8.2 The specific steps of traditional craftsmanship are as follows:

(1) Add tris(2-carboxyethyl)phosphine (TCEP, purchased from Aldrich Sigma, 0.297mM) dissolved in water to the solution of adalimumab (0.135mM) (histidine buffer, pH=6.5) medium, room temperature Reaction 2h;

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

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

(4) Ultrafiltration centrifuge tubes (purchased from Merck, Ultracel-30k, ROCB37218) purify the reaction solution (removing small molecule process impurities without affecting the different drug-loading components of the prepared ADC). Use HIC-HPLC to analyze the drug-antibody ratio (DAR) and drug distribution. Refer to Example 2 for the HIC-HPLC method. 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 the 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 by means of the drawings and examples for clear understanding, the description and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety.

Claims

A method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof, which includes the following steps:

(a) Reacting a reducing agent and an antibody in the presence of transition metal ions to selectively reduce the interchain disulfide bonds within the antibody to sulfhydryl groups;

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

(c) Step (b) obtains the antibody-drug conjugate or a pharmaceutically acceptable salt thereof without going through a quenching step and/or a re-oxidation step.
The method according to claim 1, wherein the transition metal ion in step (a) is selected from Zn 2+ , Cd 2+ , Hg 2+ or a combination thereof; preferably from Zn 2+ .
The method according to claim 1 or 2, wherein the reducing agent in step (a) is a reducing agent containing diphenylphosphine group, or a salt thereof; preferably 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-hydroxy Phenyl)diphenylphosphine, or a salt thereof; more preferably diphenylphosphinoacetic acid, or a salt thereof.
The method according to any one of claims 1 to 3, wherein step (a) is carried out at -10°C to 37°C; preferably at 0°C to 30°C; more preferably at 0°C to 25°C.
The method according to any one of claims 1 to 4, wherein the step (a) and step (b) further includes the step of adding a metal chelating agent; preferably, the metal chelating agent is selected from ethylene glycol Aminetetraacetic acid, ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid dicalcium salt, diethylenetriaminepentacetic acid or mixtures thereof.
The method according to 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 thiol group.
The method according to any one of claims 1 to 6, wherein the obtained antibody-drug conjugate or pharmaceutically acceptable salt thereof has an average drug loading of 3.0 to 5.0; preferably greater than 3.0 and less than 5.0 .
The method according to any one of claims 1 to 7, wherein said antibody is selected from monoclonal antibodies or polyclonal antibodies.
The method according to any one of claims 1 to 8, wherein the antibody is selected from the group consisting of murine antibodies, Fully human antibodies, humanized antibodies, chimeric antibodies or their antigen-binding fragments.
The method according to any one of claims 1 to 9, wherein said antibody is selected from IgG1 or IgG4 isotypes.
The method according to any one of claims 1 to 10, wherein the drug is selected from the group consisting of diagnostic agents, therapeutic agents or labeling agents.
An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, prepared by the method described in any one of claims 1 to 11.
A pharmaceutical composition comprising an effective amount of the antibody-drug conjugate according to claim 12 or a pharmaceutically acceptable salt thereof, and optional excipients, diluents or carriers.
A method of treating and/or preventing a condition or disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate according to claim 12 or a pharmaceutical method thereof. An acceptable salt, or a step of the pharmaceutical composition according to claim 13; preferably, the disorder or disease is selected from tumors, cancers, autoimmune diseases, or infectious diseases; more preferably, the autoimmune diseases Selected from rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, adult Crohn's disease, pediatric Crohn's disease, ulcerative colitis, hidradenitis suppurativa, grape inflammation, Behcet's disease, spondyloarthropathy, and psoriasis.
A method for selectively reducing an antibody includes step (a): reacting a reducing agent and an antibody in the presence of transition metal ions to selectively reduce the interchain disulfide bond within the antibody to a sulfhydryl group.
The method of selectively reducing an antibody according to claim 15, wherein step (a) is as defined in any one of claims 2 to 10.