Method for preparing antibody-drug conjugate by one-step coupling

Abstract

The present invention relates to a method for preparing an antibody-drug conjugate (ADC), which comprises incubating a mixture of an antibody, a linker-drug moiety, and a reducing agent in a buffer system, thereby obtaining an incubation mixture, wherein the linker is a substitution linker or a linker containing a carbon-carbon triple bond. The present invention further relates to an antibody-drug conjugate prepared by the method, and use of the antibody-drug conjugate in the treatment of diseases.

Description

Method for preparing antibody-drug conjugate by one-step conjugation TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to a biological conjugation method for preparing an antibody-drug conjugate by one-step conjugation, an antibody-drug conjugate prepared by the method, and the use of the antibody-drug conjugate in treating diseases. BACKGROUND

[0002] An antibody-drug conjugate (ADC) contains an antibody for targeting, a linker for drug connection, and a high-efficiency payload (e.g., a drug) as an effector. Since the U.S. Food and Drug Administration approved Adcetris (brentuximab vedotin) in 2011, the approval rate of ADC drugs has gradually increased, and has now been widely expanded for the treatment of cancer. Currently, drugs including Adcetris, Kadcyla (trastuzumab emtansine), Besponsa (inotuzumab ozagamicin), Mylotarg (gemtuzumab ozogamicin), Polivy (polatuzumab vedotin-piiq), Blenrep (margetuximab), Enhertu (deruxtecan), Padcev (enfortumab vedotin), Trodelvy (sacituzumab govitecan), Tivdak (tisotumab vedotin), Zynlonta (loncastuximab tesirine), Akalux, Lumoxiti (pixantrone), or Libtayo, etc. are included. At the same time, more drugs are in the process of research in the clinical stage.

[0003] The most widely used existing antibody-drug conjugate preparation technology is cysteine-based conjugation, which is divided into two steps:

[0004] In the first step, a reducing agent is used to reduce the disulfide bond on the interchain cysteine of the antibody to two cysteines. In the second step, an organic solvent and a linker-drug are added to the reaction system, so that the thiol group of the cysteine reacts with the linker to generate a mixture solution of antibody-drug conjugates, and the final product is obtained after purification.

[0005] The deficiency of the prior art is that: since the reactive group in the linker of the antibody-drug conjugate is usually a maleimide, the reactivity is extremely high, and in large-scale industrial production, the mixing of the organic solvent and the aqueous solution needs to spend the same or more time as the chemical reaction, which increases the risk of side reactions. At the same time, in industrial production, each additional process will increase the corresponding cost.

[0006] On the other hand, antibody-drug conjugates prepared with linkers containing maleimides release drug molecules prematurely due to the reverse Michael addition reaction in vivo, causing adverse reactions. In recent years, more linkers that can remain stable in vivo have been developed, but conjugation techniques compatible with new linkers have not been developed.

[0007] A reference preparation scheme comes from WO2020164561A1, which uses metal ions to lock the two S-S bonds of the hinge region of the antibody, so that it cannot be reduced under the action of general reducing agents. Under this action, only two disulfide bonds on one antibody can be opened, releasing four thiol groups, so it can selectively prepare products with a DAR value of 4 (D4). But it still uses two or more steps.

[0008] The prior art needs to reduce the disulfide bond of the antibody first, and then use the active drug to conjugate the antibody when modifying the thiol group of the antibody. It is also difficult to monitor the progress of the reaction in real time in the process.

[0009] Therefore, it is necessary to find a new method for preparing antibody-drug conjugates that simplifies the steps, is low in cost, and can realize real-time monitoring of the reaction. SUMMARY

[0010] The present application overcomes the deficiencies in the prior art and provides a new biological conjugation method for preparing antibody-drug conjugates. The method can effectively reduce the production cost of antibody-drug conjugates and improve the homogeneity of the final antibody-drug conjugates. Specifically, the antibody-drug conjugates prepared by the method can effectively save the operation steps in the production process and expand the time window of the operation in the production process. Compared with the conventional conjugation method involving the use of reducing agents and nucleophilic reactions of thiol groups, the present application has fewer operation steps, more relaxed time requirements for operation, and significantly improved homogeneity of the produced antibody-drug conjugates.

[0011] In one aspect, the present application provides a method for preparing an antibody-drug conjugate, comprising incubating a mixture of an antibody, a linker-drug moiety and a reducing agent in a buffer system, thereby obtaining an incubation mixture; wherein the linker is a substituted linker or a linker containing a carbon-carbon triple bond. The method can realize the reduction of the disulfide bond of the antibody while coupling with the active thiol group, thereby producing the antibody-drug conjugate in one step.

[0012] In some embodiments, the linker in the linker-drug moiety contains a leaving group connected by a linking fragment.

[0013] In some embodiments, the linker in the linker-drug moiety contains a group containing a carbon-carbon triple bond linked by a linking segment.

[0014] In some embodiments, the linking segment is selected from one or more of alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, sulfonyl, carbonyl, acyl, keto, imine, an amino acid, wherein each of the alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene is independently optionally substituted with 1-10 C 1-6 alkyl, C 1-6 heteroalkyl, C 5-10 heteroaryl, aryl, hydroxyl, amino, F, CI, Br, I, and / or cyano.

[0015] In some embodiments, the leaving group is selected from the following structures: C 1-6 alkyl-SO2-, C 1-6 alkyl-SO3-, C 1-6 alkyl-CO2-, C 1-6 alkyl-O-, (C 1-6 alkyl) 1-2 -(H) 2-1 PO3-, aryl-SO2-, aryl-SO3-, aryl-CO2-, (aryl) 1-2 -(H) 2-1 PO3-, aryl-O-, HSO2-, HSO3-, HC02-, H3PO3-, HO-, palladium addition complex, palladium oxide addition complex, platinum addition complex, benzenesulfonyl chloride, pyridinium group, sulfonyl fluoride, pentafluorophenyl ester, F, CI, Br, I, mono- or di- derivatives of disulfides, bicyclo[l. l.0]butane derivatives, highly electron-deficient aryl groups, alkenes, alkynes with electron-deficient groups, wherein each of the alkyl or aryl groups is independently optionally substituted with 1-6 C 1-6 alkyl, C 1-6 heteroalkyl, C 5-10 heteroaryl, aryl, hydroxyl, amino, F, CI, Br, I, and / or cyano. In some preferred embodiments, the leaving group is selected from sulfonyl fluoride, pentafluorophenyl ester, benzenesulfonyl chloride, Br, I, C 1-6 alkyl-SO2-.

[0016] In some embodiments, the drug in the linker-drug moiety is selected from the group consisting of a diagnostic agent, a therapeutic agent, and a labeling agent. In some embodiments, the drug in the linker-drug moiety is selected from the group consisting of a cytotoxic agent, a toxin, a radionuclide, a fluorescent agent (e.g., an amine derivatized fluorescent probe such as 5-dimethylaminonaphthalene-l-(N-(2-aminoethyl))sulfonamide-dansyl ethylenediamine, Oregon Green® 488cadaverine (catalog no. O-10465, Molecular Probes), dansyl cadaverine, N-(2-aminoethyl)-4-amino-3,6-dithio-l,8-naphthalimide, dipotassium salt (fluorophor yellow ethylenediamine), rhodamine B ethylenediamine (catalog no. L-2424, Molecular Probes), or a thiol derivatized fluorescent probe such as fluorescamine (catalog no. F-9135, Molecular Probes), a chemotherapeutic agent, an immunotherapeutic agent, an antiviral agent, an antimicrobial agent, a molecular degrading agent, an immune agonist, and a nuclear pharmaceutical chelator. In some embodiments, the drug in the linker-drug moiety is selected from the group consisting of an Auristatin, a topoisomerase inhibitor, a Maytansinoid, and a PBD, e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), exetecan, pyrrolobenzodiazepine dimers.

[0017] In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 20: 1 to 1 : 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 15: 1 to 5: 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 10: 1 to 5: 1.

[0018] In some embodiments, the reducing agent does not comprise a sulfur-based reducing agent. In some embodiments, the reducing agent is selected from the group consisting of a phosphine-based reducing agent and a metal hydride. In some embodiments, the reducing agent is selected from one or more of tris(2-carboxyethyl)phosphine hydrochloride, diphenylphosphinoacetic acid, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(diphenylphosphino)propanoic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, 1,3,5-triaza-7-phospha-tricyclo[3.3.1.13.7]decane, and n-butyldi(l-adamantyl)phosphine, sodium borohydride, and lithium aluminum hydride.

[0019] ​In some embodiments, the molar ratio of the reducing agent to the antibody is selected from the following ranges: 1 : 1 to 30: 1 ; 1 : 1 to 25: 1 ; 1 : 1 to 20: 1 ; 1 : 1 to 15: 1 ; 1 : 1 to 10: 1 ; 1 : 1 to 8: 1 ; 3: 1 to 8: 1 ; 3: 1 to 7: 1 ; 3: 1 to 6: 1 ; 3: 1 to 5: 1 ; or 4: 1.

[0020] In some embodiments, the incubation is performed in the presence of an effective amount of one or more metal ions. In some embodiments, the metal ions are selected from the ions of the metal elements in the first to third transition series and the second to fifth main groups. In some embodiments, the metal ions are selected from transition metal ions. In some embodiments, the metal ions are selected from the group consisting of zinc ions (Zn 2+ ), cadmium ions (Cd 2+ ), mercury ions (Hg 2+ ), gallium ions (Ga 3+ ), germanium ions (Ge 4+ ), indium ions (In 3+ ), tin ions (Sn 4+ ), bismuth ions (Bi 3+ ), manganese ions (Mn 2+ ), nickel ions (Ni 2+ ), ferrous ions (Fe 2+ ), ferric ions (Fe 3+ ), and copper ions (Cu 2+ ). In some embodiments, the metal ions are selected from the group consisting of zinc ions (Zn 2+ ), cadmium ions (Cd 2+ ), mercury ions (Hg 2+ ), gallium ions (Ga 3+ ), germanium ions (Ge 4+ ), indium ions (In 3+ ), tin ions (Sn 4+ ), and bismuth ions (Bi 3+ ).

[0021] In some embodiments, the molar ratio of the metal ions to the antibody is selected from the following ranges: 10: 1 to 1 : 10; 9: 1 to 1 : 9; 8: 1 to 1 : 8; 7: 1 to 1 : 7; 6: 1 to 1 : 6; 5: 1 to 1 : 5; 4: 1 to 1 : 4; 3: 1 to 1 : 3; 2: 1 to 1 : 2; 1 : 1 to 1 : 10; 1 : 1 to 1 : 9; 1 : 1 to 1 : 8; 1 : 1 to 1 : 7; 1 : 1 to 1 : 6; 1 : 1 to 1 : 5; 1 : 1 to 1 : 4; 1 : 1 to 1 : 3; 1 : 1.25 to 1 : 3; 1 : 1.25 to 1 : 2.

[0022] In embodiments where metal ions are added, a chelating agent is added to the incubation mixture after the incubation mixture is obtained to capture the metal ions for ease of removal in subsequent steps. In some embodiments, the chelating agent is EDTA, DOTA, and DPTA.

[0023] In some embodiments, the buffer system is selected from Hepes, histidine buffer, PBS, MES, and Tris. In some embodiments, the buffer system has a pH of about 5.5 to 9. In some embodiments, the buffer system has a pH of about 5.5 to 8. In some embodiments, the incubation temperature is about -10 °C to 37 °C. In some embodiments, the incubation temperature is about 0 °C to 25 °C. In some embodiments, the incubation time is 3-20 hours. In some embodiments, the incubation time is 7-20 hours.

[0024] In some embodiments, the antibody is selected from a monoclonal antibody and a polyclonal antibody. In some embodiments, the antibody is selected from a human antibody, a humanized antibody, and a chimeric antibody. In some embodiments, the antibody specifically binds to a corresponding antigen expressed on a cancer cell (also known as a tumor associated antigen (TAA)), a viral antigen, or a microbial antigen, and has antibody-dependent cell-mediated phagocytosis (ADCP) activity, in vivo anti-tumor, anti-viral, or anti-microbial activity. In some embodiments, the monoclonal antibody is selected from trastuzumab, pertuzumab, racotumomab, abciximab, adalimumab, alfaferone, alemtuzumab, basiliximab, belimumab, belotufosumab, canakinumab, pexidartinib, cetuximab, daclizumab, denosumab, efalizumab, golimumab, infliximab, ipilimumab, isibamumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, sucralfate, and ustekinumab.

[0025] In some embodiments, the method further comprises post-treatment of the incubation mixture. In some embodiments, the post-treatment comprises adding a quencher to deplete excess linker-drug moieties, and / or purifying using a desalting column and / or size exclusion chromatography. In some embodiments, the quencher is a molecule comprising a reactive thiol group and salts thereof. In some embodiments, the quencher is selected from N-acetyl cysteine, cysteine, cysteamine, and dithiothreitol.

[0026] In another aspect, the present application provides an antibody-drug conjugate prepared by the methods described herein that is more homogenous than those prepared by conventional conjugation methods.

[0027] In some embodiments, the present application produces antibody-drug conjugates having a DAR value of 2. In some embodiments, the present application produces antibody-drug conjugates having a DAR value of 4.

[0028] In some embodiments, the antibody-drug conjugates produced by the present application have a content of antibody-drug conjugates having a DAR value of 4 greater than 45%. In some embodiments, the antibody-drug conjugates produced by the present application have a content of antibody-drug conjugates having a DAR value of 4 (DAR4 or D4) greater than 60%. In some embodiments, the antibody-drug conjugates produced by the present application have a content of antibody-drug conjugates having a DAR value of 4 (DAR4 or D4) greater than 70%. In some embodiments, the antibody-drug conjugates produced by the present application have a total content of antibody-drug conjugates having a DAR value of 0 (DAR0 or D0) and a DAR value of 8 (DAR8 or D8) less than 20%. In some embodiments, the antibody-drug conjugates produced by the present application have a content of antibody-drug conjugates having a DAR value of 6 (DAR6 or D6) less than 20%.

[0029] In another aspect, the present application provides a pharmaceutical composition comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier or excipient.

[0030] In another aspect, the present application provides use of an antibody-drug conjugate described herein or a pharmaceutical composition described herein in the manufacture of a medicament for treating a disease selected from the group consisting of cancer, autoimmune disease, inflammation, and metabolic disease.

[0031] In another aspect, the present application provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate described herein or a pharmaceutical composition described herein, the disease being selected from the group consisting of cancer, autoimmune disease, inflammation, and metabolic disease.

[0032] In some embodiments, the disease is selected from the group consisting of transitional cell carcinoma of the bladder, hepatitis C, paroxysmal nocturnal hemoglobinuria (PNH), colorectal cancer, urothelial cancer, advanced breast cancer, bladder cancer, gastric cancer, elevated low-density lipoprotein cholesterol, superoxide dismutase 1-amyotrophic lateral sclerosis (SOD1-ALS), Pompe disease (acid maltase deficiency), and hypertrophic scars.

[0033] The present application has the following beneficial effects:

[0034] 1、The present application can synthesize ADCs in one step, which is more simplified than the conventional strategy of first reduction and then coupling, and the characteristics of the reduction reaction and the coupling reaction itself are not affected;

[0035] 2、The method of the present application avoids any need for protein modification or enzyme catalysis, but is based on natural interchain disulfide bonds and optional transition metal ions. Therefore, compared with conventional methods for preparing ADCs, the method of the present application has lower complexity, and the homogeneity of the antibody-drug conjugate obtained is significantly improved, and the cost is greatly reduced;

[0036] 3、By using a reducing agent and a linker-drug that do not react with each other, such as using a linker-drug containing a leaving group or using a linker-drug containing a carbon-carbon triple bond, the coupling can be carried out in a one-step method, shortening the process flow and reducing the process complexity;

[0037] 4、The reduction reaction and the coupling reaction of the present application are orthogonal to each other, and real-time monitoring of the reaction can be achieved;

[0038] 5、Using the method of the present application, an antibody conjugated drug with a DAR value of 4 can be selectively prepared. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate prepared by one-step method one in Example 3.

[0040] Figure 2 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate prepared by one-step method two in Example 3.

[0041] Figure 3 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate prepared by a conventional method in Example 3.

[0042] Figure 4 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate prepared by the method disclosed in WO2020164561A1 in Example 3.

[0043] Figure 5 is a hydrophobic interaction chromatogram of Herceptin-MC-VC-PAB-MMAE conjugate prepared by a conventional method in Example 4.

[0044] Figure 6 is a hydrophobic interaction chromatogram of Herceptin-MC-VC-PAB-MMAE conjugate prepared by one-step method one in Example 4.

[0045] Figure 7 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (12℃) prepared by one-step method one in Example 5.

[0046] Figure 8 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (12℃) prepared by one-step method two in Example 5.

[0047] Figure 9 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (12 °C) prepared by the conventional method in Example 5.

[0048] Figure 10 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (16 °C) prepared by one-step method one in Example 5.

[0049] Figure 11 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (16 °C) prepared by one-step method two in Example 5.

[0050] Figure 12 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (16 °C) prepared by the conventional method in Example 5.

[0051] Figure 13 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (22 °C) prepared by one-step method one in Example 5.

[0052] Figure 14 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (22 °C) prepared by one-step method two in Example 5.

[0053] Figure 15 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (22 °C) prepared by the conventional method in Example 5.

[0054] Figure 16 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (pH = 7) prepared by one-step method one in Example 6.

[0055] Figure 17 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (pH = 8) prepared by one-step method one in Example 6.

[0056] Figure 18 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (pH = 7) prepared by the conventional method in Example 6.

[0057] Figure 19 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate (pH = 8) prepared by the conventional method in Example 6.

[0058] Figure 20 is a hydrophobic interaction chromatogram of Herceptin-DL001 conjugate prepared by one-step method two in Example 7.

[0059] Figure 21 is a hydrophobic interaction chromatogram of Erbitux-DL001 conjugate prepared by one-step method two in Example 7.

[0060] Figure 22 is a hydrophobic interaction chromatogram of Rituximab-DL001 conjugate prepared by one-step method two in Example 7.

[0061] Figure 23 is a hydrophobic interaction chromatogram of Herceptin-DL003 conjugate (PBS buffer, 12°C, pH 7) prepared by one-step method two in Example 8.

[0062] Figure 24 is a hydrophobic interaction chromatogram of Herceptin-DL003 conjugate (PBS buffer, 12°C, pH 8) prepared by one-step method two in Example 8.

[0063] Figure 25 is a hydrophobic interaction chromatogram of Herceptin-DL003 conjugate (PBS buffer, 16°C, pH 7) prepared by one-step method two in Example 8.

[0064] Figure 26 is a hydrophobic interaction chromatogram of Herceptin-DL003 conjugate (PBS buffer, 16°C, pH 8) prepared by one-step method two in Example 8.

[0065] Figure 27 is a hydrophobic interaction chromatogram of Herceptin-DL003 conjugate (Tris buffer, 22°C, pH 8) prepared by one-step method two in Example 8.

[0066] Figure 28 is a PLRP chromatogram of Herceptin-DL004 conjugate (Hepes, 22°C, pH 7) prepared by one-step method two in Example 8.

[0067] Figure 29 is a hydrophobic interaction chromatogram of Herceptin-DL005 conjugate (Tris buffer, 22°C, pH 8) prepared by one-step method two in Example 8.

[0068] Figure 30 is a hydrophobic interaction chromatogram of Herceptin-Medilink-DL001 conjugate (2-(diphenylphosphino)-acetic acid reducing agent) prepared by one-step method two in Example 9.

[0069] Figure 31 is a hydrophobic interaction chromatogram of Herceptin-Medilink-DL002 conjugate (2-(diphenylphosphino)-acetic acid reducing agent) prepared by one-step method two in Example 9. DETAILED DESCRIPTION

[0070] The present application is further illustrated in the following specific embodiments and examples. It is to be understood that these embodiments and examples are merely illustrative of the present application and do not limit the scope of the present application.

[0071] Definitions

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event of a conflict in terminology, practice or definition between this section and a patent, application, published application or other publication, the terminology, practice or definition in this section controls.

[0073] In this document, the terms "comprise" and "comprising" can be used interchangeably with the terms "include" and "including". "Comprise" and "comprising" are to be interpreted as specifying the presence of the stated features or components, but do not preclude the presence or addition of one or more other features, components or groups thereof. In addition, "comprise" and "comprising" are intended to include examples of the term "consisting of. Thus, the term "consisting of" can be substituted for the terms "comprising" and "including" to provide a more specific embodiment.

[0074] As used herein, the terms "antibody-drug conjugate", "antibody-drug conjugate", "antibody conjugated drug" and "ADC" have the same meaning and refer to a drug composed of an antibody, a cytotoxic drug and a linker connecting the antibody and the cytotoxic drug.

[0075] As used herein, the term "drug" or "payload" refers to any cytotoxic molecule having, for example, an anti-tumor effect, an anti-infective or an anti-inflammatory effect and having at least one substituent group or moiety structure allowing connection to a linker structure. The drug can kill cells (e.g., cancer cells) and / or inhibit the growth, proliferation or metastasis of cells (e.g., cancer cells), thereby reducing, alleviating or eliminating one or more symptoms of a disease or disorder (e.g., cancer).

[0076] As used herein, the term "linker" refers to a reactive molecule containing at least two reactive groups, one of which can be covalently bound to a drug molecule and the other of which can be covalently coupled to an antibody. The term "substitution type linker" refers to a linker that can undergo an electrophilic / nucleophilic substitution reaction or an addition-elimination reaction with an antibody; from the reaction result, it is embodied that one of the reactive groups on the linker is replaced by a group on the antibody, and a reactive group on the linker is removed, which is also called a "leaving group". The reaction is represented by the following reaction formula: leaving group-linker + antibody-SH -> leaving group + antibody-S-linker.

[0077] As used herein, the term "linker-drug" refers to a reactive group in the "linker" that has covalently bound to a drug molecule.

[0078] As used herein, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains form the antigen binding site. Bispecific antibody molecules, trispecific antibody molecules, and multispecific antibody molecules are also encompassed within the antibodies according to the application. Chimeric antibodies or humanized antibodies are also encompassed within the antibodies according to the application.

[0079] As used herein, the term "antigen binding fragment" refers to a fragment of an antibody, e.g., a fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion" (see, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Antigen binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide stabilized Fv proteins ("dsFv"), single domain antibodies (sdAb, nanobodies), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.

[0080] As used herein, "Fab" of an antibody refers to the following portion of an antibody, which consists of a single light chain (both variable and constant regions) associated through disulfide bonds with the variable region and the first constant region of a single heavy chain. In some embodiments, both the first and second antigen binding portions of the antibody to be conjugated are in Fab format. Further, the constant regions of both chains of the Fab (i.e., CH1 and CL) are replaced by engineered or modified TCR constant regions.

[0081] As used herein, the "Fc" of an antibody refers to the portion of an antibody comprising the second (CH2) and third constant region (CH3) of the first heavy chain, in combination with the second and third constant regions of the second heavy chain via disulfide bonds and optionally the hinge region. The Fc portion of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not play a role in antigen binding.

[0082] As used herein, the term "leaving group" refers to an atom or group of atoms that is removed from a major or residual portion of a substrate during or in an essential step of a reaction.

[0083] As used herein, the term "DAR" also known as Drug to Antibody Ratio, is a unique quality attribute of antibody-drug conjugates. The selection of DAR value depends on the characteristics of the target antigen, the characteristics of the linker-drug / payload, which need to be considered comprehensively. Various analytical methods can be used to measure DAR, such as ultraviolet-visible (UV / Vis) spectroscopy, hydrophobic interaction chromatography (HIC), reversed-phase high-performance liquid chromatography (RP-HPLC), and liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS). Hydrophobic interaction chromatography (HIC) is a leading technology for characterizing DAR values and drug / payload distribution.

[0084] As known in the art, a mixture of antibody-drug conjugates will be produced by conventional conjugation methods. Typically, the antibody molecule can be partially or completely reduced 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 the number of drugs conjugated to a single antibody molecule is 2, 4, 6, or 8. If a disulfide-reactable linker-drug is used, the number of drugs conjugated to a single antibody molecule can be further enriched to 1, 2, 3, 4, 5, 6, 7, or 8. Depending on the number of drugs conjugated to a single antibody molecule, different antibody-drug conjugates containing different numbers of drug molecules include D0 (DAR value of 0), D1 (DAR value of 1), D2 (DAR value of 2), D3 (DAR value of 3), D4 (DAR value of 4), D5 (DAR value of 5), D6 (DAR value of 6), D7 (DAR value of 7), and D8 (DAR value of 8) antibody-drug conjugates.

[0085] As used herein, the term "homogeneity" or "homogeneity" of an antibody-drug conjugate is used to describe the property of a particular type of antibody-drug conjugate (preferably, a type of antibody-drug conjugate selected from D1, D2, D3, D4, D6) to dominate in a given mixture of antibody-drug conjugates. In the present invention, "homogeneity" or "homogeneity" of an antibody-drug conjugate means that a particular type of antibody-drug conjugate has a high level in a mixture of antibody-drug conjugates.

[0086] As used herein, the term "transition metal" refers to the elements of Groups 4B-1 IB of the Periodic Table of the Elements, having empty d orbitals available for bonding and a high charge / radius ratio, which readily form stable coordination compounds with a variety of ligands.

[0087] As used herein, the term "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" refers to a pharmaceutically-acceptable material, component or medium that is nontoxic to the subject, e.g., a liquid or solid filler, diluent, excipient, solvent or encapsulating material. In some embodiments, each component of a pharmaceutical formulation is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenecity, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0088] As used herein, the term "subject" includes any human or non-human animal, e.g., a human.

[0089] As used herein, the term "cancer" refers to any neoplastic or malignant cell growth, proliferation, or metastasis-mediated solid and non-solid tumors such as leukemias, and that gives rise to a medical condition. A "tumor" comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, penile cancer, thyroid cancer, hepatocellular cancer, anal carcinoma, penile carcinoma, and head and neck cancer.

[0090] As used herein, the terms "treatment," "treating," or "treated" generally refer to the treatment or therapy of a human or animal body with the aim of achieving a desired therapeutic effect, e.g., inhibiting the progression of a condition, and includes a reduction in the rate of progression, a halt in the rate of progression, a regression of the condition, a remission of the condition, and a cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included. For cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, or some combination thereof. For a tumor, "treatment" includes removal of all or part of the tumor, inhibiting or slowing the growth and metastasis of the tumor, inhibiting or delaying the development of the tumor, or some combination thereof.

[0091] Methods of preparing antibody-drug conjugates

[0092] In one aspect, the present application provides a method for preparing an antibody-drug conjugate, comprising incubating a mixture of an antibody, a linker-drug moiety, and a reducing agent in a buffer system, thereby obtaining an incubation mixture; wherein the linker is a substitution-type linker or a linker containing a carbon-carbon triple bond.

[0093] The present inventors have surprisingly found that incubating a mixture of an antibody to be conjugated, a substitution-type group-containing linker-drug moiety or a linker-drug moiety containing a carbon-carbon triple bond, and a reducing agent in a buffer system, conjugates the exposed active thiol sites while reducing the disulfide bonds. Such a method of preparation reduces a step from the traditional procedure, and the homogeneity of the resulting antibody-drug conjugate is significantly improved.

[0094] The antibody, substitution-type group-containing linker-drug moiety or linker-drug moiety containing a carbon-carbon triple bond, and the reducing agent can be added prior to the start of the incubation. The antibody and the reducing agent can also be added first and the incubation started, and the linker-drug moiety added before the reducing agent has completely reduced the antibody.

[0095] In some embodiments, the linker in the linker-drug moiety contains a leaving group linked by a linking fragment. In some embodiments, the linker in the linker-drug moiety contains a carbon-carbon triple bond-containing group linked by a linking fragment. In some embodiments, the linking fragment is selected from one or more of alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, sulfonyl, carbonyl, acyl, keto, imine, an amino acid, wherein each of the alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene is independently optionally substituted with 1-10 C 1-6 alkyl, C 1-6 heteroalkyl, C 5-10 heteroaryl, aryl, hydroxyl, amino, F, CI, Br, I, and / or cyano. In some embodiments, the leaving group is selected from the following structures: C1-6 alkyl-SO2-, C 1-6 alkyl-SO3-, C 1-6 alkyl-CO2-, C 1-6 alkyl-O-, (C 1-6 alkyl) 1-2 -(H) 2-1 PO3-, aryl-SO2-, aryl-SO3-, aryl-CO2-, (aryl) 1-2 -(H) 2-1 PO3-, aryl-O-, HSO2-, HSO3-, HCO2-, H3PO3-, HO-, palladium addition complex, palladium oxide addition complex, platinum addition complex, benzenesulfonyl, pyridinium group, sulfonyl fluoride, pentafluorophenyl ester, F-, Cl-, Br-, I-, mono- or di-derivatives of disulfides, bicyclo[l. l.0]butane derivatives, highly electron-deficient aryl groups, alkenes, alkynes with electron-deficient groups, wherein each of the alkyl or aryl groups is independently optionally substituted with 1-6 C 1-6 alkyl, C 1-6 heteroalkyl, C 5-10 heteroaryl, aryl, hydroxyl, amino, F-, Cl-, Br-, I-, and / or cyano. In some preferred embodiments, the leaving group is selected from sulfonyl fluoride, pentafluorophenyl ester, benzenesulfonyl, Br-, I-, C 1-6 alkyl-SO2-.

[0096] The linker contains at least two reactive groups, one of which can covalently bind a drug molecule and the other can covalently couple to an antibody. Linkers containing maleimides used in conventional ADC manufacturing methods are not applicable in the present application.

[0097] The drug in the linker-drug moiety can be any type of drug, as long as the drug molecule has the desired effect. In some embodiments, the drug in the linker-drug moiety can be a diagnostic agent, a therapeutic agent, and a labeling agent. In some embodiments, the drug in the linker-drug moiety can be a cytotoxic agent, a toxin, a radionuclide, a fluorescent agent (e.g., amine derivatized fluorescent probes such as 5-dimethylaminonaphthalene-l-(N-(2-aminoethyl))sulfonamide-dansyl ethylenediamine, Oregon Green®488 cadaverine (catalog number O-10465, Molecular Probes), dansyl cadaverine, N-(2-aminoethyl)-4-amino-3,6-dithio-l,8-naphthalimide, dipotassium salt (fluorescent yellow ethylenediamine), rhodamine B ethylenediamine (catalog number L-2424, Molecular Probes), or thiol derivatized fluorescent probes such as 488cadaverine (catalog number O-10465, Molecular Probes), dansyl cadaverine, N-(2-aminoethyl)-4-amino-3,6-dithio-l,8-naphthalimide, dipotassium salt (fluorescent yellow ethylenediamine), rhodamine B ethylenediamine (catalog number L-2424, Molecular Probes), or thiol derivatized fluorescent probes such as FLL-cysteine (Cat. No. B-20340, Molecular Probes)), chemotherapeutic agents, immunotherapeutic agents, antiviral agents, antimicrobial agents, molecular degrading agents, immune agonists, and nuclear chelators. In some embodiments, the drug in the linker-drug moiety can be an Auristatin, a topoisomerase inhibitor, a Maytansinoid, and a PBD, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), exetecan, pyrrolobenzodiazepine dimer. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 20: 1 to 1 : 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 19: 1 to 1 : 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 18: 1 to 1 : 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 17: 1 to 1 : 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 16: 1 to 1 : 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 15: 1 to 1 : 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 15: 1 to 2: 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 15: 1 to 3: 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 15: 1 to 4: 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 15: 1 to 5: 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 14: 1 to 5: 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 13: 1 to 5: 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 12: 1 to 5: 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 11 : 1 to 5: 1. In some embodiments, the molar ratio of the linker-drug moiety to the antibody is 10: 1 to 5: 1.

[0098] The reducing agent of the present application does not use sulfur-containing reducing agents used in conventional ADC manufacturing methods, such as dithiothreitol, dithioerythritol, ethane thiol, sodium sulfide, glutathione, and cysteine.

[0099] In some embodiments, the reducing agent is selected from a phosphine-based reducing agent and a metal hydride. In some embodiments, the reducing agent is selected from one or more of tris(2-carboxyethyl)phosphine hydrochloride, diphenylphosphinoacetic acid, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(diphenylphosphino)propanoic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, 1,3,5-triaza-7-phosphanorbornane, and n-butyldi(1-adamantyl)phosphine, sodium borohydride, and lithium aluminum hydride. Because phosphine-based reducing agents have some selectivity in reducing antibodies, the conjugation selectivity of the ADC can be improved in small amounts.

[0100] In some embodiments, the molar ratio of the reducing agent to the antibody is 1:1 to 30:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 1:1 to 25:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 1:1 to 20:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 1:1 to 15:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 1:1 to 10:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 1:1 to 9:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 1:1 to 8:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 2:1 to 8:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 2:1 to 7:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 2:1 to 6:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 2:1 to 5:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 3:1 to 5:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 3:1 to 6:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 3:1 to 7:1. In some embodiments, the molar ratio of the reducing agent to the antibody is 3:1 to 8:1.

[0101] In some embodiments, the incubation of step (a) is performed in the presence of an effective amount of one or more metal ions. In some embodiments, the metal ions are selected from ions of metal elements in the first to third transition series and the second to fifth main groups. In some embodiments, the metal ions are selected from transition metal ions. Transition metal ions suitable for use in the bioconjugation process of the present disclosure can include, but are not limited to, Zn 2+ , Mn 2+ , Ni 2+, Fe 2+ , Fe 3+ , Cu 2+ , etc. For example, appropriate transition metal salts can be added in step (a) as long as they are soluble in the reaction solution so as to release free transition metal ions in the reaction solution. In this regard, ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, Zn(ClO4)2, zinc formate and zinc tetrafluoroborate can be mentioned as suitable zinc salts. Likewise, other transition metal salts soluble in the reaction solution and capable of releasing free transition metal ions can also be mentioned, including but not limited to salts of Cu, Ni, Co, Fe, Mn, Cr, V, Ti, Sc, Mg, Sr, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag, Cd, Ga, Ge, etc.

[0102] In some embodiments, the metal ion is selected from the group consisting of zinc ion (Zn 2+ ), cadmium ion (Cd 2+ ), mercury ion (Hg 2+ ), gallium ion (Ga 3+ ), germanium ion (Ge 4+ ), indium ion (In 3+ ), tin ion (Sn 4+ ), bismuth ion (Bi 3+ ), manganese ion (Mn 2+ ), nickel ion (Ni 2+ ), ferrous ion (Fe 2+ ), ferric ion (Fe 3+ ), copper ion (Cu 2+ ). In some preferred embodiments, the metal ion is selected from the group consisting of zinc ion (Zn 2+ ), cadmium ion (Cd 2+ ), mercury ion (Hg 2+ ), gallium ion (Ga 3+ ), germanium ion (Ge 4+ ), indium ion (In 3+ ), tin ion (Sn 4+ ), bismuth ion (Bi 3+ ). In some more preferred embodiments, the metal ion is zinc ion (Zn 2+ ). The use of zinc ion (Zn 2+ ) as transition metal ion has the advantage of being readily available and low cost.

[0103] In some embodiments, the molar ratio of the metal ion to the antibody is 10: 1 to 1 : 10. In some embodiments, the molar ratio of the metal ion to the antibody is 9: 1 to 1 :9. In some embodiments, the molar ratio of the metal ion to the antibody is 8: 1 to 1 :8. In some embodiments, the molar ratio of the metal ion to the antibody is 7: 1 to 1 :7. In some embodiments, the molar ratio of the metal ion to the antibody is 6: 1 to 1 :6. In some embodiments, the molar ratio of the metal ion to the antibody is 5: 1 to 1 :5. In some embodiments, the molar ratio of the metal ion to the antibody is 4: 1 to 1 :4. In some embodiments, the molar ratio of the metal ion to the antibody is 3: 1 to 1 :3. In some embodiments, the molar ratio of the metal ion to the antibody is 2: 1 to 1 :2. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1 to 1 : 10. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1 to 1 :9. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1 to 1 :8. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1 to 1 :7. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1 to 1 :6. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1 to 1 :5. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1 to 1 :4. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1 to 1 :3. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1.25 to 1 :3. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1.25 to 1 :2. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1.66 to 1 :3. In some embodiments, the molar ratio of the metal ion to the antibody is 1 : 1.66 to 1 :2.

[0104] The inventors have surprisingly found that the homogeneity of the antibody can be further improved if an effective amount of a metal ion (e.g., Zn 2+ etc.) is added in step (a). Specifically, the inventors found that transition metal ions produce an additional selectivity in the reduction of disulfide bonds. In the presence of a transition metal ion, two interchain S-S bonds in the Fab region are selectively reduced. The counterion of the transition metal ion can be any counterion that remains inert in the reaction system, such as chloride (CI ), bromide (Br - ), iodide (I - ), acetate (H3CCOO -) , trifluoroacetate (F3CCOO -), sulfate ion (SO4 2- ), phosphate ion (PO4 3- ), perchlorate ion (CI04 - ), tetrafluoroborate ion (BF4 - ), hexafluorophosphate ion (PF6 - ).

[0105] In the case where a metal ion is added, a chelating agent is added after the coupling is complete to capture the metal ion for ease of removal in subsequent steps. In some embodiments, the chelating agent is EDTA. In some embodiments, the chelating agent is DOTA. In some embodiments, the chelating agent is DPTA.

[0106] Thus, in some embodiments, the method for preparing an antibody-drug conjugate comprises incubating a mixture of an antibody, a linker-drug moiety, a reducing agent, and a metal ion in a buffer system, thereby obtaining an incubation mixture, wherein the linker is a substitutional linker or a linker containing a carbon-carbon triple bond; and optionally adding a chelating agent to capture the metal ion.

[0107] In some embodiments, the linker-drug moiety is in excess relative to the antibody, i.e., the concentration of linker-drug moiety is greater than the concentration of antibody.

[0108] Thus, in some embodiments, the method for preparing an antibody-drug conjugate comprises incubating a mixture of an antibody, a linker-drug moiety, and a reducing agent in a buffer system, thereby obtaining an incubation mixture, wherein the linker is a substitutional linker or a linker containing a carbon-carbon triple bond; and optionally adding N-acetyl cysteine to deplete excess linker-drug moiety.

[0109] In some embodiments, the method for preparing an antibody-drug conjugate comprises incubating a mixture of an antibody, a linker-drug moiety, and a reducing agent in a buffer system, thereby obtaining an incubation mixture, wherein the linker is a substitutional linker or a linker containing a carbon-carbon triple bond; and optionally adding a chelating agent to capture the metal ion and adding N-acetyl cysteine to deplete excess linker-drug moiety.

[0110] One skilled in the art can determine suitable reaction conditions depending on the particular reactants employed. In some embodiments, the buffer system is selected from the group consisting of Hepes, histidine buffer, PBS, MES, and Tris. In some embodiments, the buffer system has a pH of about 5.5 to 9. In some embodiments, the buffer system has a pH of about 5.5 to 8. In some embodiments, the buffer system has a pH of about 5.5 to 7. In some embodiments, the buffer system has a pH of about 7 to 8.

[0111] In some embodiments, the incubation temperature is about -10°C to 37°C. In some embodiments, the incubation temperature is about 0°C to 25°C. In some embodiments, the incubation temperature is about 0°C to 22°C. In some embodiments, the incubation temperature is about 0°C to 16°C. In some embodiments, the incubation temperature is about 0°C to 12°C. In some embodiments, the incubation temperature is about 12°C to 22°C. In some embodiments, the incubation temperature is about 16°C to 22°C.

[0112] In some embodiments, the incubation time is 3-20 hours. In some embodiments, the incubation time is 5-20 hours. In some embodiments, the incubation time is 6-20 hours. In some embodiments, the incubation time is 7-20 hours. In some embodiments, the incubation time is 3-5 hours. In some embodiments, the incubation time is 3-6 hours. In some embodiments, the incubation time is 5-6 hours.

[0113] There is no particular restriction on the antibody that can be conjugated with the linker-drug by using the bioconjugation method of the present application. The selection of the antibody depends on the disease or disorder (e.g., cancer) to be treated by the antibody-drug conjugate. In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody. In some embodiments, the antibody is selected from the group consisting of a human antibody, a humanized antibody, and a chimeric antibody. In some embodiments, the antibody specifically binds to a corresponding antigen (also referred to as a tumor-associated antigen (TAA)) expressed on a cancer cell, a viral antigen, or a microbial antigen, and has antibody-dependent cell-mediated phagocytosis (ADCP) activity, in vivo anti-tumor, anti-viral, or anti-microbial activity. In some embodiments, the monoclonal antibody is selected from the group consisting of trastuzumab, pertuzumab, sacituzumab, abciximab, adalimumab, alefacept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, inflectra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, secukinumab, and ustekinumab. The interchain S-S bond in the antibody is the site to which the linker-drug moiety is attached.

[0114] The incubated mixture of antibody-drug conjugate produced by the methods of the application can be post-treated in any suitable manner to produce the final product. In some embodiments, the post-treatment includes adding a quencher to deplete excess linker-drug moieties, and / or purifying using a desalting column and / or size exclusion chromatography. In some embodiments, the post-treatment includes first adding N-acetyl cysteine to deplete excess linker-drug moieties, and then purifying using a desalting column and / or size exclusion chromatography. In some embodiments, the post-treatment includes adding a chelator to capture the transition metal ion, and then purifying using a desalting column and / or size exclusion chromatography. In some embodiments, the post-treatment includes first adding N-acetyl cysteine to deplete excess linker-drug moieties, and then adding a chelator to capture the transition metal ion, and then purifying using a desalting column and / or size exclusion chromatography. The post-treatment, such as quenching and purification, are all done in the same reaction vessel without intermediate separation and purification steps. In some embodiments, the quencher is a molecule comprising a reactive thiol group and salts thereof. In some embodiments, the quencher is selected from the group consisting of N-acetyl cysteine, cysteine, cysteamine, and dithiothreitol.

[0115] By using the methods of the application for preparing antibody-drug conjugates, the homogeneity of the antibody-drug conjugates is higher than those prepared by conventional conjugation methods.

[0116] Antibody-drug conjugates

[0117] In another aspect, the application provides antibody-drug conjugates prepared by the methods described herein, which have higher homogeneity than those prepared by conventional conjugation methods.

[0118] In some embodiments, the content of antibody-drug conjugates with DAR value of 4 in the antibody-drug conjugates prepared by the application is greater than 45%. In some embodiments, the content of antibody-drug conjugates with DAR value of 4 (DAR4 or D4) in the antibody-drug conjugates prepared by the application is greater than 60%. In some embodiments, the content of antibody-drug conjugates with DAR value of 4 (DAR4 or D4) in the antibody-drug conjugates prepared by the application is greater than 70%. In some embodiments with added metal ions, the sum of the content of antibody-drug conjugates with DAR value of 0 (DAR0 or D0) and DAR value of 8 (DAR8 or D8) in the antibody-drug conjugates prepared by the application is less than 20%. In some embodiments with added metal ions, the content of antibody-drug conjugates with DAR value of 6 (DAR6 or D6) in the antibody-drug conjugates prepared by the application is less than 20%. In contrast, the content of D4 in antibody-drug conjugates prepared by conventional conjugation methods is usually less than 40%.

[0119] Pharmaceutical compositions

[0120] In one embodiment, the present application provides a pharmaceutical composition comprising an antibody-drug conjugate prepared by the methods described herein and a pharmaceutically acceptable carrier or excipient.

[0121] In some embodiments, the antibody-drug conjugates provided herein are provided as part of a pharmaceutical composition comprising the antibody-drug conjugate in combination with an appropriate pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science of Pharmacy, 20th Edition; Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)). Such carriers can be selected from pharmaceutically acceptable carriers and excipients. The excipients include, but are not limited to, solubilizing agents, surfactants, emulsifying agents, and suspending agents.

[0122] Examples of solubilizing agents suitable for use in pharmaceutical compositions include, but are not limited to, alcohols and polyols such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butylene glycol and its isomers, glycerol, pentaerythritol, sorbitol, mannitol, diethylene glycol monoethyl ether, isosorbide dimethyl ether, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives, 2-pyrrolidone, N-alkyl pyrrolidones, polyvinylpyrrolidone, ethyl propionate, tributyl citrate, triethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, and propylene glycol monoacetate.

[0123] Examples of surfactants suitable for use in pharmaceutical compositions include, but are not limited to, sodium lauryl sulfate (SDS), gelatin, casein, docusate sodium, benzalkonium chloride, calcium stearate, polyethylene glycol, phosphates, polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 80, polysorbate 20), gum acacia, cholesterol, tragacanth, polyoxyethylene 20 stearyl ether, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, macrogolglycerol hydroxystearate, fatty acid sorbitan esters, vitamin E or tocopherol derivatives, tocopherol esters, lecithin, phospholipids and derivatives thereof, poloxamer, stearic acid, oleic acid, oleyl alcohol, cetyl alcohol, monoglycerides and diglycerides, propylene glycol fatty acid esters, glyceryl fatty acid esters, glycol stearate palmitate, polyoxylglycerides, propylene glycol monocaprylate, propylene glycol monolaurate, alkyl aryl polyether alcohols, and polyglyceryl oleate.

[0124] Examples of emulsifiers suitable for use in pharmaceutical compositions include, but are not limited to, acacia, tragacanth, gelatin, natural phosphatides (e.g., soybean lecithin), fatty acid esters sorbitan, polysorbates, and poloxamer.

[0125] Examples of suspending agents suitable for use in pharmaceutical compositions include, but are not limited to, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth.

[0126] In some embodiments, the provided pharmaceutical compositions can be prepared in the form of a liquid suspension or solution using liquids such as oils, water, alcohols and combinations thereof.

[0127] In some embodiments, the provided pharmaceutical compositions can be prepared in the form of a sterile injectable preparation, either as a water or oil soluble suspension. These suspensions can be prepared according to known techniques.

[0128] In some embodiments, kits comprising an antibody-drug conjugate provided herein (or a composition comprising an antibody-drug conjugate provided herein) are also provided, which are provided in a manner convenient to perform the methods described herein. In some embodiments, the kit comprises packaging an antibody-drug conjugate provided herein (or a composition comprising an antibody-drug conjugate provided herein) in a container, such as a sealed bottle or vessel, and affixing a label to the container or including a label in the kit, describing the use of the antibody-drug conjugate or composition to perform a method provided herein. In some embodiments, the antibody-drug conjugate or composition is packaged in unit dose form. In some embodiments, the kit further comprises an apparatus suitable for administering the antibody-drug conjugate or composition according to a predetermined route of administration. In some embodiments, the kit comprises an antibody-drug conjugate provided herein and instructions for administering the antibody-drug conjugate to a cancer patient.

[0129] Uses

[0130] In some embodiments, the present application provides uses of an antibody-drug conjugate prepared by the methods described herein or a pharmaceutical composition comprising the antibody-drug conjugate in the manufacture of a medicament for treating cancer, autoimmune diseases, inflammation, and / or metabolic diseases.

[0131] In some embodiments, the antibody-drug conjugate prepared by the methods described herein or a pharmaceutical composition comprising the antibody-drug conjugate can be used in any patient that can benefit from the compounds provided herein. In some embodiments, the patients can be mammals, such as humans and companion animals. In some embodiments, the patient is a human.

[0132] A therapeutically effective amount of an ADC provided herein will depend on various factors known in the art, such as body weight, age, past medical history, current medications, the health of the subject, and the likelihood of cross-reactions, allergies, sensitivities, and adverse side effects, as well as the route of administration and the extent of disease progression. The dosage can be proportionally reduced or increased as indicated by these and other conditions or requirements. In some embodiments, an ADC or pharmaceutical composition provided herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In certain of these embodiments, an ADC or pharmaceutical composition provided herein is administered at a dose of about 50 mg / kg or less, and in certain of these embodiments, the dose is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less.

[0133] In some embodiments, the dose administered can vary over the course of treatment. For example, in some embodiments, the initial dose administered can be higher than the subsequent doses administered. In some embodiments, the dose administered can vary over the course of treatment depending on the subject’s response.

[0134] In some embodiments, the disease treated by the antibody-drug conjugate described herein is selected from the group consisting of cancer, autoimmune disease, inflammation, and metabolic disease.

[0135] In some embodiments, the present application provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate described herein or a pharmaceutical composition described herein, the disease is selected from the group consisting of cancer, autoimmune disease, inflammation, and metabolic disease.

[0136] In some embodiments, the present application provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate described herein or a pharmaceutical composition described herein and another therapeutic agent for treating cancer, autoimmune disease, inflammation, and metabolic disease.

[0137] In some embodiments, the disease is selected from bladder transitional cell carcinoma, hepatitis C, paroxysmal nocturnal hemoglobinuria (PNH), colorectal cancer, urothelial cancer, advanced breast cancer, bladder cancer, gastric cancer, elevated low-density lipoprotein cholesterol, superoxide dismutase 1-amyotrophic lateral sclerosis (SOD1-ALS), Pompe disease, and hypertrophic scars.

[0138] The following examples are intended to illustrate certain methods of making the disclosed compounds and are not intended to limit the scope of reactions or reaction sequences that can be used in making the compounds provided herein.

[0139] Examples

[0140] The application will now be described in detail with reference to the following examples. However, it will be appreciated by those skilled in the art that the following examples are provided merely as illustrative and are not intended to limit the application in any way.

[0141] Unless otherwise indicated, the chemical and biological reagents used were obtained from commercial sources.

[0142] Dimethylacetamide (DMA) was commercially available from Aldrich Sigma.

[0143] Sodium dehydroascorbate (DHAA) was commercially available from Aldrich Sigma.

[0144] Herceptin, Erbitux or Rituximab was prepared by WuXi Biologies according to the published corresponding protein sequence by standard methods for preparing monoclonal antibodies.

[0145] DL001 was commercially available from WuXi Apptec. or prepared according to the following Example 1.

[0146] DL003 was commercially available from WuXi Biologics with the following structure:

[0147] DL004-0 was commercially available from MCE with the following structure:

[0148] DL004 was prepared by dissolving in DMA before use and quenching the DBCO group with 2 equivalents of 4-azidobenzoic acid at 37°C for 18 hours and used directly in the reaction. The structure is as follows:

[0149] DL005 was commercially available from WuXi Biologics with the following structure:

[0150] Example 1. Synthesis of linker-drug moiety (DL001)

[0151] DL001 chemical name: [4-[[(2S)-2-[[(2S)-3-methyl-2-[6-(2-methylsulfonyl-7-oxo- pyridine [2,3-d]pyrimidin-8-yl)hexanoylamido]butyryl]amino]-5-ureido- pentanoyl]amino]phenyl]methyl-N-[(1S)-1-[[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3- [[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxo- propyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]- methylcarbamoyl]-2-methylpropyl]carbamoyl]-2-methylpropyl]-N- methylamino

[0152] Step 1: Synthesis of DL001-2

[0153] To a solution of DL001-1 (5.00 g) and 6-bromohexanoic acid (6.00 g) in DMF (50 mL) was added potassium carbonate (7.50 g). The reaction mixture was stirred at 55-60 °C for 1 h under nitrogen atmosphere. The reaction mixture was allowed to cool to room temperature, filtered, and the filtrate was poured into ice water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The organic phase was washed with saturated sodium chloride solution (4 x 100 mL), dried over magnesium sulfate, filtered and concentrated. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 0:1-3:1) to give compound DL001-2.

[0154] LCMS: m / z [M+H] + 336.15.

[0155] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.15 (t, J = 7.09 Hz, 3 H), 1.32 (quin, J = 7.61 Hz, 2 H), 1.53 - 1.68 (m, 4 H), 2.28 (t, J = 7.34 Hz, 2 H), 2.58 (s, 3 H), 4.03 (q, J = 7.09 Hz, 2 H), 4.24 - 4.30 (m, 2 H), 6.62 (d, J = 9.41 Hz, 1 H), 7.93 (d, J = 9.54 Hz, 1 H), 8.88 (s, 1 H).

[0156] Step 2: Synthesis of DL001-3

[0157] To a solution of DL001-2 (7.50 g) in tetrahydrofuran (30 mL), methanol (30 mL) and water (30 mL) was added lithium hydroxide (1.00 g) and stirred at 15-20 °C for 1 hour. The reaction solution was diluted with water (50 mL) and the pH of the solution was adjusted to 3 by dropwise addition of hydrochloric acid (2 M) under ice-water bath. A solid was precipitated and was filtered. The filter cake was washed with methyl tert-butyl ether and dried to give compound DL001-3.

[0158] LCMS: m / z [M+H] + 308.10.

[0159] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.28 - 1.37 (m, 2 H), 1.54 (quin, J = 7.46 Hz, 2 H), 1.63 (quin, J = 7.52 Hz, 2 H), 2.20 (t, J = 7.27 Hz, 2 H), 2.58 (s, 3 H), 4.22 - 4.29 (m, 2 H), 6.62 (d, J = 9.41 Hz, 1 H), 7.92 (d, J = 9.41 Hz, 1 H), 8.87 (s, 1 H), 12.00 (br s, 1 H).

[0160] Step 3: Synthesis of DL001-4

[0161] To a solution of DL001-3 (7.00 g) in acetic acid (70 mL) and water (1.4 mL) was added N-chlorosuccinimide (9.10 g) and triethylamine (7.00 g). The reaction was stirred at 15-20 °C for 1 hour under nitrogen atmosphere. The reaction solution was concentrated and the crude product was diluted with water (150 mL). The pH of the solution was adjusted to about 2 by dropwise addition of hydrochloric acid (2 M) under ice-water bath. A solid was precipitated and was filtered to give compound DL001-4.

[0162] LCMS: m / z [M+H] + 340.08.

[0163] 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.33 (quin, J = 7.58 Hz, 2 H), 1.54 (quin, J = 7.49 Hz, 2 H), 1.67 (quin, J = 7.52 Hz, 2 H), 2.20 (t, J = 7.34 Hz, 2 H), 3.47 (s, 3 H), 4.30 (t, J = 7.40 Hz, 2 H), 6.93 (d, J = 9.54 Hz, 1 H), 8.12 (d, J = 9.54 Hz, 1 H), 9.30 (s, 1 H), 11.98 (br s, 1 H).

[0164] Step 4: Synthesis of DL001-6

[0165] To a solution of DL001-5 (0.40 g) in DMF (4 mL) was added triethylamine (0.36 g). Stirring at 15-20 °C for 16 h, directly used in the next step.

[0166] LCMS: m / z [M+H] + 1123.66.

[0167] Step 5: Synthesis of DL001

[0168] To a solution of DL001-4 (0.10 g) and HATU (0.12 g) in DMF (1 mL). Stirring at 15-20 °C for 0.2 h. Added DL001-6 (4 mL solution in DMF from step 4). Stirring at 15-20 °C for 7 h. The reaction was diluted with acetonitrile (10 mL) and purified by preparative HPLC (DAC-50, YMC-C18, 7 um, mobile phase: water (0.1% trifluoroacetic acid) / acetonitrile) to give compound DL001.

[0169] LCMS: m / z [(M+2H) / 2] + 723.18.

[0170] 1H NMR (400 MHz, DMSO-d6) δ ppm 0.70 - 0.90 (m, 25 H), 0.95 - 1.07 (m, 7 H), 1.21 - 1.60 (m, 11 H), 1.61 - 1.84 (m, 7 H), 1.89 - 2.35 (m, 9 H), 2.41 (br d, J=14.43 Hz, 1 H), 2.86 (br d, J=15.04 Hz, 3 H), 2.91 - 3.07 (m, 4 H), 3.11 (s, 2 H), 3.15 - 3.26 (m, 8 H), 3.31 (br d, J=9.90 Hz, 1 H), 3.47 (s, 3 H), 3.90 - 4.07 (m, 3 H), 4.14 - 4.22 (m, 1 H), 4.28 (br t, J=7.34 Hz, 3 H), 4.33 - 4.51 (m, 4 H), 4.58 - 4.80 (m, 1 H), 4.91 - 5.12 (m, 3 H), 5.41 (br s, 2 H), 5.98 (br s, 1 H), 6.92 (d, J=9.54 Hz, 1 H), 7.17 (br t, J=6.97 Hz, 1 H), 7.22 - 7.36 (m, 7 H), 7.57 (br d, J=8.56 Hz, 2 H), 7.64 (d, J=8.19 Hz, 0.5 H), 7.79 (br d, J=8.93 Hz, 1 H), 7.90 (br d, J=8.80 Hz, 0.5 H), 8.04 - 8.17 (m, 2.5 H), 8.32 (br s, 0.5 H), 9.29 (s, 1 H), 9.99 (br s, 1 H).

[0171] Example 2. Synthesis of linker-drug moiety (DL002)

[0172] DL002 Chemical Name: N-((S)-10-benzyl-l-(((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzopyran[3',4':6,7]indolizine[l,2- b]quinoline-l-yl)amino)-l,6,9,12,15-pentaoxo-3-oxa-5,8,l l,14-tetraazahexadecan-16-yl)-6-(2- methylsulfonyl-7-oxopyrido[2,3-d]pyrimidin-8(7H)-yl)hexanamide

[0173] Step 1: Synthesis of DL002-2

[0174] To a solution of DL002-1 (0.10 g) in DMF (0.2 mL) and acetonitrile (0.8 mL) was added DBU (26 mg). Stirring at 0-5 °C for 0.2 h, the reaction solution was used directly in the next step.

[0175] LCMS: m / z [M+H] + 841.25.

[0176] Step 2: Synthesis of DL002

[0177] To a solution of DL001-4 (32 mg) and HATU (36 mg) in DMF (0.2 mL) and acetonitrile (0.8 mL) was added DIEA (40 mg). Stirring at 15-20 °C for 0.4 h, DL002-2 (80 mg, 1 mL solution) was added. Stirring at 0-5 °C for 0.2 h, the reaction solution was diluted with acetonitrile (10 mL) and purified by preparative high performance liquid chromatography (DAC-50, YMC-C18, 7 um, mobile phase: water (0.1% trifluoroacetic acid) / acetonitrile) to give compound DL002.

[0178] LCMS: m / z [M+H] + 1162.38.

[0179] 1H NMR (400 MHz, DMSO-d6) δ ppm 0.86 (br t, J=7.09 Hz, 3 H), 1.26 - 1.33 (m, 2 H), 1.47 - 1.56 (m, 2 H), 1.59 - 1.67 (m, 2 H), 1.85 (tt, J=13.97, 7.00 Hz, 2 H), 2.05 - 2.24 (m, 4 H), 2.15 - 2.22 (m, 1 H), 2.37 (s, 3 H), 2.76 (br dd, J=13.69, 9.66 Hz, 1 H), 3.01 (br dd, J=13.63, 4.22 Hz, 1 H), 3.09 - 3.21 (m, 2 H), 3.45 (s, 3 H), 3.55 - 3.76 (m, 6 H), 4.01 (s, 2 H), 4.27 (br t, J=7.21 Hz, 2 H), 4.42 - 4.50 (m, 1 H), 4.63 (br d, J=6.97 Hz, 2 H), 5.12 - 5.24 (m, 2 H), 5.36 - 5.45 (m, 2 H), 5.59 (br d, J=6.97 Hz, 1 H), 6.52 (br s, 1 H), 6.90 (d, J=9.41 Hz, 1 H), 7.15 - 7.25 (m, 5 H), 7.30 (s, 1 H), 7.76 (br d, J=10.88 Hz, 1 H), 8.02 (dt, J=17.61, 5.50 Hz, 2 H), 8.07 - 8.14 (m, 2 H), 8.30 (br t, J=5.56 Hz, 1 H), 8.50 (br d, J=8.68 Hz, 1 H), 8.63 (br t, J=6.48 Hz, 1 H), 9.27 (s, 1 H).

[0180] Example 3. Preparation of Herceptin-DL001 conjugate (drug-antibody ratio about 4) by using Method one or Method two of the present application or conventional method or WO2020164561A1 method

[0181] Preparation of Herceptin-DL001 conjugate in one-step Method one reaction:

[0182] DL001 (0.3 mM) and TCEP (0.12 mM) in DMA were added to a solution of Herceptin (0.03 mM in phosphate buffer, pH 7, 20 mM) and the reaction mixture was allowed to react at 22 °C for 3 hours to give unpurified Herceptin-DL001 conjugate with drug-antibody ratio about 4.

[0183] Preparation of Herceptin-DL001 conjugate in one-step Method two reaction:

[0184] DL001 (0.3 mM) in DMA, ZnCl2 (0.018 mM) and TCEP (0.15 mM) in DMA were added to a solution of Herceptin (0.03 mM in phosphate buffer, pH 7, 20 mM) and the reaction mixture was allowed to react at 12 °C for 20 hours to give unpurified Herceptin-DL001 conjugate with a drug-antibody ratio of about 4.

[0185] As a control, Herceptin-DL001 conjugate with a drug-antibody ratio of about 4 was prepared using the conventional method.

[0186] (1) TCEP (0.075 mM) was added to a solution of Herceptin (0.03 mM in phosphate buffer, pH 7, 20 mM) and the reaction mixture was allowed to react at 22 °C for 3 hours to give a reduced antibody solution;

[0187] (2) DL001 (0.3 mM) in DMA was added to the reduced antibody solution, mixed well, and the mixture was allowed to react at 22 °C for 1 hour to give unpurified Herceptin-DL001 conjugate with a drug-antibody ratio of about 4.

[0188] The mixtures obtained by the above three methods were post-treated to remove excess linker-drug via the following general procedure:

[0189] (1) N-acetyl cysteine (0.24 mM) was added to deplete excess DL001;

[0190] (2) The reaction mixture was purified with a desalting column (type: 40K, 0.5 mL, REF: 87766, Lot# SJ251704, manufacturer: Thermo).

[0191] As a control, Herceptin-DL001 conjugate with a drug-antibody ratio of about 4 was prepared using the WO2020164561 method.

[0192] (1) ZnCl2 (0.12 mM) and TCEP (0.105 mM) were added to a solution of Herceptin (0.03 mM in phosphate buffer, pH 7, 20 mM) and the reaction mixture was allowed to stand at 12 °C overnight;

[0193] (2) DL001 (0.12 mM) in DMA was introduced, along with EDTA (0.09 mM) and the reaction was continued at 12 °C for 1 hour;

[0194] (3) N-acetyl cysteine (0.24 mM) was added to deplete excess DL001;

[0195] (4) EDTA (0.24 mM) was added to capture Zn 2+ and DHAA (0.24 mM) was added to oxidize excess thiol;

[0196] (5) The reaction mixture was purified using a desalting column (Type: 40K, 0.5 mL, REF: 87766, Lot# SJ251704, Manufacturer: Thermo).

[0197] All the conjugates prepared above were subjected to homogeneity assay respectively to compare their quality difference. HIC-HPLC was used to analyze drug / antibody ratio (DAR) and product distribution. Purification analysis of various components was performed by hydrophobic interaction chromatography (HIC) on Tosoh TSKgel Butyl-NPR 4.6 mm I.D. x 3.5 cm, 2.5 μm at ambient temperature with a flow rate of 0.6 mL / min. The injection amount was 50 μg, solvent A was 1.5 M Na2SO4 and 50 mM sodium phosphate pH 7. Solvent B was 75% v / v 50 mM sodium phosphate pH 7 and 25% v / v isopropanol. Different drug-loaded species were eluted by sequential fractional gradient.

[0198] The results are shown in Table 1 and Figures 1-4.

[0199] Table 1

[0200] It can be found that the drug-antibody ratio of the conjugates prepared by several methods can effectively prepare a mixture with a drug-antibody ratio close to 4, but the proportion of the mixture is different. It is generally recognized in the art that the higher the content of D4 component in the conjugate with a DAR value close to 4, the higher the quality of the mixture.

[0201] In the mixture prepared by the conventional method, the D4 content is only about 42%, and using the one-step method one, the proportion of D4 in the mixture prepared can be increased to 64% by adjusting the synthesis steps only, without adding any additional components. If the one-step method two is used on this basis, a small amount of zinc ions is added to the reaction solution and removed after the reaction is completed, the content of D4 component can be increased to 82%, and the increase is close to 100%. This result is also better than the method of WO2020164561 which also adds zinc ions.

[0202] Both of these improved coupling strategies result in a more homogeneous mixture of components with higher quality. At the same time, the ratio of D6+D8 in the mixture prepared by the conventional method is 33%, while the ratio of D6+D8 in the mixture prepared by one-step method one is only about 23%, and the ratio of D6+D8 in the mixture prepared by one-step method two is only about 13%. D6 and D8 components are high-toxic components in the mixture, and reducing their ratio helps to increase the safety of the final product.

[0203] Example 4. Preparation of Herceptin-MC-VC-PAB-MMAE conjugate (drug-antibody ratio about 4) by using the method one of the present application or the conventional method

[0204] Preparation of Herceptin-MC-VC-PAB-MMAE conjugate by one-step method one reaction:

[0205] Add MC-VC-PAB-MMAE (purchased from WuXi Biotechnology, 0.3 mM) and TCEP (0.12 mM) in DMA to the solution of Herceptin (0.03 mM in phosphate buffer, pH 7, 20 mM) and allow the reaction mixture to react at 22 °C for 3 hours to obtain unpurified Herceptin-MC-VC-PAB-MMAE conjugate with a drug-antibody ratio of about 4.

[0206] As a control, Herceptin-MC-VC-PAB-MMAE conjugate with a drug-antibody ratio of about 4 was prepared using the conventional method.

[0207] (1) Add TCEP (0.075 mM) to the solution of Herceptin (0.03 mM in phosphate buffer, pH 7, 20 mM) and allow the reaction mixture to react at 22 °C for 3 hours to obtain a reduced antibody solution.

[0208] (2) Add MC-VC-PAB-MMAE (synthesized by ourselves, 0.3 mM) dissolved in DMA to the reduced antibody solution, mix well, and allow the mixture to react at 22 °C for 1 hour to obtain unpurified Herceptin-MC-VC-PAB-MMAE conjugate with a drug-antibody ratio of about 4.

[0209] The mixtures obtained by the above two methods are post-treated by the following general steps to remove excess linker-drugs:

[0210] (1) Add N-acetyl cysteine (0.24 mM) to consume excess MC-VC-PAB-MMAE;

[0211] (2) The reaction mixture was purified using a desalting column (Type: 40K, 0.5 mL, REF: 87766, Lot # SJ251704, Manufacturer: Thermo).

[0212] All conjugates prepared above were subjected to homogeneity assay respectively to compare the quality difference. HIC-HPLC was used to analyze the drug / antibody ratio (DAR) and product distribution. Purification analysis of various components was performed by hydrophobic interaction chromatography (HIC) on Tosoh TSKgel Butyl-NPR 4.6 mm I.D. x 3.5 cm, 2.5 pm at ambient temperature with a flow rate of 0.6 mL / min. The injection amount was 50 pg, and solvent A was 1.5 M Na2S04and 50 mM sodium phosphate pH 7. Solvent B was 75% v / v 50 mM sodium phosphate pH 7 and 25% v / v isopropanol. Different drug-loaded species were eluted by sequential fractional gradient.

[0213] The results are shown in Table 2 and Figures 5-6.

[0214] Table 2

[0215] From the results, it can be found that when using MC-VC-PAB-MMAE as the linker-drug, the results obtained using the method of the present application will be far inferior to those obtained using conventional methods. This result is due to the reaction between TCEP and maleimide derivatives, which will be consumed by each other when coexisting in the solution, and the expected reaction result cannot be achieved. Similarly, the method in WO2020164561A1 belongs to an improvement on the traditional method, which cannot avoid this problem. Therefore, the related patent cannot provide any hint to the present patent.

[0216] Example 5. Preparation of Herceptin-DL001 conjugate (drug-antibody ratio about 4) using the method of the present application at different temperatures

[0217] In order to prove that the method of the present application has an advantage over the conventional method at different temperatures, the conjugates prepared using the method of the present application and the conventional method were compared at different temperatures.

[0218] Preparation of Herceptin-DL001 conjugate in one-step method:

[0219] DL001 in DMA (0.3 mM) and TCEP (0.09 mM) were added to a solution of Herceptin (0.03 mM in phosphate buffer, pH 7, 20 mM) and the reaction mixture was allowed to react at different temperatures (12°C, 16°C, 22°C) for 3 hours; Herceptin-DL001 conjugates with a drug-antibody ratio of about 4 were obtained without purification.

[0220] Herceptin-DL001 conjugates were prepared in a two-step process in one pot:

[0221] DL001 in DMA (0.3 mM), ZnCl2 (0.015 mM) and TCEP (0.14 mM) were added to a solution of Herceptin (0.03 mM in phosphate buffer, pH 7, 20 mM) and the reaction mixture was allowed to react at different temperatures (12°C, 16°C, 22°C) for 5 hours; Herceptin-DL001 conjugates with a drug-antibody ratio of about 4 were obtained without purification.

[0222] As a control, Herceptin-DL001 conjugates with a drug-antibody ratio of about 4 were prepared using a conventional method.

[0223] (1) TCEP (0.075 mM) was added to a solution of Herceptin (0.03 mM in phosphate buffer, pH 7, 20 mM) and the reaction mixture was allowed to react at 22°C for 3 hours to obtain a reduced antibody solution;

[0224] (2) DL001 (0.3 mM) dissolved in DMA was added to the reduced antibody solution, mixed well, and the mixture was allowed to react at different temperatures (12°C, 16°C, 22°C) for 1 hour to obtain Herceptin-DL001 conjugates with a drug-antibody ratio of about 4 without purification.

[0225] In general, the mixtures obtained in the above three steps were post-treated to remove excess linker-drug via the following general steps:

[0226] (1) N-acetyl cysteine (0.24 mM) was added to deplete excess DL001;

[0227] (2) The reaction mixture was purified using a desalting column (type: 40K, 0.5 mL, REF: 87766, Lot# SJ251704, manufacturer: Thermo).

[0228] The prepared conjugates were subjected to homogeneity assay to compare the quality difference. HIC-HPLC was used to analyze the drug / antibody ratio (DAR) and product distribution. Purification analysis of various components was performed by hydrophobic interaction chromatography (HIC) on Tosoh TSKgel Butyl-NPR 4.6 mm I.D. x 3.5 cm, 2.5 μm at ambient temperature with a flow rate of 0.6 mL / min. The injection amount was 50 μg, solvent A was 1.5 M Na2SO4and 50 mM sodium phosphate pH 7. Solvent B was 75% v / v 50 mM sodium phosphate pH 7 and 25% v / v isopropanol. Different drug-loaded species were eluted by sequential stepwise gradient.

[0229] The results are shown in Table 3 and Figures 7-15.

[0230] Table 3

[0231] In summary, the two methods proposed in the present application can be applied to a variety of different temperatures without causing the quality of the resulting product to decrease.

[0232] Example 6. Preparation of Herceptin-DL001 conjugate (drug-antibody ratio about 4) using the method of the present application at different pH

[0233] In order to verify whether the method of the present application is applicable to different pH conditions, attempts were made to perform conjugation at different pH conditions in this example.

[0234] Preparation of Herceptin-DL001 conjugate in one-step method I reaction:

[0235] DL001 (0.3 mM) and TCEP (0.09 mM) in DMA were added to a solution of Herceptin (0.03 mM in phosphate buffer, pH 7 / 8, 20 mM) and the reaction mixture was allowed to react at 22°C for 3 hours to obtain unpurified Herceptin-DL001 conjugate with a drug-antibody ratio of about 4.

[0236] As a control, Herceptin-DL001 conjugate with a drug-antibody ratio of about 4 was prepared using the conventional method.

[0237] (1) TCEP (0.075 mM) was added to a solution of Herceptin (0.03 mM in phosphate buffer, pH 7 / 8, 20 mM) and the reaction mixture was allowed to react at 22°C for 3 hours to obtain a reduced antibody solution.

[0238] (2) DL001 (0.3 mM) dissolved in DMA was added to the reduced antibody solution, mixed well, and the mixture was allowed to react at 22 °C for 1 hour to obtain unpurified Herceptin-DL001 conjugate with a drug-antibody ratio of about 4.

[0239] In general, the mixture obtained from the above two steps was post-treated to remove excess linker-drug via the following general procedure:

[0240] (1) N-acetyl cysteine (0.24 mM) was added to deplete excess DL001;

[0241] (2) The reaction mixture was purified using a desalting column (Type: 40K, 0.5 mL, REF: 87766, Lot# SJ251704, Manufacturer: Thermo).

[0242] The conjugates prepared were each subjected to homogeneity assay to compare the quality difference. HIC-HPLC was used to analyze the drug / antibody ratio (DAR) and product profile. Purification analysis of the various components was performed by hydrophobic interaction chromatography (HIC) on Tosoh TSKgel Butyl-NPR 4.6 mm I.D. x 3.5 cm, 2.5 μm at ambient temperature with a flow rate of 0.6 mL / min. The injection amount was 50 μg, solvent A was 1.5 M Na2S04and 50 mM sodium phosphate pH 7. Solvent B was 75% v / v 50 mM sodium phosphate pH 7 and 25% v / v isopropanol. Different drug-loaded species were eluted by sequential stepwise gradient.

[0243] The results are shown in Table 4 and Figures 16-19.

[0244] Table 4

[0245] The above data demonstrate that the quality of the conjugates prepared by the method of the present application is significantly better than that of the conjugates prepared by the conventional method at various pH.

[0246] Example 7. Preparation of DL001 conjugates (drug-antibody ratio of about 4) of various antibodies by using the one-step method two of the present application

[0247] To demonstrate the universality of the method of the present application, three commercially available antibodies (Herceptin / Erbitux / Rituximab) were used as examples in this example to prepare the DL001 conjugates (drug-antibody ratio of about 4) of the corresponding antibodies.

[0248] The DL001 conjugates of the antibodies were prepared by the one-step method two reaction:

[0249] DL001 (0.3 mM), ZnCl2(0.015 mM) and TCEP (0.14 mM) in DMA were added to a solution of antibody (Herceptin / Erbitux / Rituximab, 0.03 mM in phosphate buffer, pH 7, 20 mM) and the reaction mixture was allowed to react at 12 °C for 6 hours to give unpurified Herceptin / Erbitux / Rituximab-DL001 conjugate with a drug-antibody ratio of about 4.

[0250] The resulting mixture was post-treated to remove excess linker-drug via the following general procedure:

[0251] (1) N-acetyl cysteine (0.24 mM) was added to deplete excess DL001;

[0252] (2) The reaction mixture was purified using a desalting column (Type: 40K, 0.5 mL, REF: 87766, Lot # SJ251704, Manufacturer: Thermo).

[0253] The prepared conjugates were each subjected to homogeneity assay to compare the quality difference. The drug / antibody ratio (DAR) and product profile were analyzed using HIC-HPLC. Purification analysis of the various components was performed by hydrophobic interaction chromatography (HIC) on Tosoh TSKgel Butyl-NPR 4.6 mm I.D. x 3.5 cm, 2.5 μm at ambient temperature with a flow rate of 0.6 mL / min. The injection amount was 50 μg, solvent A was 1.5 M Na2S04and 50 mM sodium phosphate pH 7. Solvent B was 75% v / v 50 mM sodium phosphate pH 7 and 25% v / v isopropanol. Different drug-loaded species were eluted by sequential fractional gradient.

[0254] The results are shown in Table 5 and Figures 20-22.

[0255] Table 5

[0256] The above results show that for the most common antibodies on the market, the present method can play a good role in controlling the quality of the product, and the proportion of D4 component in the product is greater than 75%.

[0257] Example 8. Preparation of conjugates of different linker-drugs of Herceptin (drug-antibody ratio of about 4) under different conditions by using Method Two of the present application

[0258] To demonstrate that the method continues to work for different linker-drugs, we reacted Herceptin with DL003, DL004 or DL005 in a one-pot method two reactions.

[0259] The load in DMA, ZnCl2 and TCEP were added to the solution of Herceptin (0.03 mM in buffer, 20 mM) as shown in Table 6 below and the mixture was allowed to react thoroughly; Herceptin-DL003 or Herceptin-DL004 conjugates with drug-antibody ratio of about 4 were obtained without purification.

[0260] Table 6

[0261] The resulting mixture was post-treated to remove excess linker-drugs via the following general procedure:

[0262] (1) N-acetyl cysteine (0.24 mM) was added to deplete excess DL003 / DL004 / DL005;

[0263] (2) The reaction mixture was purified using a desalting column (Type: 40K, 0.5 mL, REF: 87766, Lot# SJ251704, Manufacturer: Thermo).

[0264] The prepared conjugates were subjected to homogeneity assay to compare their quality difference. HIC-HPLC was used to analyze the drug / antibody ratio (DAR) and product profile of Herceptin-DL003. Purification analysis of various components was performed by hydrophobic interaction chromatography (HIC) on Tosoh TSKgel Butyl-NPR 4.6 mm I.D. x 3.5 cm, 2.5 μm at ambient temperature with a flow rate of 0.6 mL / min. The injection amount was 50 μg, solvent A was 1.5 M Na2S04and 50 mM sodium phosphate pH 7. Solvent B was 75% v / v 50 mM sodium phosphate pH 7 and 25% v / v isopropanol. Different drug-loaded species were eluted by sequential stepwise gradient. PLRP-HPLC was used to analyze the product profile of Herceptin-DL004, purification analysis of various components was performed by reverse phase chromatography (PLRP) on Agilent PLRP-S-8um Serial No. 000S644787-150 at ambient temperature with a flow rate of 0.6 mL / min. The injection amount was 10 μg, solvent A was 0.05% (v / v) trifluoroacetic acid in water, solvent B was 0.05% (v / v) trifluoroacetic acid in acetonitrile. Different drug-loaded species were eluted by sequential stepwise gradient and DAR value was calculated accordingly, where H1% represents the proportion of D4% component.

[0265] HIC results are shown in Table 7, Figures 23-27 and 29, and PLRP results are shown in Table 8 and Figure 28.

[0266] Table 7

[0267] Table 8

[0268] This result shows that due to different reactivity of different linker-drugs, the optimal reaction temperature, pH, and buffer type are different. The commonality is that the method two of the present application is suitable for a variety of different linker-drugs, and can obtain the corresponding ADC crude product after one-step operation, but whether the phenomenon of rising D4 proportion occurs or not depends on the different types of linkers.

[0269] Example 9. Preparation of conjugates of different linker-drugs of Herceptin (drug-antibody ratio about 4) by using the method two of the present application under different reducing agent conditions

[0270] In order to prove that the present method continues to be suitable for different reducing agents and linker-drugs, we use Herceptin and DL001 and DL002 to react by one-step method two, using 2-(diphenylphosphino)-acetic acid as a reducing agent.

[0271] A solution of Herceptin (0.03 mM in phosphate buffer (pH 7), 20 mM) was added to DL001 or DL002 (0.3 mM) in DMA, ZnCl2(0.018 mM) and 2-(diphenylphosphino)-acetic acid (0.15 mM) and the reaction mixture was allowed to react at 12 °C for 20 hours to give unpurified Herceptin-DL001 / DL002 conjugate with a drug-antibody ratio of about 4.

[0272] The resulting mixture was post-treated to remove excess linker-drug via the following general procedure:

[0273] (1) N-acetyl cysteine (0.24 mM) was added to deplete excess linker-drug;

[0274] (2) The reaction mixture was purified using a desalting column (Type: 40K, 0.5 mL, REF: 87766, Lot# SJ251704, Manufacturer: Thermo).

[0275] The prepared conjugates were each subjected to homogeneity assay to compare the quality difference. HIC-HPLC was used to analyze the drug / antibody ratio (DAR) and product distribution. Purification analysis of the various components was performed by hydrophobic interaction chromatography (HIC) on Tosoh TSKgel Butyl-NPR 4.6 mm I.D. x 3.5 cm, 2.5 μm at ambient temperature with a flow rate of 0.6 mL / min. The injection amount was 50 μg, solvent A was 1.5 M Na2S04and 50 mM sodium phosphate pH 7. Solvent B was 75% v / v 50 mM sodium phosphate pH 7 and 25% v / v isopropanol. Different drug-loaded species were eluted by sequential fractional gradient.

[0276] The results are shown in Table 9 and Figures 30-31.

[0277] Table 9

[0278] This result shows that the present method can be applied to different reducing agents.

[0279] The above results show that the novel conjugation method provided by the present application has extremely broad application range. The present application has proved that the temperature, reaction time, solvent type, pH, reducing agent type, antibody type, linker type, drug type, etc. in the process are all within the scope that can be adjusted.

[0280] It is to be understood that the present description is not limited to the technical solutions above illustrated and that various modifications and changes can be made to the various technical solutions without departing from the scope thereof. The present description is intended to embrace any and all modifications and variations, including such as come within the scope of the present description and that are otherwise simple substitutions of known or routine techniques by a person of the ordinary skill in the art. The present description and embodiments are to be considered merely exemplary, the scope of the application being limited only by the claims that follow.

Claims

1. A method for preparing an antibody-drug conjugate comprising incubating a mixture of an antibody, a linker-drug moiety and a reducing agent in a buffer system, thereby obtaining an incubation mixture; wherein the linker is a substitutional linker or a linker containing a carbon-carbon triple bond.

2. The method of claim 1, wherein the linker in the linker-drug moiety contains a leaving group connected by a linking fragment or a group containing a carbon-carbon triple bond connected by a linking fragment; the leaving group is selected from the following structures: ###0001### ###0002### ###0003### ###0004### ###0005### ###0006### ###0007### ###0008### ###0009### ###0010### ###0011### ###0012### ###0013### ###0014### ###0015### ###0016### ###0017### ###0018### ###0019### ###0020### ###0021### ###0022### ###0023### ###0024### ###0025### ###0026### ###0027### ###0028### ###0029### ###0030### ###0031### ###0032### ###0033### ###0034### ###0035### ###0036### ###0037### ###0038### ###0039### ###0040### ###0041### ###0042### ###0043### ###0044### ###0045### ###0046### ###0047### ###0048### ###0049### ###0050### ###0051### ###0052### ###0053### ###0054### ###0055### ###0056### ###0057### ###0058### ###0059### ###0060### ###0061### ###0062### ###0063### ###0064### ###0065### ###0066### ###0067### ###0068### ###0069### ###0070### ###0071### ###0072### ###0073### ###0074### ###0075### ###0076### ###0077### ###0078### ###0079### ###0080### ###0081### ###0082### ###0083### ###0084### ###0085### ###0086### ###0087### ###0088### ###0089### ###0090### ###0091### ###0092### ###0093### ###0094### ###0095### ###0096### ###0097### ###0098### ###0099### ###0100### ###0101### ###0102### ###0103### ###0104### ###0105### ###0106### ###0107### ###0108### ###0109### ###0110### ###0111### ###0112### ###0113### ###0114### ###0115### ###0116### ###0117### ###0118### ###0119### ###0120### ###0121### ###0122### ###0123### ###0124### ###0125### ###0126### ###0127### ###0128### ###0129### ###0130### ###0131### ###0132### ###0133### ###0134### ###0135### ###0136### ###0137### ###0138### ###0139### ###0140### ###0141### ###0142### ###0143### ###0144### ###0145### ###0146### ###0147### ###0148### ###0149### ###0150### ###0151### ###0152### ###0153### ###0154### ###0155### ###0156### ###0157### ###0158### ###0159### ###0160### ###0161### ###0162### ###0163### ###0164### ###0165### ###0166### ###0167### ###0168### ###0169### ###0170### ###0171### ###0172### ###0173### ###0174### ###0175### ###0176### ###0177### ###0178### ###0179### ###0180### ###0181### ###0182### ###0183### ###0184### ###0185### ###0186### ###0187### ###0188### ###0189### ###0190### ###0191### ###0192### ###0193### ###0194### ###0195### ###0196### ###0197### ###0198### ###0199### ###0200### ###0201### ###0202### ###0203### ###0204### ###0205### ###0206### ###0207### ###0208### ###0209### ###0210### ###0211### ###0212### ###0213### ###0214### ###0215### ###0216### ###0217### ###0218### ###0219### ###0220### ###0221### ###0222### ###0223### ###0224### ###0225### ###0226### ###0227### ###0228### ###0229### ###0230### ###0231### ###0232### ###0233### ###0234### ###0235### ###0236### ###0237### ###0238### ###0239### ###0240### ###0241### ###0242### ###0243### ###0244### ###0245### ###0246### ###0247### ###0248### ###0249### ###0250### ###0251### ###0252### ###0253### ###0254### ###0255### ###0256### ###0257### ###0258### ###0259### ###0260### ###0261### ###0262### ###0263### ###0264### ###0265### ###0266### ###0267### ###0268### ###0269### ###0270### ###0271### ###0272### ###0273### ###0274### ###0275### ###0276### ###0277### ###0278### ###0279### ###0280### ###0281### ###0282### ###0283### ###0284### ###0285### ###0286### ###0287### ###0288### ###0289### ###0290### ###0291### ###0292### ###0293### ###0294### ###0295### ###0296### ###0297### ###0298### ###0299### ###0300### ###0301### ###0302### ###0303### ###0304### ###0305### ###0306### ###0307### ###0308### ###0309### ###0310### ###0311### ###0312### ###0313### ###0314### ###0315### ###0316### ###0317### ###0318### ###0319### ###0320### ###0321### ###0322### ###0323### ###0324### ###0325### ###0326### ###0327### ###0328### ###0329### ###0330### ###0331### ###0332### ###0333### ###0334### ###0335### ###0336### ###0337### ###0338### ###0339### ###0340### ###0341### ###0342### ###0343### ###0344### ###0345### ###0346### ###0347### ###0348### ###0349### ###0350### ###0351### ###0352### ###0353### ###0354### ###0355### ###0356### ###0357### ###0358### ###0359### ###0360### ###0361### ###0362### ###0363### ###0364### ###0365### ###0366### ###0367### ###0368### ###0369### ###0370### ###0371### ###0372### ###0373### ###0374### ###0375### ###0376### ###0377### ###0378### ###0379### ###0380### ###0381### ###0382### ###0383### ###0384### ###0385### ###0386### ###0387### ###0388### ###0389### ###0390### ###0391### ###0392### ###0393### ###0394### ###0395### ###0396### ###0397### ###0398### ###0399### ###0400### ###0401### ###0402### ###0403### ###0404### ###0405### ###0406### ###0407### ###0408### ###0409### ###0410### ###0411### ###0412### ###0413### said linker segment is selected from one or more of alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, sulfonyl, carbonyl, acyl, keto, imine, an amino acid, wherein each of said alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene is independently optionally substituted with 1-10 C 1-6 alkyl, C 1-6 heteroalkyl, C 5-10 heteroaryl, aryl, hydroxyl, amino, F, CI, Br, I, and / or cyano; ​ C 1-6 alkyl-SO2-, C 1-6 alkyl-SO3-, C 1-6 alkyl-CO2-, C 1-6 alkyl-O-, (C 1-6 alkyl) 1-2 -(H) 2- 1PO3-, aryl-SO2-, aryl-SO3-, aryl-CO2-, (aryl) 1-2 -(H) 2-1 PO3-, aryl-O-, HSO2-, HSO3-, HCO2-, H3PO3-, HO-, palladium addition complex, palladium oxide addition complex, platinum addition complex, benzenesulfochloride, pyridinium group, sulfonyl fluoride, pentafluorophenyl ester, F-, Cl-, Br-, I-, mono- or di-derivatives of disulfides, bicyclo[1.1.0]butane derivatives, highly electron-deficient aryl groups, alkenes, alkynes with electron-deficient groups, wherein each of the alkyl or aryl groups is independently optionally substituted with 1-6 C 1-6 alkyl, C 1-6 heteroalkyl, C 5-10 heteroaryl, aryl, hydroxyl, amino, F-, Cl-, Br-, I- and / or cyano; Preferably, the leaving group is selected from sulfonyl fluoride, pentafluorophenol ester, benzenesulfonyl chloride, Br, I, C 1-6 alkyl-SO2-; The drug in the linker-drug moiety is selected from the group consisting of diagnostic agents, therapeutic agents, and marker agents, such as cytotoxic agents, toxins, radionuclides, fluorescent agents, chemotherapeutic agents, immunotherapeutic agents, antiviral agents, antimicrobial agents, molecular degradation agents, immune stimulants, and nuclear pharmaceutical chelators; preferably, the drug is an Auristatin, a topoisomerase inhibitor, a Maytansinoid, and a PBD, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), exetecan, pyrrolobenzodiazepine ​ ​ ​ ​ 4. The method of any one of claims 1 to 3, wherein the incubation is performed in the presence of an effective amount of one or more metal ions; preferably the metal ions are selected from the group consisting of ions of the metal elements of the first to third transition series and the second to fifth main groups; more preferably the metal ions are selected from the group consisting of zinc ions (Zn 2+ ), cadmium ions (Cd 2+ ), mercury ions (Hg 2+ ), gallium ions (Ga 3+ ), germanium ions (Ge 4+ ), indium ions (In 3+ ), tin ions (Sn 4+ ), bismuth ions (Bi 3+ ), manganese ions (Mn 2+ ), nickel ions (Ni 2+ ), ferrous ions (Fe 2+ ), ferric ions (Fe 3+ ) and copper ions (Cu 2+ ); further preferably the metal ions are selected from the group consisting of zinc ions (Zn 2+ ), cadmium ions (Cd 2+ ), mercury ions (Hg 2+ ), gallium ions (Ga 3+ ), germanium ions (Ge 4+ ), indium ions (In 3+ ), tin ions (Sn 4+ ) and bismuth ions (Bi 3+ ). ​ ​ ​ ​ Preferably, the pH value of the buffer system is about 5.5 to 9, preferably about 5.5 to 8; Preferably, the incubation temperature is about -10°C to 37°C; preferably about 0°C to 25°C; Preferably, the incubation time is 3-20 hours, preferably 7-20 hours.

7. The method of any one of claims 1 to 6, wherein the antibody is selected from the group consisting of a monoclonal antibody and a polyclonal antibody; preferably, the antibody is selected from the group consisting of a human antibody, a humanized antibody and a chimeric antibody; more preferably, the antibody specifically binds to a tumor-associated antigen, a viral antigen or a microbial antigen and has an anti-tumor, anti-viral or anti-microbial activity in vivo; preferably, the monoclonal antibody is selected from the group consisting of trastuzumab, pertuzumab, racotumomab, abciximab, adalimumab, alfaferone, alemtuzumab, basiliximab, belimumab, belotufosumab, canakinumab, pexidartinib, cetuximab, daclizumab, denosumab, efalizumab, golimumab, infliximab, ipilimumab, isibamumab, natalizumab, nivolumab, olaratumab, ocrelizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, sucralfate and ustekinumab.

8. The method of any one of claims 1 to 7, wherein the method further comprises post-treatment of the incubation mixture; the post-treatment comprises addition of a quencher to deplete excess linker-drug moieties, and / or purification using a desalting column and / or size exclusion chromatography; preferably, the quencher is a molecule comprising a reactive thiol group and salts thereof; more preferably, the quencher is selected from the group consisting of N-acetyl cysteine, cysteine, cysteamine and dithiothreitol.

9. An antibody-drug conjugate prepared by the method of any one of claims 1 to 8, wherein the content of antibody-drug conjugates having a DAR value of 4 is greater than 45%, for example greater than 60% or greater than 70%; and / or the sum of the content of antibody-drug conjugates having a DAR value of 0 and 8 is less than 20%; and / or the content of antibody-drug conjugates having a DAR value of 6 is less than 20%.

10. A pharmaceutical composition comprising the antibody-drug conjugate of claim 9 and a pharmaceutically acceptable carrier or excipient.

11. Use of the antibody-drug conjugate prepared by the method of any one of claims 1 to 8, the antibody-drug conjugate of claim 9 or the pharmaceutical composition of claim 10 for the manufacture of a medicament for the treatment of a disease selected from the group consisting of cancer, autoimmune disease, inflammation and metabolic disease.

12. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody-drug conjugate prepared by the method of any one of claims 1 to 8, the antibody-drug conjugate of claim 9, or the pharmaceutical composition of claim 10, the disease selected from the group consisting of cancer, autoimmune disease, inflammation, and metabolic disease; preferably, the disease is selected from the group consisting of transitional cell carcinoma of the bladder, hepatitis C, paroxysmal nocturnal hemoglobinuria (PNH), colorectal cancer, urothelial cancer, advanced breast cancer, bladder cancer, gastric cancer, elevated low-density lipoprotein cholesterol, superoxide dismutase 1-amyotrophic lateral sclerosis (SOD1-ALS), Pompe disease, and hypertrophic scars.

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