Preparation method of antibody drug conjugate

Abstract

Belongs to the technical field of biology, and relates to a preparation method of an antibody drug conjugate, which comprises the following steps: (i) reacting an antibody or an antigen binding fragment thereof with a reducing agent in a buffer system to reduce an inter-chain disulfide bond of the antibody or the antigen binding fragment thereof; (ii) reacting a linker-payload with the thiol group-containing antibody or antigen-binding fragment thereof obtained in step (i); wherein the reaction temperature in the step (i) is more than 10 DEG C.

Description

[0001] This application is a divisional application. The application date of the original application is November 03, 2023, the application number is 202311458310.3, and the name of the invention is “Method for preparing antibody-drug conjugates”. Technical Field

[0002] The present invention belongs to the field of biotechnology and relates to a method for preparing an antibody-drug conjugate, and an antibody-drug conjugate prepared by the method. Background Art

[0003] Antibody-Drug Conjugate (ADC) is a class of drugs that combines the high specificity of therapeutic antibodies and the high killing activity of cytotoxic drugs, in which the therapeutic antibody part and the cytotoxic drug part are connected through the intermediate linker part. Compared with traditional chemotherapy drugs, antibody-drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells. At present, since the first antibody-drug conjugate Mylotarg was launched in the United States in 2000, at least ten ADCs have been launched on the market worldwide. At the same time, there are also literature reports on the research of various new antibody-drug conjugates in the anti-tumor field.

[0004] Antibodies typically have four interchain disulfide bonds, which can serve as binding sites for the antibody and the linker-payload. Methods for producing antibody-drug conjugates of DAR4 are known, however, some conjugation methods often result in high heterogeneity of antibody-drug conjugates and low D4 content, and some methods have harsh reaction conditions and the presence of toxic reagents, etc. Therefore, there is still a need to develop new methods for conjugating antibodies to cytotoxic drugs. Summary of the invention

[0005] Preparation method

[0006] The present disclosure provides a method for preparing an antibody-drug conjugate, which comprises:

[0007] (i) reacting the antibody or antigen-binding fragment thereof with a reducing agent in a buffer to reduce interchain disulfide bonds of the antibody or antigen-binding fragment thereof;

[0008] (ii) reacting the linker-payload with the antibody or antigen-binding fragment thereof having a thiol group obtained in step (i);

[0009] in,

[0010] The reaction temperature in step (i) is greater than 10°C.

[0011] In some embodiments, the reaction temperature in step (i) is 10.5°C to 20°C. In some embodiments, the reaction temperature in step (i) is 10.5°C to 15°C. In some embodiments, the reaction temperature in step (i) is 10.5°C to 14°C. In some embodiments, the reaction temperature in step (i) is 10.5°C to 11°C, 11°C to 14°C, or 11°C to 13°C. In some embodiments, the reaction temperature in step (i) is 10.5°C, 11°C, 11.5°C, 12°C, 12.5°C, 13°C, 13.5°C, 14°C, 14.5°C, 15°C, or a range formed by any of the above values.

[0012] In some embodiments, the ratio of the amount of the reducing agent to the amount of the antibody or antigen-binding fragment thereof is 1:1 to 4:1. In some embodiments, the ratio of the amount of the reducing agent to the amount of the antibody or antigen-binding fragment thereof is 2:1 to 3:1. In some embodiments, the ratio of the amount of the reducing agent to the amount of the antibody or antigen-binding fragment thereof is 2.4:1 to 2.5:1. In some embodiments, the ratio of the amount of the reducing agent to the amount of the antibody or antigen-binding fragment thereof is 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, or a range formed by any of the above values.

[0013] In some embodiments, the reducing agent can be tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof, dithiothreitol or 2-mercaptoethanol. In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine or a salt thereof, preferably tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl).

[0014] In some embodiments, the buffer can be a HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, a histidine buffer, a phosphate buffer, a borate buffer, or an acetate buffer. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is a histidine-hydrochloric acid buffer. In some embodiments, the concentration of the buffer is 1 mM to 30 mM, such as 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM, preferably 20 mM.

[0015] In some embodiments, the time of reaction in the step (i) is 1 to 10 hours. In some embodiments, the time of reaction in the step (i) is 1 to 5 hours. In some embodiments, the time of reaction in the step (i) is 2 to 4 hours. In some embodiments, the time of reaction in the step (i) is 2 to 3 hours. In some embodiments, the time of reaction in the step (i) is 2.5 to 3 hours. In some embodiments, the time of reaction in the step (i) is 2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3 hours, 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, 4 hours or the range formed by any of the above values.

[0016] In some embodiments, the pH of the reaction in step (i) is 5 to 9. In some embodiments, the pH of the reaction in step (i) is 6 to 8. In some embodiments, the pH of the reaction in step (i) is 6.5 to 7.5. In some embodiments, the pH of the reaction in step (i) is 6.9 to 7.2. In some embodiments, the pH of the reaction in step (i) is 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or a range formed by any of the above values.

[0017] In some embodiments, sucrose is further added to the buffer. In some embodiments, the concentration of the sucrose in the buffer is 1% to 10% (w / v). In some embodiments, the concentration of the sucrose in the buffer is 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v) or 10% (w / v), preferably 5% (w / v).

[0018] In some embodiments, the ratio of the amount of linker-payload to the amount of antibody or antigen-binding fragment thereof in step (ii) is 2: 1 to 10: 1. In some embodiments, the ratio of the amount of linker-payload to the amount of antibody or antigen-binding fragment thereof in step (ii) is 3: 1 to 7: 1. In some embodiments, the ratio of the amount of linker-payload to the amount of antibody or antigen-binding fragment thereof in step (ii) is 4: 1 to 6: 1. In some embodiments, the ratio of the amount of linker-payload to the amount of antibody or antigen-binding fragment thereof in step (ii) is 4.5: 1 to 5.5: 1. In some embodiments, the ratio of the amount of linker-payload to the amount of antibody or antigen-binding fragment thereof in step (ii) is 4.7: 1 to 4.8: 1. In some embodiments, the ratio of the amount of linker-payload to the amount of antibody or antigen-binding fragment thereof in step (ii) is 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1, 5.1:1, 5.2:1, 5.3:1, 5.4:1, 5.5:1, or a range formed by any of the above values.

[0019] In some embodiments, for step (ii), more specifically, a solution containing a linker-payload is added to a buffer containing the antibody or antigen-binding fragment thereof having a thiol group obtained in step (i), so that the linker-payload can react with the antibody or antigen-binding fragment thereof.

[0020] In some embodiments, in the solution containing the linker-payload, examples of solvents for dissolving the linker-payload include organic solvents, which can be aqueous acetone (e.g., 50% aqueous acetone), aqueous ethanol (e.g., 80% aqueous ethanol), aqueous methanol (e.g., 80% aqueous methanol), aqueous isopropanol (e.g., 80% aqueous isopropanol), aqueous dimethyl sulfoxide (e.g., 80% aqueous dimethyl sulfoxide), acetone, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA) or N-methyl-2-pyrrolidone (NMP). In some embodiments, the organic solvent as a solvent is an acetone solution, preferably a 50% aqueous acetone solution. In some embodiments, the organic solvent as a solvent is acetone. In some embodiments, the organic solvent as a solvent is a DMSO solution or DMSO, preferably DMSO.

[0021] In some embodiments, the reaction temperature in step (ii) is 10.5°C to 20°C. In some embodiments, the reaction temperature in step (ii) is 10.5°C to 15°C. In some embodiments, the reaction temperature in step (ii) is 10.5°C to 14°C. In some embodiments, the reaction temperature in step (ii) is 10.5°C to 11°C, 11°C to 14°C, or 11°C to 13°C. In some embodiments, the reaction temperature in step (ii) is 10.5°C, 11°C, 11.5°C, 12°C, 12.5°C, 13°C, 13.5°C, 14°C, 14.5°C, 15°C, or a range formed by any of the above values.

[0022] In some embodiments, the time of reaction in the step (ii) is 1 to 10 hours. In some embodiments, the time of reaction in the step (ii) is 1 to 5 hours. In some embodiments, the time of reaction in the step (ii) is 2 to 4 hours. In some embodiments, the time of reaction in the step (ii) is 2 to 3 hours. In some embodiments, the time of reaction in the step (ii) is 2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3 hours, 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, 4 hours or the range formed by any of the above values.

[0023] In some embodiments, the reaction in step (ii) can be terminated by inactivating the unreacted linker-payload with a sulfhydryl-containing reagent. In some embodiments, the sulfhydryl-containing reagent can be cysteine ​​or N-acetylcysteine, preferably N-acetylcysteine. In some specific embodiments, the reaction in step (ii) can be terminated by adding N-acetylcysteine ​​to the reaction system containing the linker-payload and reacting for 10 to 40 minutes, preferably 25 to 40 minutes. In some embodiments, the ratio of the amount of N-acetylcysteine ​​to the amount of the antibody or its antigen-binding fragment is 1: 1 to 8: 1, preferably 4: 1.

[0024] In some embodiments, in step (ii), before adding the solution containing the linker-payload, an organic solvent is added to the buffer containing the antibody or antigen-binding fragment thereof having a thiol group obtained in step (i) and the concentration of the organic solvent is 1% to 10% (v / v), preferably 2% to 5% (v / v). In some embodiments, the concentration of the organic solvent is 1% (v / v), 2% (v / v), 3% (v / v), 4% (v / v), 5% (v / v), 6% (v / v), 7% (v / v), 8% (v / v), 9% (v / v) or 10% (v / v). In some embodiments, the organic solvent can be acetone, ethanol, methanol, isopropanol, DMSO, DMF, DMA or NMP, preferably acetone or DMSO.

[0025] The reaction in step (ii) can couple the linker-payload to the antibody or its antigen-binding fragment to produce an antibody drug conjugate. After the coupling is completed, the antibody drug conjugate is separated and purified from the system, for example, a commercially available ultrafiltration membrane can be used for separation and purification. As this ultrafiltration membrane, an appropriate ultrafiltration membrane can be used, for example, an ultrafiltration membrane with a molecular weight of 1 kDa to 100 kDa can be used, and preferably an ultrafiltration membrane with a molecular weight of 30 kDa can be used.

[0026] In some specific embodiments, the preparation method comprises:

[0027] (i) reacting the antibody or antigen-binding fragment thereof with a reducing agent in a buffer to reduce the interchain disulfide bonds of the antibody or antigen-binding fragment thereof, wherein the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride;

[0028] (ii) reacting a linker-payload with the antibody or antigen-binding fragment thereof having a thiol group obtained in step (i), wherein the linker-payload is

[0029]

[0030] in,

[0031] The reaction temperature in step (i) is 10.5°C to 14°C, and the reaction time in step (i) is 2 to 3 hours;

[0032] And the DAR value of the prepared antibody drug conjugate is 3.5-4.5.

[0033] In some specific embodiments, the preparation method comprises:

[0034] (i) reacting the antibody or antigen-binding fragment thereof with a reducing agent in a buffer to reduce the interchain disulfide bonds of the antibody or antigen-binding fragment thereof, wherein the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride, the buffer is a histidine buffer, and preferably, sucrose is further added to the buffer; preferably, the antibody or antigen-binding fragment thereof is an anti-TROP-2 antibody or antigen-binding fragment thereof;

[0035] (ii) reacting a linker-payload with the antibody or antigen-binding fragment thereof having a thiol group obtained in step (i), wherein the linker-payload is

[0036]

[0037] in,

[0038] The reaction temperature in step (i) is 10.5° C. to 14° C., the reaction time in step (i) is 2 to 3 hours, and the ratio of the amount of the reducing agent to the amount of the antibody or antigen-binding fragment thereof in step (i) is 2:1 to 3:1, preferably 2.5:1;

[0039] The ratio of the amount of linker-payload to the amount of antibody or antigen-binding fragment thereof in step (ii) is 4.5:1 to 5.5:1, preferably 4.8:1, the reaction temperature in step (ii) is 10.5°C to 14°C, the reaction time in step (ii) is 2 to 4 hours, and before adding the solution containing the linker-payload in step (ii), acetone or DMSO is added to the buffer containing the antibody or antigen-binding fragment thereof having a thiol group obtained in step (i) and the concentration of the organic solvent is 2% to 5% (v / v), preferably 5% (v / v); and

[0040] The DAR value of the prepared antibody drug conjugate is 3.5-4.5, wherein the content of D4 is 55% or more, preferably 60% or more.

[0041] The preparation method disclosed in the present invention has mild reaction conditions, low cost and is easy to operate.

[0042] The linker-payload is formed by connecting the cytotoxic drug to the linker. The number of cytotoxic drugs connected to the antibody or its antigen-binding fragment can vary, so that the antibody-drug conjugate can be uniform or heterogeneous, and the heterogeneous antibody-drug conjugate includes antibodies or their antigen-binding fragments connected to different numbers of cytotoxic drugs, such as 1 molecule of antibody or its antigen-binding fragment connected to 0 (i.e., without cytotoxic drugs), 1, 2, 3, 4, 5, 6, 7, 8 or more molecules of cytotoxic drugs. The antibody-drug conjugates in which 1 molecule of antibody or its antigen-binding fragment is connected to 0, 1, 2, 3, 4, 5, 6, 7 or 8 molecules of cytotoxic drugs are respectively referred to as D0, D1, D2, D3, D4, D5, D6, D7, and D8.

[0043] By controlling the ratio of the above-mentioned antibodies or their antigen-binding fragments to which different numbers of cytotoxic drugs are connected, antibody-drug conjugates with different drug-antibody ratios (DAR) can be produced. In this article, "DAR" and "n" are used interchangeably. It should be understood that the DAR or n is the average number of connections of cytotoxic drugs per molecule of antibody or its antigen-binding fragment in the antibody-drug conjugate. For example, "DAR is 4" refers to such an antibody-drug conjugate, comprising a heterogeneous mixture of cytotoxic drugs (e.g., each antibody or its antigen-binding fragment molecule is connected to 0, 1, 2, 3, 4, 5, 6, 7 and / or 8 cytotoxic drugs) per antibody or its antigen-binding fragment molecule, but the average number of connections of cytotoxic drugs per molecule of antibody or its antigen-binding fragment is 4. Similarly, "DAR is 8" refers to the average number of connections of cytotoxic drugs per molecule of antibody or its antigen-binding fragment in the antibody-drug conjugate is 8.

[0044] D8 is excellent in terms of treatment (e.g., anti-tumor) effect, but in some cases, it may cause problems in terms of safety, such as toxicity. Therefore, in order to improve safety, while maintaining the therapeutic effect, there is a situation where an antibody drug conjugate with a DAR value of less than 8 (e.g., DAR of 4) is used. Antibody drug conjugates are generally composed of one or more of D0, D1, D2, D3, D4, D5, D6, D7, and D8. Therefore, there may be such a situation, wherein, even if different antibody drug conjugates have the same DAR value as each other, if the distribution of the number of cytotoxic drugs connected to them is different, their efficacy and / or toxicity are different from each other. That is to say, an antibody drug conjugate with a DAR of 4 having a higher D0 and D8 content may have a reduced therapeutic effect compared to an antibody drug conjugate with a higher D4 content, and may show strong toxicity. The antibody drug conjugate produced by the preparation method disclosed herein has a DAR value of 3-5 or 3.5-4.5. In some embodiments, the antibody drug conjugate produced by the preparation method of the present disclosure has a DAR value of 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 or 4.5. In some embodiments, the content of D4 in the antibody drug conjugate produced by the preparation method of the present disclosure is 50% or more, 55% or more, 60% or more, or 65% or more, or 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 70%, or 55% to 65%. In some embodiments, the content of D4 in the antibody drug conjugate produced by the preparation method of the present disclosure is 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65%. In some embodiments, the content of D8 in the antibody drug conjugate produced by the preparation method of the present disclosure is 5% or less, 2% or less, or 1% or less, or 0%. Therefore, the antibody drug conjugate produced by the preparation method of the present disclosure has excellent safety.

[0045] In some embodiments, the structure of the antibody drug conjugate produced by the preparation method of the present disclosure is shown in Formula I below:

[0046]

[0047] Wherein Ab is an antibody or an antigen-binding fragment thereof, n is selected from 3 to 5, R 1 is selected from hydrogen atom, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclyl, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl, R 2is selected from hydrogen atom, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclyl, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl; or, R 1 and R 2 The atoms to which they are attached form an optionally substituted 5- to 8-membered heterocyclic group; and the 3-position of -(succinimidyl-3-yl-N)- in Formula I is connected to Ab. In some embodiments, the R 1 and R 2 are independently selected from a hydrogen atom or C 1-5 Alkyl (preferably C 1-4 Alkyl groups, such as C 1-3 In some embodiments, the R 1 and R 2 Each is independently selected from a hydrogen atom, a methyl group, an ethyl group, a propyl group or an isopropyl group. 1 and R 2 is a hydrogen atom. In some embodiments, the 3-position of -(succinimidyl-3-yl-N)- in the structure shown in Formula I is connected to the sulfhydryl group after the disulfide bond reduction of Ab. In some embodiments, n is 3.5-4.5. In some embodiments, n is 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 or 4.5.

[0048] In some specific embodiments, the structure of the antibody drug conjugate produced by the preparation method of the present disclosure is shown in the following formula I-1.

[0049]

[0050] Wherein, Ab is an antibody or an antigen-binding fragment thereof, and n is selected from 3 to 5. In some embodiments, the 3-position of -(succinimidyl-3-yl-N)- in the structure shown in Formula I-1 is connected to the sulfhydryl group after the disulfide bond reduction of Ab. In some embodiments, n is 3.5-4.5. In some embodiments, n is 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 or 4.5.

[0051] Antibodies or antigen-binding fragments thereof

[0052] The antibodies or antigen-binding fragments thereof used in the present disclosure include, but are not limited to, monoclonal antibodies, monospecific antibodies, bispecific antibodies, multispecific antibodies, nanobodies, or antigen-binding fragments of the above antibodies, wherein the antigen-binding fragments include, but are not limited to, Fab fragments, F(ab') 2fragment, Fd fragment, Fv fragment, dAb, single-chain Fv (scFv) molecule, single-chain Fab (scFab), V H H. The antibody or its antigen-binding fragment may be any class of IgG, IgE, IgM, IgD or IgA, preferably IgG. In addition, the antibody or its antigen-binding fragment may be any subclass of IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1 or IgG4. The antibody or its antigen-binding fragment may be derived from any species, including but not limited to humans, rats, mice and rabbits; when derived from species other than humans, it is preferably chimerized or humanized using known techniques. In some embodiments, the antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody. The antibody or its antigen-binding fragment may bind to any disease-associated antigen known in the art. When the disease is a tumor, the antigen can be selected from any tumor-associated antigen, including but not limited to HER2, HER3, EGFR, ROR1, CLDN18.2, B7-H3, TROP-2, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, PD-1 or PD-L1. In some embodiments, the antibody or antigen-binding fragment thereof is an anti-HER2 antibody or an antigen-binding fragment thereof, an anti-HER3 antibody or an antigen-binding fragment thereof, an anti-EGFR antibody or an antigen-binding fragment thereof, an anti-ROR1 antibody or an antigen-binding fragment thereof, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, an anti-B7-H3 antibody or an antigen-binding fragment thereof, an anti-TROP-2 antibody or an antigen-binding fragment thereof, an anti-CD20 antibody or an antigen-binding fragment thereof, an anti-CD22 antibody or an antigen-binding fragment thereof, an anti-CD30 antibody or an antigen-binding fragment thereof, an anti-CD33 antibody or an antigen-binding fragment thereof, an anti-CD47 antibody or an antigen-binding fragment thereof, an anti-CD56 antibody or an antigen-binding fragment thereof, an anti-CD70 antibody or an antigen-binding fragment thereof, an anti-CD79b antibody or an antigen-binding fragment thereof, an anti-VEGF antibody or an antigen-binding fragment thereof, an anti-VEGFR antibody or an antigen-binding fragment thereof, an anti-MUC1 antibody or an antigen-binding fragment thereof, an anti-c-MET antibody or an antigen-binding fragment thereof, an anti-RET antibody or an antigen-binding fragment thereof, an anti-LIV-1 antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, or an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0053] In some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises:

[0054] (1) HCDR1 of the amino acid sequence set forth in SEQ ID NO:1, HCDR2 of the amino acid sequence set forth in SEQ ID NO:2, HCDR3 of the amino acid sequence set forth in SEQ ID NO:3, LCDR1 of the amino acid sequence set forth in SEQ ID NO:4, LCDR2 of the amino acid sequence set forth in SEQ ID NO:5, and LCDR3 of the amino acid sequence set forth in SEQ ID NO:6;

[0055] (2) a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8, and comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described in (1) above;

[0056] (3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8;

[0057] (4) a heavy chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:9, and a light chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:10, and comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described in (1) above; or

[0058] (5) A heavy chain comprising the amino acid sequence shown in SEQ ID NO:9, and a light chain comprising the amino acid sequence shown in SEQ ID NO:10.

[0059] In some embodiments, the C-terminal lysine of the heavy chain constant region of the anti-TROP-2 antibody or antigen-binding fragment thereof may be present or absent, and the absence of the C-terminal lysine of the heavy chain constant region usually occurs during recombinant expression. In some embodiments, the C-terminal lysine of the amino acid sequence shown in SEQ ID NO:9 is absent, as shown in SEQ ID NO:11.

[0060] In some embodiments, the amino acid sequence of the heavy chain of the anti-TROP-2 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain is shown in SEQ ID NO: 10. In some embodiments, the amino acid sequence of the heavy chain of the anti-TROP-2 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain is shown in SEQ ID NO: 10.

[0061] The sequence information of exemplary anti-TROP-2 antibodies or antigen-binding fragments thereof disclosed herein is shown in Table S1 below.

[0062] Table S1. Sequence information of anti-TROP-2 antibodies or antigen-binding fragments thereof

[0063]

[0064] Method for producing an antibody or antigen-binding fragment thereof having a thiol group

[0065] The present disclosure also provides a method for producing an antibody or an antigen-binding fragment thereof having a thiol group, comprising: reacting the antibody or the antigen-binding fragment thereof with a reducing agent in a buffer to reduce the interchain disulfide bonds of the antibody or the antigen-binding fragment thereof, wherein the reaction temperature is greater than 10°C.

[0066] In some embodiments, the reaction temperature is 10.5°C to 20°C. In some embodiments, the reaction temperature is 10.5°C to 15°C. In some embodiments, the reaction temperature is 10.5°C to 14°C. In some embodiments, the reaction temperature is 10.5°C to 11°C, 11°C to 14°C, or 11°C to 13°C. In some embodiments, the reaction temperature is 10.5°C, 11°C, 11.5°C, 12°C, 12.5°C, 13°C, 13.5°C, 14°C, 14.5°C, 15°C, or a range formed by any of the above values.

[0067] In some embodiments, the ratio of the amount of the reducing agent to the amount of the antibody or antigen-binding fragment thereof is 1:1 to 4:1. In some embodiments, the ratio of the amount of the reducing agent to the amount of the antibody or antigen-binding fragment thereof is 2:1 to 3:1. In some embodiments, the ratio of the amount of the reducing agent to the amount of the antibody or antigen-binding fragment thereof is 2.4:1 to 2.5:1. In some embodiments, the ratio of the amount of the reducing agent to the amount of the antibody or antigen-binding fragment thereof is 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, or a range formed by any of the above values.

[0068] In some embodiments, the reducing agent can be tris(2-carboxyethyl)phosphine or a salt thereof, dithiothreitol or 2-mercaptoethanol. In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine or a salt thereof, preferably tris(2-carboxyethyl)phosphine hydrochloride.

[0069] In some embodiments, the buffer can be a HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, a histidine buffer, a phosphate buffer, a borate buffer, or an acetate buffer. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is a histidine-hydrochloric acid buffer. In some embodiments, the concentration of the buffer is 1 mM to 30 mM, such as 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM, preferably 20 mM.

[0070] In some embodiments, the reaction time is 1 to 10 hours. In some embodiments, the reaction time is 1 to 5 hours. In some embodiments, the reaction time is 2 to 4 hours. In some embodiments, the reaction time is 2 to 3 hours. In some embodiments, the reaction time is 2.5 to 3 hours. In some embodiments, the reaction time is 2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3 hours, 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, 4 hours, or the range formed by any of the above values.

[0071] In some embodiments, the pH of the reaction is 5 to 9. In some embodiments, the pH of the reaction is 6 to 8. In some embodiments, the pH of the reaction is 6.5 to 7.5. In some embodiments, the pH of the reaction is 6.9 to 7.2. In some embodiments, the pH of the reaction is 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or a range formed by any of the above values.

[0072] In some embodiments, the antibody or antigen-binding fragment thereof having a thiol group is used to prepare an antibody-drug conjugate, and the DAR value of the antibody-drug conjugate is 3-5, or 3.5-4.5. In some embodiments, the DAR value is 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 or 4.5.

[0073] In some embodiments, the antibody or antigen-binding fragment thereof having a thiol group is used to prepare an antibody drug conjugate, and the content of D4 in the antibody drug conjugate is 50% or more, 55% or more, 60% or more, or 65% or more, or in the range of 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 70%, or 55% to 65%. In some embodiments, the content of D4 in the antibody drug conjugate is 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65%. In some embodiments, the content of D8 in the antibody drug conjugate is 5% or less, 2% or less, or 1% or less, or 0%.

[0074] Cytotoxic drugs

[0075] The cytotoxic drug used in the present disclosure is not particularly limited as long as it has an anti-tumor effect and has a substituent or structure that can be linked to the linker of the present disclosure.

[0076] The cytotoxic drugs used in the present disclosure can be, for example, alkaloids, antimetabolites, antitumor antibiotics, alkylating agents, and platinums. In some embodiments, the cytotoxic drugs are tubulin inhibitor cytotoxic drugs or cytotoxic drugs acting on DNA. In some embodiments, the tubulin inhibitor cytotoxic drugs include but are not limited to Maytansine (Maytansine) class, Auristatin (Auristatin) class and, Dolastatin (Dolastatin), Tubulysins (Tubulysins), Cryptomycins (Cryptomycins), and Eribulin (Eribulin) or its derivatives. In some embodiments, the cytotoxic drugs acting on DNA include but are not limited to Calichemicin (Calicheamicin) class, Duocarmycin (Duocarmycin) class, Anthramycin derivative PBD (Pyrrolobenzodiazepine), topoisomerase I inhibitors and topoisomerase II inhibitors.

[0077] In some embodiments, the cytotoxic drug is a tubulin inhibitor.

[0078] In some specific embodiments, the cytotoxic drug is eribulin or a derivative thereof.

[0079] Connector-Payload

[0080] The cytotoxic drug used in the present disclosure can be linked to the antibody or antigen-binding fragment thereof via a linker. The linker used in the present disclosure preferably has an N-substituted maleimide group.

[0081] The linker-payload used in the present disclosure is not particularly limited as long as it is a compound that can react with the sulfhydryl group generated by reducing the interchain disulfide bonds of the antibody or its antigen-binding fragment. The linker-payload used in the present disclosure is preferably a linker-payload having an N-substituted maleimide group, which can react with the sulfhydryl group generated by reducing the interchain disulfide bonds of the antibody or its antigen-binding fragment, thereby connecting to the antibody or its antigen-binding fragment. The linker-payload used in the present disclosure is more preferably a compound of the structure shown in the following formula II:

[0082]

[0083] Among them, R 1 is selected from hydrogen atom, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclyl, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl, R 2is selected from hydrogen atom, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclyl, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl; or, R 1 and R 2 Together with the atoms to which they are attached, they form an optionally substituted 5- to 8-membered heterocyclyl. 1 and R 2 are independently selected from a hydrogen atom or C 1-5 Alkyl (preferably C 1-4 Alkyl groups, such as C 1-3 In some embodiments, the R 1 With R 2 Each is independently selected from a hydrogen atom, a methyl group, an ethyl group, a propyl group or an isopropyl group. 1 and R 2 A hydrogen atom.

[0084] In some specific embodiments, the linker-payload is a compound of the structure shown in the following formula II-1:

[0085]

[0086] It should be understood that the linker-payload used in the present disclosure is not limited to the above-mentioned compounds, and all types of linker-payloads can be applied to the preparation method of the present disclosure as long as they have a functional group for reacting with the sulfhydryl group generated after the reduction of the interchain disulfide bonds of the antibody or its antigen-binding fragment.

[0087] Pharmaceutical composition

[0088] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody drug conjugate produced by the preparation method of the present disclosure, or an isomer of the antibody drug conjugate, a pharmaceutically acceptable salt, or a solvate of the antibody drug conjugate, its isomer, or its pharmaceutically acceptable salt. In some embodiments, the pharmaceutical composition of the present disclosure further comprises a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other agents.

[0089] use

[0090] The present disclosure also provides uses of the antibody-drug conjugate produced by the preparation method of the present disclosure or the pharmaceutical composition.

[0091] In one aspect, the present disclosure provides the use of an antibody drug conjugate or the pharmaceutical composition produced by the preparation method of the present disclosure in the preparation of a drug for treating a disease expressing an antigen bound by the antibody or its antigen-binding fragment. In some embodiments, the present disclosure provides the use of an antibody drug conjugate or the pharmaceutical composition produced by the preparation method of the present disclosure in the preparation of a drug for treating a tumor. In some embodiments, the present disclosure provides the use of an antibody drug conjugate or the pharmaceutical composition produced by the preparation method of the present disclosure in the preparation of a drug for treating an autoimmune disease.

[0092] In one aspect, the present disclosure provides a method for treating a disease that expresses an antigen bound by the antibody or its antigen-binding fragment, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody-drug conjugate or the pharmaceutical composition produced by the preparation method of the present disclosure. In some embodiments, the present disclosure provides a method for treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody-drug conjugate or the pharmaceutical composition produced by the preparation method of the present disclosure. The present disclosure provides a method for treating an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody-drug conjugate or the pharmaceutical composition produced by the preparation method of the present disclosure.

[0093] In some embodiments, the tumor is a TROP-2 expressing tumor.

[0094] In some embodiments, the tumor is biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, melanoma, pharyngeal cancer, oral cancer, skin cancer, lung cancer, urethral cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, liver cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, or myeloma.

[0095] Explanation and Definitions

[0096] Unless otherwise indicated, the following terms used in this disclosure have the following meanings. A particular term should not be considered as indefinite or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. When a trade name appears in this article, it is intended to refer to the corresponding commodity or its active ingredient.

[0097] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo (i.e., =O), it means that two hydrogen atoms are replaced, and the oxo will not occur on the aromatic group. "Optionally substituted" means that it may be substituted or not substituted, and unless otherwise specified, the type and number of the substituent can be any on the basis of chemically feasible.

[0098] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 2 R's, each R has an independent choice.

[0099] The term "mercapto" refers to a -SH group.

[0100] As used herein, the structure of "-(succinimidyl-3-yl-N)-" is as follows:

[0101]

[0102] As used herein, the structure of an "N-substituted maleimido group" is as follows:

[0103]

[0104] Unless otherwise specified, the key is a solid wedge. and dotted wedge key Indicates the absolute configuration of a stereocenter.

[0105] Unless otherwise specified, when a group has a linkable site, the link between that site and other groups can be represented by a wavy line. express.

[0106] As used herein, compounds formed by substitution of atoms or atomic groups in a molecule of a parent compound with other atoms or atomic groups are referred to as "derivatives" of the parent compound.

[0107] The compounds of the present disclosure may exist in specific geometric isomer or stereoisomer forms. The present disclosure contemplates all such compounds, including cis and trans isomers, levorotatory and dextrorotatory isomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present disclosure.

[0108] Unless otherwise indicated, the term "cis-trans isomers" or "geometric isomers" arises from the inability of a ring to rotate freely about double bonds or single bonds of ring carbon atoms.

[0109] Unless otherwise indicated, the term "enantiomer" refers to stereoisomers that are mirror images of one another.

[0110] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

[0111] The compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations. A specific example of a proton tautomer is an imidazole moiety, in which a proton can migrate between two ring nitrogens. Valence tautomers include interconversions by reorganization of some bonding electrons.

[0112] "Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine-hydrochloric acid buffer, histidine-acetate buffer, histidine-phosphate buffer, etc. Histidine-hydrochloric acid buffer can be prepared using histidine (e.g., L-histidine) and further adjusted with hydrochloric acid to pH; or prepared using histidine (e.g., L-histidine) and histidine hydrochloride or its hydrate (e.g., monohydrated histidine hydrochloride). Histidine-acetate buffer can be prepared using histidine (e.g., L-histidine) and further adjusted with acetic acid to pH. Histidine-phosphate buffer can be prepared using histidine (e.g., L-histidine) and further adjusted with phosphoric acid to pH.

[0113] The term "treatment" means administering the compounds or pharmaceutical compositions of the present disclosure to prevent, improve or eliminate a disease or one or more symptoms associated with the disease, and includes but is not limited to:

[0114] (i) preventing a disease or disease state from occurring in a mammal, particularly where such mammal is susceptible to such disease state but has not yet been diagnosed as having such disease state;

[0115] (ii) inhibiting a disease or disease state, i.e., arresting its development;

[0116] (iii) ameliorating a disease or condition, even if such disease or condition regresses;

[0117] (iv) reduce any direct or indirect pathological consequence of the disease or disease state.

[0118] The term "therapeutically effective amount" means an amount of the disclosed compound that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the disclosed compound or pharmaceutical composition that constitutes a "therapeutically effective amount" may vary according to a number of factors, such as the compound or pharmaceutical composition and its ability to elicit a desired response in an individual, the disease state and its severity, the mode of administration, and the age, sex, and weight of the mammal to be treated. The therapeutically effective amount may also be routinely determined by those skilled in the art based on their own knowledge and the present disclosure.

[0119] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0120] The term "pharmaceutically acceptable salt" refers to a salt of a compound (e.g., the antibody drug conjugate of the present disclosure), which is safe and effective when used in mammals and has the desired biological activity, for example, a metal salt, an ammonium salt, a salt formed with an organic base, a salt formed with an inorganic acid, a salt formed with an organic acid, a salt formed with a basic or acidic amino acid, etc.

[0121] The term "excipient" refers to any ingredient other than an active ingredient (e.g., an antibody drug conjugate of the present disclosure). The choice of excipient will largely depend on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0122] The term "solvate" refers to an association of a compound with solvent molecules.

[0123] The term "antibody" is used in the broadest sense and includes, but is not limited to, various antibody structures including monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies, trispecific antibodies), so long as they exhibit the desired antigen-binding activity.

[0124] The "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the function of specifically binding to an antigen (e.g., a TROP-2 protein). It has been demonstrated that the antigen-binding function of an antibody can be implemented by a fragment of a full-length antibody. Examples encompassed within the term "antigen-binding fragment" of an antibody include: (i) Fab fragment: a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) dAb fragment consisting of a VH domain (see Ward et al., Nature. 341: 544-546 (1989)); and (vi) nanobodies, an antibody comprising a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, VH and VL can be connected into a single protein chain by a recombinant method through a linker, wherein VL and VH are paired to form a monovalent molecule called single-chain Fv (scFv) (see Bird et al., Science. 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. 85: 5879-5883 (1988)), and these single-chain antibodies are also included in the term antigen-binding fragment. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened by the same method as full-length antibodies.

[0125] The antibodies or antigen-binding fragments thereof of the present disclosure may be of IgG1, IgG2, IgG3 or IgG4 isotypes. The term "isotype" refers to the class of antibodies encoded by the heavy chain constant region gene. In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are of IgG1 isotype. The antibodies or antigen-binding fragments thereof of the present disclosure may be derived from any species, including but not limited to mice, rats, rabbits, non-human primates (such as chimpanzees, cynomolgus monkeys, spider monkeys, macaques), llamas and humans. The antibodies or antigen-binding fragments thereof of the present disclosure may be murine antibodies, chimeric antibodies, humanized antibodies or human antibodies.

[0126] The term "mouse antibody" or "murine antibody" refers to an antibody in which the framework region and CDR region in the variable region are both derived from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from a mouse germline immunoglobulin sequence. The murine antibody of the present disclosure may include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo), but "mouse antibody" or "murine antibody" does not include antibodies in which CDR sequences derived from other mammalian germlines are inserted into mouse framework sequences.

[0127] "Chimeric antibodies" are antibodies formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by mouse antibodies. To create chimeric antibodies, you must first create a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region genes from the hybridoma cells, and then clone the constant region genes of human antibodies as needed. The mouse variable region genes and human constant region genes are connected into chimeric genes and inserted into an expression vector, and finally the chimeric antibodies are expressed in a eukaryotic or prokaryotic system.

[0128] A "humanized antibody" is an antibody that contains complementarity determining regions (CDRs) derived from a non-human antibody and framework and constant regions derived from a human antibody.

[0129] The term “CDR” (complementarity determining region), also known as “hypervariable region.” A natural four-chain antibody usually contains six CDRs, three in the heavy chain variable region and three in the light chain variable region.

[0130] The term "variable region" refers to a domain of about 100 to 110 or more amino acids defined by the N-terminal domain of the light or heavy chain of an antibody that is primarily responsible for antigen recognition. The terms light chain variable region (VL) and heavy chain variable region (VH) refer to these light chain domains and heavy chain domains, respectively.

[0131] The term "identity" is also known as consistency. The "percentage (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in the sequence to be compared that are identical to the amino acid residues in the specific amino acid sequence shown in this article, after comparing the sequence to be compared with the specific amino acid sequence shown in this article and introducing spaces if necessary to achieve the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity. The amino acid sequence alignment of identity can be carried out in a variety of ways within the scope of the art, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required to obtain the maximum alignment in the full length of the comparison sequence.

[0132] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Preferably, the subject according to the present disclosure is a human. Unless otherwise indicated, the terms "patient" or "subject" can be used interchangeably. "Subjects in need" include those subjects who already have a disease or condition, subjects who are at risk of developing a disease or condition, and subjects who may have a disease or condition and whose purpose is to prevent, delay or attenuate the disease or condition.

[0133] As used herein, "about" means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation as practiced in the art. Alternatively, "about" may mean a range of up to ±5%, such as fluctuations within ±2%, within ±1%, or within ±0.5% of a given specific numerical range. When a specific value is given in the scope of the present disclosure, unless otherwise indicated, the meaning of "about" should be considered to be within the acceptable error range for that specific value. In this document, unless otherwise indicated, the values ​​of step parameters or conditions are modified by "about" by default.

[0134] The terms "comprise," "comprises," or "comprising" and their equivalents (for example, contain, contains, containing, include, includes, and including) should be understood as "including but not limited to," meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be included.

[0135] Herein, singular terms include plural referents and vice versa unless the context clearly dictates otherwise. DETAILED DESCRIPTION

[0136] The present disclosure also provides the following specific implementation schemes, but the protection scope of the present disclosure is not limited thereto:

[0137] Embodiment 1. A method for preparing an antibody drug conjugate, comprising:

[0138] (i) reacting the antibody or antigen-binding fragment thereof with a reducing agent in a buffer to reduce interchain disulfide bonds of the antibody or antigen-binding fragment thereof;

[0139] (ii) reacting the linker-payload with the antibody or antigen-binding fragment thereof having a thiol group obtained in step (i);

[0140] in,

[0141] The reaction temperature in step (i) is greater than 10°C.

[0142] Embodiment 2. The preparation method according to embodiment 1, wherein the DAR value of the produced antibody drug conjugate is 3-5.

[0143] Embodiment 3. The preparation method according to Embodiment 2, wherein the DAR value of the produced antibody drug conjugate is 3.5-4.5.

[0144] Embodiment 4. The preparation method according to embodiment 3, wherein the DAR value of the antibody drug conjugate produced is 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 or 4.5.

[0145] Embodiment 5. The preparation method according to any one of Embodiments 1-4, wherein the D4 content of the produced antibody drug conjugate is 50% or more, 55% or more, 60% or more, or 65% or more.

[0146] Embodiment 6. The preparation method according to any one of Embodiments 1-4, wherein the D4 content of the produced antibody drug conjugate is in the range of 50% to 90%.

[0147] Embodiment 7. The preparation method according to Embodiment 6, wherein the content of D4 in the produced antibody drug conjugate is in the range of 50% to 80%.

[0148] Embodiment 8. The preparation method according to Embodiment 7, wherein the content of D4 in the produced antibody drug conjugate is in the range of 50% to 70%.

[0149] Embodiment 9. The preparation method according to embodiment 8, wherein the D4 content of the produced antibody drug conjugate is in the range of 50% to 60%, 60% to 70%, or 55% to 65%.

[0150] Embodiment 10. The preparation method according to any one of Embodiments 1-9, wherein the reaction temperature in step (i) is 10.5°C to 20°C.

[0151] Embodiment 11. The preparation method according to Embodiment 10, wherein the reaction temperature in step (i) is 10.5°C to 15°C.

[0152] Embodiment 12. The preparation method according to Embodiment 11, wherein the reaction temperature in step (i) is 10.5°C to 14°C.

[0153] Embodiment 13. The preparation method according to Embodiment 12, wherein the reaction temperature in step (i) is 10.5°C to 11°C.

[0154] Embodiment 14. A preparation method according to any one of Embodiments 1-13, wherein the molar ratio of the reducing agent to the antibody or its antigen-binding fragment is 1:1 to 4:1, 2:1 to 3:1, or 2.4:1 to 2.5:1.

[0155] Embodiment 15. A preparation method according to any one of Embodiments 1 to 14, wherein the reducing agent is tris(2-carboxyethyl)phosphine or a salt thereof, dithiothreitol or 2-mercaptoethanol, preferably tris(2-carboxyethyl)phosphine or a salt thereof.

[0156] Embodiment 16. The preparation method according to Embodiment 15, wherein the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride.

[0157] Embodiment 17. The preparation method according to any one of Embodiments 1-16, wherein the buffer is HEPES buffer, histidine buffer, phosphate buffer, borate buffer or acetate buffer, preferably histidine buffer.

[0158] Embodiment 18. A preparation method according to any one of Embodiments 1-17, wherein the reaction time in step (i) is 1 to 10 hours, 1 to 5 hours, 2 to 4 hours, 2 to 3 hours, or 2.5 to 3 hours.

[0159] Embodiment 19. A preparation method according to any one of Embodiments 1-18, wherein the reaction temperature in step (ii) is 10°C to 20°C, 10.5°C to 15°C, 10.5°C to 14°C, or 10.5°C to 11°C.

[0160] Embodiment 20. A preparation method according to any one of Embodiments 1-19, wherein the ratio of the amount of linker-payload to the amount of antibody or antigen-binding fragment thereof in step (ii) is 2:1 to 10:1, 3:1 to 7:1, 4:1 to 6:1, 4.5:1 to 5.5:1, or 4.7:1 to 4.8:1.

[0161] Embodiment 21. The preparation method according to any one of Embodiments 1-20, wherein the reaction time in step (ii) is 1 to 5 hours, 2 to 4 hours, or 2 to 3 hours.

[0162] Embodiment 22. A preparation method according to any one of Embodiments 1-21, wherein the antibody or its antigen-binding fragment is an anti-TROP-2 antibody or its antigen-binding fragment, an anti-HER2 antibody or its antigen-binding fragment, an anti-HER3 antibody or its antigen-binding fragment, an anti-ROR1 antibody or its antigen-binding fragment, an anti-B7-H3 antibody or its antigen-binding fragment, an anti-CD79b antibody or its antigen-binding fragment, an anti-CLDN18.2 antibody or its antigen-binding fragment, an anti-c-MET antibody or its antigen-binding fragment, or an anti-LIV-1 antibody or its antigen-binding fragment.

[0163] Embodiment 23. The preparation method according to Embodiment 22, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises HCDR1 of the amino acid sequence shown in SEQ ID NO:1, HCDR2 of the amino acid sequence shown in SEQ ID NO:2, HCDR3 of the amino acid sequence shown in SEQ ID NO:3, LCDR1 of the amino acid sequence shown in SEQ ID NO:4, LCDR2 of the amino acid sequence shown in SEQ ID NO:5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:6.

[0164] Embodiment 24. The preparation method according to Embodiment 23, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:8.

[0165] Embodiment 25. The preparation method according to embodiment 23 or 24, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:9 or 11, and a light chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:10.

[0166] Embodiment 26. A preparation method according to any one of Embodiments 1-25, wherein the linker-payload has an N-substituted maleimide group.

[0167] Embodiment 27. The preparation method according to Embodiment 26, wherein the structure of the linker-payload is shown in Formula II below:

[0168]

[0169] in,

[0170] R 1 is selected from hydrogen atom, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclyl, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl,

[0171] R 2 is selected from hydrogen atom, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclyl, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl;

[0172] Or, R 1 and R 2 Together with the atoms to which it is attached, it forms an optionally substituted 5- to 8-membered heterocyclyl.

[0173] Embodiment 28. The preparation method according to Embodiment 27, wherein the R 1 With R 2 are each independently selected from a hydrogen atom, a methyl group, an ethyl group, a propyl group or an isopropyl group; preferably, R 1 and R 2 A hydrogen atom.

[0174] Embodiment 29. An antibody drug conjugate produced by the preparation method of any one of Embodiments 1-28, wherein the DAR value of the antibody drug conjugate is 3-5, or 3.5-4.5, and the content of D4 is 50% or more, 55% or more, 60% or more, or 65% or more.

[0175] Embodiment 30. The antibody drug conjugate according to Embodiment 29, wherein the content of D4 in the antibody drug conjugate is in the range of 50% to 90%.

[0176] Embodiment 31. The antibody drug conjugate according to Embodiment 30, wherein the content of D4 in the antibody drug conjugate is in the range of 50% to 80%.

[0177] Embodiment 32. The antibody drug conjugate according to Embodiment 31, wherein the content of D4 in the antibody drug conjugate is in the range of 50% to 70%.

[0178] Embodiment 33. The antibody drug conjugate according to Embodiment 32, wherein the content of D4 of the antibody drug conjugate is in the range of 50% to 60%, 60% to 70%, or 55% to 65%.

[0179] Embodiment 34. An antibody-drug conjugate according to any one of Embodiments 29-33, wherein the antibody or antigen-binding fragment thereof is an anti-TROP-2 antibody or an antigen-binding fragment thereof, an anti-HER2 antibody or an antigen-binding fragment thereof, an anti-HER3 antibody or an antigen-binding fragment thereof, an anti-ROR1 antibody or an antigen-binding fragment thereof, an anti-B7-H3 antibody or an antigen-binding fragment thereof, an anti-CD79b antibody or an antigen-binding fragment thereof, an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, an anti-c-MET antibody or an antigen-binding fragment thereof, or an anti-LIV-1 antibody or an antigen-binding fragment thereof.

[0180] Embodiment 35. The antibody-drug conjugate of Embodiment 34, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises HCDR1 of the amino acid sequence shown in SEQ ID NO:1, HCDR2 of the amino acid sequence shown in SEQ ID NO:2, HCDR3 of the amino acid sequence shown in SEQ ID NO:3, LCDR1 of the amino acid sequence shown in SEQ ID NO:4, LCDR2 of the amino acid sequence shown in SEQ ID NO:5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:6.

[0181] Embodiment 36. The antibody drug conjugate of Embodiment 35, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8.

[0182] Embodiment 37. The antibody drug conjugate of embodiment 35 or 36, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 9 or 11, and a light chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10.

[0183] Embodiment 38. The antibody-drug conjugate according to any one of Embodiments 29-37, wherein the structure of the antibody-drug conjugate is shown in Formula I below:

[0184]

[0185] in,

[0186] Ab is an antibody or its antigen-binding fragment,

[0187] n is selected from 3-5, preferably 3.5-4.5,

[0188] R 1 is selected from hydrogen atom, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclyl, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl,

[0189] R 2 is selected from hydrogen atom, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclyl, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl;

[0190] Or, R 1 and R 2 together with the atoms to which they are attached form an optionally substituted 5- to 8-membered heterocyclyl; and

[0191] The 3-position of -(succinimidyl-3-yl-N)- in Formula I is linked to Ab.

[0192] Embodiment 39. The antibody drug conjugate according to embodiment 38, wherein the R 1 With R2 are each independently selected from a hydrogen atom, a methyl group, an ethyl group, a propyl group or an isopropyl group; preferably, R 1 and R 2 A hydrogen atom.

[0193] Embodiment 40. A pharmaceutical composition comprising the antibody drug conjugate according to any one of Embodiments 29-39, or an isomer, a pharmaceutically acceptable salt, or a solvate of the antibody drug conjugate, its isomer, or a pharmaceutically acceptable salt thereof; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0194] Embodiment 41. Use of the antibody drug conjugate of any one of Embodiments 29-39 or the pharmaceutical composition of Embodiment 40 in the preparation of a medicament for treating a tumor.

[0195] Embodiment 42. The use according to embodiment 41, wherein the tumor is biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, melanoma, pharyngeal cancer, oral cancer, skin cancer, lung cancer, urethral cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, liver cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma or myeloma.

[0196] Embodiment 43. A method for treating a tumor, comprising administering a therapeutically effective amount of the antibody drug conjugate of any one of Embodiments 29-39 or the pharmaceutical composition of Embodiment 40 to a subject in need thereof.

[0197] Embodiment 44. The method according to embodiment 43, wherein the tumor is biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, melanoma, pharyngeal cancer, oral cancer, skin cancer, lung cancer, urethral cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, liver cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma or myeloma.

[0198] Embodiment 45. A method for producing an antibody or an antigen-binding fragment thereof having a thiol group, comprising:

[0199] Allowing the antibody or antigen-binding fragment thereof to react with a reducing agent in a buffer to reduce interchain disulfide bonds of the antibody or antigen-binding fragment thereof, wherein the reaction temperature is greater than 10°C;

[0200] The produced antibody or antigen-binding fragment thereof having a thiol group is used to prepare an antibody-drug conjugate having a DAR value of 3-5, or 3.5-4.5, and a D4 content of 50% or more, 55% or more, 60% or more, or 65% or more.

[0201] Embodiment 46. The method according to Embodiment 45, wherein the content of D4 in the antibody drug conjugate is in the range of 50% to 90%.

[0202] Embodiment 47. The method according to Embodiment 46, wherein the content of D4 in the antibody drug conjugate is in the range of 50% to 80%.

[0203] Embodiment 48. The method according to Embodiment 47, wherein the content of D4 in the antibody drug conjugate is in the range of 50% to 70%.

[0204] Embodiment 49. The method according to Embodiment 48, wherein the content of D4 in the antibody drug conjugate is in the range of 50% to 60%, 60% to 70%, or 55% to 65%.

[0205] Embodiment 50. The method according to any one of Embodiments 45-49, wherein the reaction temperature is 10.5°C to 20°C.

[0206] Embodiment 51. The method according to Embodiment 50, wherein the reaction temperature is 10.5°C to 15°C.

[0207] Embodiment 52. The method according to Embodiment 51, wherein the reaction temperature is 10.5°C to 14°C.

[0208] Embodiment 53. A method according to Embodiment 52, wherein the reaction temperature is 10.5°C to 11°C.

[0209] Embodiment 54. A method according to any one of Embodiments 45-53, wherein the ratio of the amount of the reducing agent to the amount of the antibody or its antigen-binding fragment is 1:1 to 4:1, 2:1 to 3:1, or 2.4:1 to 2.5:1.

[0210] Embodiment 55. The method according to any one of Embodiments 45-54, wherein the reducing agent is tris(2-carboxyethyl)phosphine or a salt thereof, dithiothreitol or 2-mercaptoethanol, preferably tris(2-carboxyethyl)phosphine or a salt thereof.

[0211] Embodiment 56. A method according to Embodiment 55, wherein the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride.

[0212] Embodiment 57. A method according to any one of Embodiments 45-56, wherein the buffer is HEPES buffer, histidine buffer, phosphate buffer, borate buffer or acetate buffer, preferably histidine buffer.

[0213] Embodiment 58. The method according to any one of Embodiments 45-57, wherein the reaction time is 1 to 10 hours, 1 to 5 hours, 2 to 4 hours, 2 to 3 hours, or 2.5 to 3 hours.

[0214] Embodiment 59. A method according to any one of Embodiments 45-58, wherein the antibody or its antigen-binding fragment is an anti-TROP-2 antibody or its antigen-binding fragment, an anti-HER2 antibody or its antigen-binding fragment, an anti-HER3 antibody or its antigen-binding fragment, an anti-ROR1 antibody or its antigen-binding fragment, an anti-B7-H3 antibody or its antigen-binding fragment, an anti-CD79b antibody or its antigen-binding fragment, an anti-CLDN18.2 antibody or its antigen-binding fragment, an anti-c-MET antibody or its antigen-binding fragment, or an anti-LIV-1 antibody or its antigen-binding fragment.

[0215] Embodiment 60. A method according to Embodiment 59, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises HCDR1 of the amino acid sequence shown in SEQ ID NO:1, HCDR2 of the amino acid sequence shown in SEQ ID NO:2, HCDR3 of the amino acid sequence shown in SEQ ID NO:3, LCDR1 of the amino acid sequence shown in SEQ ID NO:4, LCDR2 of the amino acid sequence shown in SEQ ID NO:5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:6.

[0216] Embodiment 61. A method according to embodiment 60, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8.

[0217] Embodiment 62. The method of embodiment 60 or 61, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:9 or 11, and a light chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:10.

[0218] For the sake of clarity, the present disclosure is further illustrated by examples, but the examples do not limit the scope of the present disclosure. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce substantially similar results.

[0219] The anti-TROP-2 antibody hz-Ab35-1.3 used in the example and its preparation method are derived from patent application document WO2023104080, and its heavy chain amino acid sequence is shown in SEQ ID NO:70 in WO2023104080, and its light chain amino acid sequence is shown in SEQ ID NO:71 in WO2023104080.

[0220] The linker-payload MC-GGFG-Eribulin used in the embodiment and its preparation method are derived from patent application document patent application document WO2023041006, and its structure is shown in Formula II-1 in the present disclosure.

[0221] Example 1: Preparation of Antibody Drug Conjugates

[0222] Reagents:

[0223] The antibody was hz-Ab35-1.3 and the linker-payload was MC-GGFG-eribulin.

[0224] Experimental process:

[0225] (1) The concentration of antibody hz-Ab35-1.3 was adjusted to about 10 g / L using 20 mM histidine-hydrochloric acid buffer (containing 1.43 mg / mL L-histidine, 2.27 mg / mL L-histidine hydrochloride monohydrate, pH 6.0), 0.3 M Na 2 HPO 4The pH of the aqueous solution was adjusted to 7.0, and sucrose was added thereto to make the concentration of sucrose 5% (w / v). The temperature of the above solution was adjusted to 10.5-14.0°C, and a 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution was added to make the molar ratio of TCEP·HCl to the antibody 2.5:1, and the reaction was carried out at 10.5-14.0°C and 20-100 rpm with stirring for 2.5 hours in the dark.

[0226] (2) DMSO was added to a final concentration of 2% (v / v), and then 10 g / L of linker-payload (MC-GGFG-Eribulin) dissolved in DMSO was added to make the molar ratio of linker-payload to antibody 4.8:1. The reaction was allowed to proceed at 10.5-14.0°C and 20-100 rpm with stirring for 2 hours in the dark.

[0227] (3) Add 10 mM N-acetylcysteine ​​so that the ratio of its amount to the antibody substance is 4:1, and react for 25 to 40 minutes. Then, use a 30 kDa ultrafiltration membrane to replace the reaction solution into 20 mM histidine-hydrochloric acid buffer (pH 6.0) to obtain an antibody drug conjugate.

[0228] According to the specific conditions in Table 1 and Table 2, antibody drug conjugates ADC1-ADC6 were synthesized, and the structures were as follows:

[0229]

[0230] By the method of Example 2, the number distribution and average connection number (n) of cytotoxic drugs in each antibody molecule of ADC1-ADC6 were measured. The specific results are shown in Table 3.

[0231] Table 1. Specific parameters of step (1)

[0232] ADC Number Antibody concentration (g / L) Reaction temperature in dark environment (℃) ADC1 9.7 11.0 ADC2 10.0 11.0 ADC3 9.9 10.5 ADC4 9.9 12.0 ADC5 9.9 13.0 ADC6 9.9 14.0

[0233] Table 2. Specific parameters of step (2)

[0234] ADC Number Reaction temperature in dark environment (℃) ADC1 11.0 ADC2 11.0 ADC3 10.5 ADC4 12.0 ADC5 13.0 ADC6 14.0

[0235] Table 3. Peak area percentages containing D0-D8 and DAR values ​​of ADC (i.e. n)

[0236] ADC Number D0(％) D2(％) D3(％) D4(％) D5(％) D6(％) DAR value ADC1 1.75 15.63 2.15 60.07 1.60 18.80 3.99 ADC2 1.33 13.03 2.21 61.07 1.80 20.56 4.09 ADC3 1.78 17.75 2.70 61.18 1.41 15.18 3.86 ADC4 2.07 18.76 2.94 58.47 1.42 16.34 3.85 ADC5 2.35 20.02 3.12 58.39 1.39 14.74 3.78 ADC6 1.93 18.11 2.97 58.61 1.53 16.86 3.88

[0237] Note: The peak area percentages of D1, D7 and D8 are all 0%.

[0238] Example 2: Determination of DAR value of antibody drug conjugate

[0239] The DAR value of the antibody-drug conjugate was determined by the hydrophobic interaction chromatography (HIC) method. A neutral high-salt mobile phase was used to increase the hydrophobic properties of the protein molecules, thereby combining with the hydrophobic bonds in the chromatographic column. The salt concentration was then gradually reduced and the proportion of isopropanol was gradually increased to elute the substance. The less hydrophobic molecules were eluted first, and the more hydrophobic molecules were eluted later.

[0240] The components of the antibody drug conjugate were separated using a non-porous PS / DVB filler bonded with butyl. The chromatographic column specifications were Sepax, Proteomix HIC Butyl-NP5 (5μm, 4.6mm×100mm), and the column temperature was 25°C. The mobile phase A was 1.5M ammonium sulfate-50mM phosphate buffer (pH 7.0): isopropanol = 95:5 (v / v) (188.30g of ammonium sulfate and 7.41g of sodium dihydrogen phosphate dihydrate were weighed, 750mL of ultrapure water was added, and the mixture was stirred until fully dissolved, the pH was adjusted to 7.0 with 50% sodium hydroxide solution, ultrapure water was added to 950mL, and 50mL of isopropanol was added, and the mixture was filtered through a 0.22μm filter membrane). Mobile phase B is 50mM phosphate buffer (pH7.0): isopropanol = 80:20 (v / v) (weigh 6.24g of sodium dihydrogen phosphate dihydrate, add 750mL of ultrapure water, stir until fully dissolved, adjust pH to 7.0 with 50% sodium hydroxide solution, add ultrapure water to 800mL, add 200mL of isopropanol, mix well and filter through a 0.22μm filter membrane to obtain the result). Dilute the ADC sample with ultrapure water to about 2.0mg of protein per 1mL as the test solution, inject 20μg of protein, and detect at a wavelength of 214nm. The flow rate is 1.0mL / min, and the gradient elution parameters are shown in Table 4.

[0241] Table 4. Gradient elution parameters

[0242]

[0243]

[0244] Data processing, using area normalization method for quantitative analysis of the results. Calculate the peak area percentages of D0, D1, D2, D3, D4, D5, D6, D7 and D8 respectively, and calculate the DAR value results. The calculation formula is: DAR value = (D0 peak area percentage × 0 + D1 peak area percentage × 1 + D2 peak area percentage × 2 + D3 peak area percentage × 3 + D4 peak area percentage × 4 + D5 peak area percentage × 5 + D6 peak area percentage × 6 + D7 peak area percentage × 7 + D8 peak area percentage × 8) / 100%.

[0245] All patents, patent applications and other identified publications are expressly incorporated herein by reference for the purposes of description and disclosure. These publications are provided solely because their disclosure predates the filing date of the present disclosure. All statements regarding the dates of these documents or representations of the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any reference to these publications herein does not constitute an admission that the publications become part of the common general knowledge in the art in any country.

[0246] Although the present disclosure has been described in detail above with general descriptions and specific implementation schemes, it is obvious to those skilled in the art that some modifications or improvements can be made to the present disclosure. Therefore, these modifications or improvements made without departing from the spirit of the present disclosure are within the scope of protection claimed by the present disclosure.

Claims

1. A method for preparing an antibody-drug conjugate, comprising: (i) reacting the antibody or antigen-binding fragment thereof with a reducing agent in a buffer to reduce the interchain disulfide bonds of the antibody or antigen-binding fragment thereof, wherein the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride; (ii) reacting a linker-payload with the antibody or antigen-binding fragment thereof having a thiol group obtained in step (i), wherein the linker-payload is in, The reaction temperature in step (i) is 10.5°C to 14°C, and the reaction time in step (i) is 2 to 3 hours; And the DAR value of the prepared antibody drug conjugate is 3.5-4.

5.

2. The preparation method according to claim 1, wherein The resulting antibody drug conjugates had a DAR value of 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 or 4.

5.

3. The preparation method according to claim 1, wherein The D4 content of the resulting ADCs was in the range of 55% to 65%.

4. The preparation method according to claim 1, wherein The reaction temperature in step (i) is 10.5°C to 11°C.

5. The preparation method according to any one of claims 1 to 4, wherein The molar ratio of the reducing agent to the antibody or antigen-binding fragment thereof is 2:1 to 3:

1.

6. The preparation method according to any one of claims 1 to 4, wherein The buffer is a histidine buffer.

7. The preparation method according to any one of claims 1 to 4, wherein The reaction temperature in step (ii) is 10.5° C. to 14° C., and the reaction time in step (ii) is 2 to 3 hours.

8. The preparation method according to any one of claims 1 to 4, wherein The ratio of the amount of linker-payload to the amount of antibody or antigen-binding fragment thereof in step (ii) is 4:1 to 6:

1.

9. The preparation method according to any one of claims 1 to 4, wherein The antibody or antigen-binding fragment thereof is an anti-TROP-2 antibody or antigen-binding fragment thereof.

10. The preparation method according to claim 9, wherein: The anti-TROP-2 antibody or its antigen-binding fragment comprises HCDR1 of the amino acid sequence shown in SEQ ID NO:1, HCDR2 of the amino acid sequence shown in SEQ ID NO:2, HCDR3 of the amino acid sequence shown in SEQ ID NO:3, LCDR1 of the amino acid sequence shown in SEQ ID NO:4, LCDR2 of the amino acid sequence shown in SEQ ID NO:5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

6.

11. The preparation method according to claim 10, wherein: The anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region whose amino acid sequence is at least 85% identical to the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region whose amino acid sequence is at least 85% identical to the amino acid sequence shown in SEQ ID NO:

8.

12. The preparation method according to claim 10 or 11, wherein: The anti-TROP-2 antibody comprises a heavy chain having an amino acid sequence at least 85% identical to the amino acid sequence shown in SEQ ID NO:9, and a light chain having an amino acid sequence at least 85% identical to the amino acid sequence shown in SEQ ID NO:10.

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