Preparation method for antibody-drug conjugate

Abstract

The present invention relates to a preparation method for an antibody-drug conjugate. The preparation method comprises: (i) reacting an antibody or an antigen-binding fragment thereof with a reducing agent in a buffer solution to reduce interchain disulfide bonds of the antibody or the antigen-binding fragment thereof; and (ii) reacting a linker-payload with the antibody or the antigen-binding fragment thereof with mercapto obtained in step (i), wherein the reaction temperature in step (i) is greater than 10 °C.

Description

Method for preparing antibody-drug conjugates Technical Field

[0001] The present disclosure 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 preparation method. Background Art

[0002] Antibody-Drug Conjugates (ADCs) are a class of drugs that combine the high specificity of therapeutic antibodies with the high cytotoxic activity of cytotoxic drugs. The therapeutic antibody and cytotoxic drug are connected by a linker. Compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells while reducing the impact on normal cells. Since the first antibody-drug conjugate, Mylotarg, was launched in the United States in 2000, at least ten ADCs have been launched globally. There are also reports of research on a variety of new antibody-drug conjugates in the anti-tumor field.

[0003] Antibodies typically have four interchain disulfide bonds, which serve as binding sites for the antibody and linker-payload. Methods for producing DAR4 antibody-drug conjugates exist, but some methods often result in high heterogeneity and low D4 content in the antibody-drug conjugates. Other methods also have harsh reaction conditions and the presence of toxic reagents. Therefore, there is a need to develop new methods for conjugating antibodies to cytotoxic drugs.

[0004] Summary of the Invention

[0005] Preparation method

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

[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 2.6:1. 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 foregoing 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, for example, 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 4.9:1. 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 acetone aqueous solution (e.g., 50% acetone aqueous solution), ethanol aqueous solution (e.g., 80% ethanol aqueous solution), methanol aqueous solution (e.g., 80% methanol aqueous solution), isopropanol aqueous solution (e.g., 80% isopropanol aqueous solution), dimethyl sulfoxide aqueous solution (e.g., 80% dimethyl sulfoxide aqueous solution), acetone, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA) or N-methyl-2-pyrrolidone (NMP). In some embodiments, the organic solvent as a solvent is acetone aqueous solution, preferably 50% acetone aqueous solution. In some embodiments, the organic solvent as a solvent is acetone. In some embodiments, the organic solvent as a solvent is DMSO aqueous 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 scope formed by above-mentioned arbitrary value.

[0023] In some embodiments, the reaction in step (ii) can be terminated by inactivating 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 antigen-binding fragment thereof 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 sulfhydryl 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, using a commercially available ultrafiltration membrane. 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, 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 solution to reduce 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] in,

[0030] 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;

[0031] The DAR value of the prepared antibody-drug conjugate is 3.5-4.5.

[0032] In some specific embodiments, the preparation method comprises:

[0033] (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, wherein the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride and the buffer is a histidine buffer. 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;

[0034] (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

[0035] in,

[0036] 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 molar ratio of the reducing agent to the antibody or antigen-binding fragment thereof in step (i) is 2:1 to 3:1, preferably 2.5:1;

[0037] In step (ii), the ratio of the amount of linker-payload to the amount of antibody or antigen-binding fragment thereof 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 in step (ii), before adding the solution containing the linker-payload, 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

[0038] 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.

[0039] The preparation method disclosed herein has mild reaction conditions, low cost, and is easy to operate.

[0040] The linker-payload is formed by connecting a cytotoxic drug to a 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, for example, 1 molecule of antibody or its antigen-binding fragment is connected to 0 (i.e., without cytotoxic drugs), 1, 2, 3, 4, 5, 6, 7, 8 or other 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.

[0041] 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 cytotoxic drug connections per molecule of antibody or its antigen-binding fragment in the antibody-drug conjugate. For example, "DAR of 4" refers to an antibody-drug conjugate comprising a heterogeneous mixture of cytotoxic drugs (e.g., each antibody or its antigen-binding fragment is connected to 0, 1, 2, 3, 4, 5, 6, 7, and / or 8 cytotoxic drugs) per molecule of antibody or its antigen-binding fragment, but the average number of cytotoxic drug connections per molecule of antibody or its antigen-binding fragment is 4. Similarly, "DAR of 8" refers to an antibody-drug conjugate in which the average number of cytotoxic drug connections per molecule of antibody or its antigen-binding fragment is 8.

[0042] D8 is excellent in terms of treatment (e.g., anti-tumor) effect, but in some cases 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 they are connected to 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 lower therapeutic effect than an antibody drug conjugate with a higher D4 content, and may exhibit strong toxicity. The antibody drug conjugate produced by the preparation method of the present invention 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.

[0043] 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:

[0044] Wherein, Ab is an antibody or an antigen-binding fragment thereof, n is selected from 3-5, R 1 selected from hydrogen atoms, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclic group, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl, R 2 selected from hydrogen atoms, optionally substituted C 1-6Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclic group, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl; or, R 1 and R 2 and the atoms to which they are attached form an optionally substituted 5- to 8-membered heterocyclic group; and the 3-position of -(succinimide-3-yl-N)- in Formula I is connected to Ab. In some embodiments, the R 1 and R 2 Each 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 of Formula I is connected to the sulfhydryl group of Ab after disulfide bond reduction. 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.

[0045] 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.

[0046] Wherein, Ab is an antibody or 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 of Formula I-1 is linked to the sulfhydryl group of Ab after disulfide bond reduction. 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.

[0047] Antibodies or antigen-binding fragments thereof

[0048] 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')2 fragments, Fd fragments, Fv fragments, dAbs, single-chain Fv (scFv) molecules, single-chain Fab (scFab), V HH. The antibody or its antigen-binding fragment may be any class among IgG, IgE, IgM, IgD or IgA, preferably IgG. In addition, the antibody or its antigen-binding fragment may be any subclass among 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, CLDN6, CLDN18.2, B7-H3, B7-H4, 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 antigen-binding fragment thereof, an anti-HER3 antibody or antigen-binding fragment thereof, an anti-EGFR antibody or antigen-binding fragment thereof, an anti-ROR1 antibody or antigen-binding fragment thereof, an antibody or antigen-binding fragment targeting CLDN6, an anti-CLDN18.2 antibody or antigen-binding fragment thereof, an anti-B7-H3 antibody or antigen-binding fragment thereof, an anti-B7-H4 antibody or antigen-binding fragment thereof, an anti-TROP-2 antibody or antigen-binding fragment thereof, an anti-CD20 antibody or antigen-binding fragment thereof, an anti-CD22 antibody or antigen-binding fragment thereof, an anti-CD30 antibody or antigen-binding fragment thereof Antigen-binding fragment, anti-CD33 antibody or antigen-binding fragment thereof, anti-CD47 antibody or antigen-binding fragment thereof, anti-CD56 antibody or antigen-binding fragment thereof, anti-CD70 antibody or antigen-binding fragment thereof, anti-CD79b antibody or antigen-binding fragment thereof, anti-VEGF antibody or antigen-binding fragment thereof, anti-VEGFR antibody or antigen-binding fragment thereof, anti-MUC1 antibody or antigen-binding fragment thereof, anti-c-MET antibody or antigen-binding fragment thereof, anti-RET antibody or antigen-binding fragment thereof, anti-LIV-1 antibody or antigen-binding fragment thereof, anti-PD-1 antibody or antigen-binding fragment thereof or anti-PD-L1 antibody or antigen-binding fragment thereof.

[0049] In some embodiments, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises:

[0050] (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;

[0051] (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;

[0052] (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;

[0053] (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, and LCDR3 described in (1) above; or

[0054] (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.

[0055] In some embodiments, the C-terminal lysine in the heavy chain constant region of the anti-TROP-2 antibody or antigen-binding fragment thereof may be present or absent. Deletion of the C-terminal lysine in the heavy chain constant region typically occurs during recombinant expression. In some embodiments, the C-terminal lysine in the amino acid sequence set forth in SEQ ID NO:9 is absent, as shown in SEQ ID NO:11.

[0056] 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.

[0057] The sequence information of exemplary anti-TROP-2 antibodies or antigen-binding fragments thereof disclosed herein is shown in Table S1 below.

[0058] Table S1. Sequence information of anti-TROP-2 antibodies or antigen-binding fragments thereof

[0059] Method for producing an antibody or antigen-binding fragment thereof having a thiol group

[0060] The present disclosure also provides a method for producing an antibody or an antigen-binding fragment thereof having a sulfhydryl 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.

[0061] In some embodiments, the reaction temperature is between 10.5°C and 20°C. In some embodiments, the reaction temperature is between 10.5°C and 15°C. In some embodiments, the reaction temperature is between 10.5°C and 14°C. In some embodiments, the reaction temperature is between 10.5°C and 11°C, 11°C and 14°C, or 11°C and 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 foregoing values.

[0062] 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 2.6:1. 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 foregoing values.

[0063] 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.

[0064] 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, for example, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM, preferably 20 mM.

[0065] 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 a range formed by any of the above values.

[0066] In some embodiments, the pH of the reaction is between 5 and 9. In some embodiments, the pH of the reaction is between 6 and 8. In some embodiments, the pH of the reaction is between 6.5 and 7.5. In some embodiments, the pH of the reaction is between 6.9 and 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 thereof.

[0067] 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.

[0068] 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%.

[0069] Cytotoxic drugs

[0070] 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.

[0071] 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 that act 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), tubulysin (Tubulysins), cryptomycin (Cryptomycins) and Eribulin (Eribulin) or its derivatives. In some embodiments, the cytotoxic drugs that act on DNA include but are not limited to calicheamicin (Calicheamicin) class, duocarmycin (Duocarmycin) class, anthramycin derivatives PBD (Pyrrolobenzodiazepine), topoisomerase I inhibitors and topoisomerase II inhibitors.

[0072] In some embodiments, the cytotoxic drug is a tubulin inhibitor.

[0073] In some embodiments, the cytotoxic drug is eribulin or a derivative thereof.

[0074] Connector-Payload

[0075] 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.

[0076] 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 being linked to the antibody or its antigen-binding fragment. The linker-payload used in the present disclosure is more preferably a compound represented by the structure of the following formula II:

[0077] Among them, R 1 selected from hydrogen atoms, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclic group, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl, R 2 selected from hydrogen atoms, optionally substituted C 1-6 Alkyl, optionally substituted C3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclic group, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl; or, R 1 and R 2 and the atoms to which they are attached form an optionally substituted 5- to 8-membered heterocyclyl. 1 and R 2 Each 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.

[0078] In some specific embodiments, the linker-payload is a compound represented by the following formula II-1:

[0079] It should be understood that the linker-payloads used in the present disclosure are 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.

[0080] Pharmaceutical composition

[0081] 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, a pharmaceutically acceptable salt of the antibody drug conjugate, 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.

[0082] use

[0083] The present disclosure also provides uses of the antibody-drug conjugate produced by the preparation method of the present disclosure or the pharmaceutical composition.

[0084] In one aspect, the present disclosure provides the use of an antibody-drug conjugate or pharmaceutical composition produced by the preparation method of the present disclosure in the preparation of a medicament for treating a disease expressing an antigen to which the antibody or antigen-binding fragment thereof binds. In some embodiments, the present disclosure provides the use of an antibody-drug conjugate or pharmaceutical composition produced by the preparation method of the present disclosure in the preparation of a medicament for treating a tumor. In some embodiments, the present disclosure provides the use of an antibody-drug conjugate or pharmaceutical composition produced by the preparation method of the present disclosure in the preparation of a medicament for treating an autoimmune disease.

[0085] In one aspect, the present disclosure provides a method for treating a disease that expresses an antigen to which the antibody or antigen-binding fragment thereof binds, 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.

[0086] In some embodiments, the tumor is a TROP-2 expressing tumor.

[0087] 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, urethra 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.

[0088] Explanation and Definition

[0089] Unless otherwise indicated, the following terms used in this disclosure have the following meanings. A particular term should not be construed as undefined or unclear unless specifically defined, but rather should be understood according to its ordinary meaning in the art. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0090] 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 compound after substitution is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are replaced, and oxo does not occur on an aromatic group. "Optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be any on the basis of chemically feasible.

[0091] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition on each occurrence is independent. Thus, for example, if a group is substituted with two R's, each R has an independent alternative.

[0092] The term "mercapto" refers to a -SH group.

[0093] As used herein, the structure of "-(succinimidyl-3-yl-N)-" is as follows:

[0094] As used herein, the structure of an "N-substituted maleimido group" is as follows:

[0095] Unless otherwise specified, use a solid wedge key. and dotted wedge key Indicates the absolute configuration of a stereocenter.

[0096] 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.

[0097] As used herein, a compound formed by replacing an atom or a group of atoms in a molecule of a parent compound with another atom or a group of atoms is referred to as a "derivative" of the parent compound.

[0098] The compounds of the present disclosure may exist in specific geometric isomer or stereoisomeric 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 enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present disclosure.

[0099] Unless otherwise indicated, the term "cis-trans isomers" or "geometric isomers" arises from the inability to rotate freely about double bonds or single bonds forming ring carbon atoms.

[0100] Unless otherwise indicated, the term "enantiomer" refers to stereoisomers that are mirror images of one another.

[0101] 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.

[0102] 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 the imidazole moiety, in which a proton can migrate between two ring nitrogens. Valence tautomers include interconversions by reorganization of some bonding electrons.

[0103] "Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine-hydrochloric acid buffer, histidine-acetate buffer, histidine-phosphate buffer, and the like. Histidine-hydrochloric acid buffer can be prepared using histidine (e.g., L-histidine) and further adjusted with hydrochloric acid for pH; or can be prepared using histidine (e.g., L-histidine) and histidine hydrochloride or a hydrate thereof (e.g., histidine hydrochloride monohydrate). Histidine-acetate buffer can be prepared using histidine (e.g., L-histidine) and further adjusted with acetic acid for pH. Histidine-phosphate buffer can be prepared using histidine (e.g., L-histidine) and further adjusted with phosphoric acid for pH.

[0104] The term "treatment" means administering a compound or pharmaceutical composition 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:

[0105] (i) preventing a disease or disease state from occurring in a mammal, particularly where such mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state;

[0106] (ii) inhibiting the disease or disease state, i.e., curbing its development;

[0107] (iii) alleviate the disease or condition, even if the disease or condition regresses;

[0108] (iv) reducing any direct or indirect pathological consequences of the disease or disease state.

[0109] The term "therapeutically effective amount" means an amount of a compound of the present disclosure that (i) treats or prevents a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. The amount of a compound of the present disclosure or pharmaceutical composition that constitutes a "therapeutically effective amount" may vary depending on 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. A therapeutically effective amount can also be routinely determined by one skilled in the art based on their own knowledge and this disclosure.

[0110] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, 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.

[0111] The term "pharmaceutically acceptable salt" refers to a salt of a compound (e.g., the antibody-drug conjugates of the present disclosure) that is safe and effective when used in mammals and has the desired biological activity. For example, the salt may be 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.

[0112] The term "excipient" refers to any ingredient other than the active ingredient (e.g., the 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.

[0113] The term "solvate" refers to an association of a compound with solvent molecules.

[0114] 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.

[0115] An "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) a Fab fragment: a monovalent fragment consisting of a VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of a VH and CH1 domain; (iv) an Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) a dAb fragment consisting of a VH domain (see Ward et al., Nature. 341: 544-546 (1989)); and (vi) a nanobody, 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 encompassed by 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.

[0116] 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.

[0117] 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 antibodies of the present disclosure may include amino acid residues that are 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.

[0118] Chimeric antibodies are antibodies created by fusing the variable region of a mouse antibody with the constant region of a human antibody. These antibodies can mitigate the immune response induced by mouse antibodies. To create chimeric antibodies, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies. The variable region genes are then cloned from the hybridoma cells. Furthermore, the constant region genes of human antibodies are cloned as needed. The mouse variable region genes and human constant region genes are then linked to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody is expressed in a eukaryotic or prokaryotic system.

[0119] 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.

[0120] The term “CDR” (complementarity determining region), also known as “hypervariable region.” Natural four-chain antibodies typically contain six CDRs, three in the heavy chain variable region and three in the light chain variable region.

[0121] The term "variable region" refers to the 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.

[0122] 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 aligned that are identical to the amino acid residues in the specific amino acid sequence shown in this article, after comparing the sequence to be aligned and, if necessary, introducing gaps to achieve maximum sequence identity, and not considering any conservative substitutions as part of sequence identity. The alignment of amino acid sequences for identity can be performed 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 aligning sequences, including any algorithm needed to obtain maximum alignment over the full length of the comparison sequence.

[0123] 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, cows, 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 thereof" include those who already have a disease or condition, those who are at risk of developing a disease or condition, and those who may develop a disease or condition and for which the purpose is to prevent, delay, or attenuate the disease or condition.

[0124] 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" can mean within 1 or more than 1 standard deviation as practiced in the art. Alternatively, "about" can mean a range of up to ±5%, for example, fluctuations within ±2%, within ±1%, or within ±0.5% of the specific numerical range given. 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.

[0125] 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.

[0126] Herein, singular terms encompass plural referents and vice versa unless the context clearly dictates otherwise. DETAILED DESCRIPTION

[0127] The present disclosure also provides the following specific implementation schemes, but the protection scope of the present disclosure is not limited thereto:

[0128] Embodiment 1. A method for preparing an antibody drug conjugate, comprising:

[0129] (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;

[0130] (ii) reacting the linker-payload with the antibody or antigen-binding fragment thereof having a thiol group obtained in step (i);

[0131] in,

[0132] The reaction temperature in step (i) is greater than 10°C.

[0133] Embodiment 2. The preparation method according to embodiment 1, wherein the DAR value of the produced antibody drug conjugate is 3-5.

[0134] Embodiment 3. The preparation method according to embodiment 2, wherein the DAR value of the produced antibody drug conjugate is 3.5-4.5.

[0135] 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.

[0136] 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.

[0137] 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%.

[0138] Embodiment 7. The preparation method according to embodiment 6, wherein the D4 content of the produced antibody drug conjugate is in the range of 50% to 80%.

[0139] Embodiment 8. The preparation method according to embodiment 7, wherein the D4 content of the produced antibody drug conjugate is in the range of 50% to 70%.

[0140] 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%.

[0141] 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.

[0142] Embodiment 11. The preparation method according to Embodiment 10, wherein the reaction temperature in step (i) is 10.5°C to 15°C.

[0143] Embodiment 12. The preparation method according to Embodiment 11, wherein the reaction temperature in step (i) is 10.5°C to 14°C.

[0144] Embodiment 13. The preparation method according to Embodiment 12, wherein the reaction temperature in step (i) is 10.5°C to 11°C.

[0145] Embodiment 14. The preparation method according to any one of Embodiments 1-13, wherein the molar ratio of the reducing agent to the antibody or antigen-binding fragment thereof is 1:1 to 4:1, 2:1 to 3:1, 2.4:1 to 2.6:1, or 2.4:1 to 2.5:1.

[0146] Embodiment 15. The preparation method according to any one of Embodiments 1-14, 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.

[0147] Embodiment 16. The preparation method according to Embodiment 15, wherein the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride.

[0148] 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.

[0149] Embodiment 18. The 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.

[0150] Embodiment 19. The 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.

[0151] Embodiment 20. The 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, 4.7:1 to 4.9:1, or 4.7:1 to 4.8:1.

[0152] 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.

[0153] Embodiment 22. The preparation method according to any one of Embodiments 1-21, 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-B7-H4 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 antibody or an antigen-binding fragment thereof targeting CLDN6, an anti-c-MET antibody or an antigen-binding fragment thereof, or an anti-LIV-1 antibody or an antigen-binding fragment thereof.

[0154] 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.

[0155] Embodiment 24. The method of 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 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.

[0156] Embodiment 25. The method of 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 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.

[0157] Embodiment 26. The preparation method of any one of Embodiments 1-25, wherein the linker-payload has an N-substituted maleimide group.

[0158] Embodiment 27. The preparation method according to embodiment 26, wherein the structure of the linker-payload is shown in the following formula II:

[0159] in,

[0160] R 1 selected from hydrogen atoms, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclic group, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl,

[0161] R 2 selected from hydrogen atoms, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclic group, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl;

[0162] 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.

[0163] 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.

[0164] 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.

[0165] Embodiment 30. The antibody drug conjugate according to embodiment 29, wherein the D4 content of the antibody drug conjugate is in the range of 50% to 90%.

[0166] Embodiment 31. The antibody drug conjugate according to embodiment 30, wherein the D4 content of the antibody drug conjugate is in the range of 50% to 80%.

[0167] Embodiment 32. The antibody drug conjugate according to embodiment 31, wherein the D4 content of the antibody drug conjugate is in the range of 50% to 70%.

[0168] Embodiment 33. The antibody drug conjugate according to embodiment 32, wherein the D4 content of the antibody drug conjugate is in the range of 50% to 60%, 60% to 70%, or 55% to 65%.

[0169] Embodiment 34. The 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 antigen-binding fragment thereof, an anti-HER2 antibody or antigen-binding fragment thereof, an anti-HER3 antibody or antigen-binding fragment thereof, an anti-ROR1 antibody or antigen-binding fragment thereof, an anti-B7-H3 antibody or antigen-binding fragment thereof, an anti-B7-H4 antibody or antigen-binding fragment thereof, an anti-CD79b antibody or antigen-binding fragment thereof, an anti-CLDN18.2 antibody or antigen-binding fragment thereof, an antibody or antigen-binding fragment targeting CLDN6, an anti-c-MET antibody or antigen-binding fragment thereof, or an anti-LIV-1 antibody or antigen-binding fragment thereof.

[0170] Embodiment 35. The antibody-drug conjugate of Embodiment 34, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 1, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 3, a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 4, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 5, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 6.

[0171] 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.

[0172] 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.

[0173] 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:

[0174] in,

[0175] Ab is an antibody or its antigen-binding fragment,

[0176] n is selected from 3-5, preferably 3.5-4.5,

[0177] R 1 selected from hydrogen atoms, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted 3 to 7 membered heterocyclic group, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl,

[0178] R 2 selected from hydrogen atoms, optionally substituted C 1-6 Alkyl, optionally substituted C 3-7cycloalkyl, optionally substituted 3 to 7 membered heterocyclic group, optionally substituted C 6-10 aryl, optionally substituted 5- to 12-membered heteroaryl;

[0179] 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

[0180] The 3-position of -(succinimidyl-3-yl-N)- in Formula I is linked to Ab.

[0181] Embodiment 39. The antibody drug conjugate according to embodiment 38, wherein the R 1 With R 2 are independently selected from hydrogen, methyl, ethyl, propyl or isopropyl; preferably, R 1 and R 2 A hydrogen atom.

[0182] 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 its pharmaceutically acceptable salt; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0183] 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 tumors.

[0184] 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.

[0185] 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.

[0186] Embodiment 44. The method of 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.

[0187] Embodiment 45. A method for producing an antibody or antigen-binding fragment thereof having a sulfhydryl group, comprising:

[0188] 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, wherein the reaction temperature is greater than 10° C.;

[0189] The resulting 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.

[0190] Embodiment 46. The method according to embodiment 45, wherein the D4 content of the antibody drug conjugate is in the range of 50% to 90%.

[0191] Embodiment 47. The method according to embodiment 46, wherein the D4 content of the antibody drug conjugate is in the range of 50% to 80%.

[0192] 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%.

[0193] Embodiment 49. The method according to embodiment 48, wherein the D4 content of the antibody drug conjugate is in the range of 50% to 60%, 60% to 70%, or 55% to 65%.

[0194] Embodiment 50. The method of any one of Embodiments 45-49, wherein the reaction temperature is 10.5°C to 20°C.

[0195] Embodiment 51. The method of Embodiment 50, wherein the reaction temperature is 10.5°C to 15°C.

[0196] Embodiment 52. The method of Embodiment 51, wherein the reaction temperature is 10.5°C to 14°C.

[0197] Embodiment 53. The method of Embodiment 52, wherein the reaction temperature is 10.5°C to 11°C.

[0198] Embodiment 54. The method of any one of Embodiments 45-53, wherein the ratio of the amount of reducing agent to the amount of the antibody or antigen-binding fragment thereof is 1:1 to 4:1, 2:1 to 3:1, 2.4:1 to 2.6:1, or 2.4:1 to 2.5:1.

[0199] 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.

[0200] Embodiment 56. The method of embodiment 55, wherein the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride.

[0201] Embodiment 57. The 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.

[0202] 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.

[0203] Embodiment 59. The method according to any one of Embodiments 45-58, wherein the antibody or antigen-binding fragment thereof is an anti-TROP-2 antibody or antigen-binding fragment thereof, an anti-HER2 antibody or antigen-binding fragment thereof, an anti-HER3 antibody or antigen-binding fragment thereof, an anti-ROR1 antibody or antigen-binding fragment thereof, an anti-B7-H3 antibody or antigen-binding fragment thereof, an anti-B7-H4 antibody or antigen-binding fragment thereof, an anti-CD79b antibody or antigen-binding fragment thereof, an anti-CLDN18.2 antibody or antigen-binding fragment thereof, an antibody or antigen-binding fragment targeting CLDN6, an anti-c-MET antibody or antigen-binding fragment thereof, or an anti-LIV-1 antibody or antigen-binding fragment thereof.

[0204] Embodiment 60. The method of embodiment 59, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 1, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 3, a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 4, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 5, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 6.

[0205] Embodiment 61. The method of 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.

[0206] 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.

[0207] 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.

[0208] The anti-TROP-2 antibody hz-Ab35-1.3 used in the examples and its preparation method are derived from patent application document WO2023104080. Its heavy chain amino acid sequence is shown in SEQ ID NO: 70 of WO2023104080, and its light chain amino acid sequence is shown in SEQ ID NO: 71 of WO2023104080. The amino acid sequence of HCDR1 of the anti-TROP-2 antibody hz-Ab35-1.3 used in the examples is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6. The amino acid sequence of the heavy chain of the anti-TROP-2 antibody hz-Ab35-1.3 is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain is shown in SEQ ID NO: 10.

[0209] The linker-payload MC-GGFG-eribulin and its preparation method used in the embodiment are derived from patent application document WO2023041006, and its structure is shown in Formula II-1 in the present disclosure.

[0210] Example 1: Preparation of Antibody Drug Conjugates

[0211] Reagents:

[0212] The antibody is hz-Ab35-1.3, and the linker-payload is MC-GGFG-eribulin.

[0213] Experimental process:

[0214] (1) The concentration of antibody hz-Ab35-1.3 was adjusted to approximately 10 g / L using 20 mM histidine-hydrochloric acid buffer (containing 1.43 mg / mL L-histidine and 2.27 mg / mL L-histidine hydrochloride monohydrate, pH 6.0). The pH was adjusted to 7.0 using a 0.3 M Na2HPO4 aqueous solution, and sucrose was added to a sucrose concentration of 5% (w / v). The temperature of the 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 a molar ratio of TCEP·HCl to antibody of 2.5:1. The reaction was incubated at 10.5-14.0°C and stirred at 20-100 rpm in the dark for 2.5 hours.

[0215] (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 incubated at 10.5-14.0°C and 20-100 rpm in the dark for 2 hours.

[0216] (3) Add 10 mM N-acetylcysteine ​​at a molar ratio of 4:1 to the antibody, and allow to react for 25 to 40 minutes. The reaction solution is then filtered into 20 mM histidine-hydrochloric acid buffer (pH 6.0) using a 30 kDa ultrafiltration membrane to obtain an antibody-drug conjugate.

[0217] According to the specific conditions in Table 1 and Table 2, antibody drug conjugates ADC1-ADC6 were synthesized, and the structures are as follows:

[0218] The distribution of the number of cytotoxic drugs and the average number of connections (n) per antibody molecule of ADC1-ADC6 were measured using the method of Example 2. The specific results are shown in Table 3.

[0219] Table 1. Specific parameters of step (1)

[0220] Table 2. Specific parameters of step (2)

[0221] Table 3. Peak area percentages containing D0-D8 and DAR values ​​(i.e., n) of ADCs Note: The peak area percentages of D1, D7 and D8 are all 0%.

[0222] Example 2: Determination of DAR value of antibody drug conjugates

[0223] The DAR value of the antibody drug conjugate was determined by hydrophobic interaction chromatography (HIC). 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 substance was then eluted by gradually decreasing the salt concentration and increasing the proportion of isopropanol. The less hydrophobic molecules were eluted first, and the more hydrophobic molecules were eluted later.

[0224] The antibody-drug conjugate components were separated using a butyl-bonded, non-porous PS / DVB packing. The column specifications were Sepax, Proteomix HIC Butyl-NP5 (5μm, 4.6mm×100mm), and the column temperature was 25°C. Mobile phase A consisted of 1.5M ammonium sulfate-50mM phosphate buffer (pH 7.0):isopropanol (95:5 v / v). (Weigh 188.30g of ammonium sulfate and 7.41g of sodium dihydrogen phosphate dihydrate, add 750mL of ultrapure water, stir until fully dissolved, adjust the pH to 7.0 with 50% sodium hydroxide solution, add ultrapure water to 950mL, then add 50mL of isopropanol, mix thoroughly, and filter through a 0.22μm filter. Mobile phase B consisted of 50 mM phosphate buffer (pH 7.0): isopropanol (80:20 v / v). (Weigh 6.24 g of sodium dihydrogen phosphate dihydrate, add 750 mL of ultrapure water, stir until fully dissolved, adjust the pH to 7.0 with 50% sodium hydroxide solution, add ultrapure water to 800 mL, add 200 mL of isopropanol, mix thoroughly, and filter through a 0.22 μm filter. The ADC sample was diluted with ultrapure water to approximately 2.0 mg of protein per mL as the test solution. 20 μg of protein was injected and detected at a wavelength of 214 nm. The flow rate was 1.0 mL / min, and the gradient elution parameters were shown in Table 4.

[0225] Table 4. Gradient elution parameters

[0226] Data processing and quantitative analysis of the results were performed using the area normalization method. The peak area percentages of D0, D1, D2, D3, D4, D5, D6, D7, and D8 were calculated, and the DAR value was calculated. 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%.

[0227] Example 3: Preparation of Antibody Drug Conjugates and Determination of DAR Values

[0228] Reagents:

[0229] The antibody is an anti-ROR1 antibody, and the linker-payload is MC-GGFG-eribulin.

[0230] Experimental process:

[0231] (1) The antibody concentration was adjusted to approximately 10.0 g / L using 20 mM histidine-hydrochloric acid buffer (containing 1.43 mg / mL L-histidine and 2.27 mg / mL L-histidine hydrochloride monohydrate, pH 6.0). The pH was adjusted to approximately 7.0 using 0.3 M Na2HPO4 aqueous solution. The temperature was adjusted to 11.0°C. 10 mM TCEP·HCl aqueous solution was then added to adjust the molar ratio of TCEP·HCl to antibody to 2.6:1. The reaction was carried out in the dark for 1 to 4 hours while stirring at 20 to 100 rpm and maintaining the adjusted temperature.

[0232] (2) DMSO was added to a final concentration of 5% (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 4.85:1. The reaction was incubated at 11°C and stirred at 20-100 rpm in the dark for 1-4 hours.

[0233] (3) Add 10 mM N-acetylcysteine ​​at a molar ratio of 4:1 to the antibody, and allow to react for 25 to 40 minutes. The reaction solution is then filtered into 20 mM histidine-hydrochloric acid buffer (pH 6.0) using a 30 kDa ultrafiltration membrane to obtain an antibody-drug conjugate.

[0234] According to the specific conditions in Table 5 and Table 6, the antibody drug conjugate ADC7-ADC14 was synthesized, and the structure is as follows:

[0235] The distribution of the number of cytotoxic drugs and the average number of connections (n) per antibody molecule of ADC7-ADC14 were measured using the method of Example 2. The specific results are shown in Table 7.

[0236] Table 5. Specific parameters of step (1)

[0237] Table 6. Specific parameters of step (2)

[0238] Table 7. Peak area percentages containing D0-D8 and DAR values ​​(i.e., n) of ADCs Note: The peak area percentages of D1, D3, D5 and D8 are all 0%.

[0239] Example 4: Preparation of Antibody Drug Conjugates and Determination of DAR Values

[0240] Reagents:

[0241] The antibody is an anti-B7-H3 antibody, and the linker-payload is MC-GGFG-eribulin.

[0242] Experimental process:

[0243] (1) The antibody concentration was adjusted to approximately 9.0 g / L using 20 mM histidine-hydrochloric acid buffer (containing 1.43 mg / mL L-histidine and 2.27 mg / mL L-histidine hydrochloride monohydrate, pH 6.0). The pH was adjusted to 7.0 using a 0.3 M Na2HPO4 aqueous solution, and sucrose was added to a sucrose concentration of 5% (w / v). The temperature was adjusted to 11.0°C, and a 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution was added to a molar ratio of TCEP·HCl to antibody of 2.4:1. The reaction was maintained at the adjusted temperature and stirred at 20-100 rpm in the dark for 2.5 hours.

[0244] (2) DMSO was added to a final concentration of 5% (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 incubated at 11.0°C with stirring at 20-100 rpm for 2 hours in the dark.

[0245] (3) 10 mM N-acetylcysteine ​​was added at a molar ratio of 4:1 to the antibody, and the reaction was allowed to proceed for 25 to 40 minutes. The reaction solution was then replaced with a 20 mM histidine-hydrochloric acid buffer (pH 6.0) using a 30 kDa ultrafiltration membrane to obtain ADC15. The DAR value of ADC15 was measured using the method of Example 2 and was found to be 4.24. The distribution of the amount of cytotoxic drug per antibody molecule is shown in Table 8.

[0246] The structure of ADC15 is as follows:

[0247] Table 8. Peak area percentages containing D0-D8 and DAR values ​​(i.e., n) of ADCs

[0248] Note: The peak area percentages of D1 and D7 are both 0%.

[0249] Example 5: Preparation of Antibody Drug Conjugates and Determination of DAR Values

[0250] Reagents:

[0251] The antibody is hz-Ab35-1.3, and the linker-payload is MC-GGFG-eribulin.

[0252] Experimental process:

[0253] (1) The concentration of the antibody hz-Ab35-1.3 was adjusted to approximately 10 g / L using a 20 mM histidine-hydrochloric acid buffer (containing 1.43 mg / mL L-histidine and 2.27 mg / mL L-histidine hydrochloride monohydrate, pH 6.0). The pH was adjusted to 7.0 using a 0.3 M Na2HPO4 aqueous solution, and sucrose was added thereto to a sucrose concentration of 5% (w / v). The temperature of the solution was adjusted to 4.0°C, 20.0°C, or 25.0°C, and a 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution was added to a molar ratio of TCEP·HCl to the antibody of 2.5:1. The reaction was maintained at the adjusted temperature and stirred at 20 to 100 rpm in the dark for 2.5 hours.

[0254] (2) DMSO was added to a final concentration of 4% (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 maintained at 4.0°C, 20.0°C, or 25.0°C and stirred at 20-100 rpm in the dark for 2 hours.

[0255] (3) Add 10 mM N-acetylcysteine ​​at a molar ratio of 4:1 to the antibody, and allow to react for 25 to 40 minutes. The reaction solution is then filtered into 20 mM histidine-hydrochloric acid buffer (pH 6.0) using a 30 kDa ultrafiltration membrane to obtain an antibody-drug conjugate.

[0256] According to the specific conditions in Tables 9 and 10, antibody-drug conjugate ADC16-ADC18 was synthesized, and the structure is as follows:

[0257] The distribution of the number of cytotoxic drugs and the average number of connections (n) per antibody molecule of ADC16, ADC17, and ADC18 were measured. The specific results are shown in Table 11.

[0258] Table 9. Specific parameters of step (1)

[0259] Table 10. Specific parameters of step (2)

[0260] Table 11. Peak area percentages containing D0-D8 and DAR values ​​(i.e., n) of ADCs Note: The peak area percentages of D1 and D8 are both 0%.

[0261] Example 6: Preparation of Antibody Drug Conjugates and Determination of DAR Values

[0262] Reagents:

[0263] The antibody is an antibody targeting CLDN6, and the linker-payload is MC-GGFG-eribulin.

[0264] Experimental process:

[0265] (1) The antibody concentration was adjusted to approximately 10.0 g / L using 20 mM histidine-hydrochloric acid buffer (containing 1.43 mg / mL L-histidine and 2.27 mg / mL L-histidine hydrochloride monohydrate, pH 6.0), the pH was adjusted to approximately 7.0 using 0.3 M Na2HPO4 aqueous solution, the temperature was adjusted to 10.5-14.0°C, and a 10 mM TCEP·HCl aqueous solution was added to adjust the molar ratio of TCEP·HCl to antibody to 2.4:1 to 2.6:1. The reaction was carried out in the dark for 1-4 hours while maintaining the adjusted temperature and stirring at 20-100 rpm.

[0266] (2) DMSO was added to a final concentration of about 2-5% (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 about 4.8:1. The reaction was carried out in the dark for 1-4 hours while maintaining the temperature adjusted in step (1) and stirring at 20-100 rpm.

[0267] (3) 10 mM N-acetylcysteine ​​was added at a molar ratio of 4:1 to the antibody, and the mixture was allowed to react for 25 to 40 minutes. The reaction solution was then replaced with 20 mM histidine-hydrochloric acid buffer (pH 6.0) using a 30 kDa ultrafiltration membrane to obtain an antibody-drug conjugate targeting CLDN6, the structure of which is shown below:

[0268] For the purposes of description and disclosure, all patents, patent applications, and other identified publications are expressly incorporated herein by reference. These publications are provided solely for their disclosure prior to the filing date of the present disclosure. All statements regarding the dates of these documents or representations of their contents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents. Furthermore, any citation of these publications herein does not constitute an admission that the publications form part of the common general knowledge in the art, in any country.

[0269] Although the present disclosure has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications or improvements may be made based on this disclosure. Therefore, such modifications or improvements, which do not depart from the spirit of this disclosure, are within the scope of protection claimed by this 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 interchain disulfide bonds of the antibody or antigen-binding fragment thereof; (ii) reacting the linker-payload with the antibody or antigen-binding fragment thereof having a thiol group obtained in step (i); in, The reaction temperature in step (i) is greater than 10°C.

2. The preparation method according to claim 1, wherein The resulting antibody-drug conjugates had a DAR value of 3-5; Preferably, the DAR value of the antibody drug conjugate produced is 3.5-4.5; Preferably, the antibody drug conjugate produced 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.

3. The preparation method according to claim 1 or 2, wherein The resulting antibody drug conjugate has a D4 content of 50% or more, 55% or more, 60% or more, or 65% or more; Preferably, the D4 content of the produced antibody drug conjugate is in the range of 50% to 90%; Preferably, the D4 content of the produced antibody drug conjugate is in the range of 50% to 80%; Preferably, the D4 content of the produced antibody drug conjugate is in the range of 50% to 70%; Preferably, the D4 content of the produced antibody drug conjugate is in the range of 50% to 60%, 60% to 70%, or 55% to 65%.

4. The preparation method according to any one of claims 1 to 3, wherein The reaction temperature in step (i) is 10.5°C to 20°C; Preferably, the reaction temperature in step (i) is 10.5°C to 15°C; Preferably, the reaction temperature in step (i) is 10.5°C to 14°C; Preferably, 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 1:1 to 4:1, 2:1 to 3:1, 2.4:1 to 2.6:1, or 2.4:1 to 2.5:

1.

6. The preparation method according to any one of claims 1 to 5, 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; Preferably, the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride.

7. The preparation method according to any one of claims 1 to 6, wherein The buffer is HEPES buffer, histidine buffer, phosphate buffer, borate buffer or acetate buffer, preferably histidine buffer.

8. The preparation method according to any one of claims 1 to 7, 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.

9. The preparation method according to any one of claims 1 to 8, 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.

10. The preparation method according to any one of claims 1 to 9, 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, 4.7:1 to 4.9:1, or 4.7:1 to 4.8:

1.

11. The preparation method according to any one of claims 1 to 10, wherein The reaction time in step (ii) is 1 to 5 hours, 2 to 4 hours, or 2 to 3 hours.

12. The preparation method according to any one of claims 1 to 11, wherein The antibody or its antigen-binding fragment 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 antibody or an antigen-binding fragment targeting CLDN6, 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.

13. The preparation method according to claim 12, 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; Preferably, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region whose amino acid sequence 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 heavy chain variable region whose amino acid sequence 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. a light chain variable region that is 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; Preferably, 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.

14. The preparation method according to any one of claims 1 to 13, wherein The linker-payload has an N-substituted maleimido group; Preferably, the structure of the linker-payload is shown in Formula II below: in, 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 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; Optionally, R 1 and R 2 together with the atoms to which they are attached, form an optionally substituted 5- to 8-membered heterocyclyl; Preferably, 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.

15. A method for producing an antibody or an antigen-binding fragment thereof having a thiol group, comprising: 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; The produced antibody or antigen-binding fragment thereof having a thiol group is used to prepare an antibody-drug conjugate, 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; Preferably, the content of D4 in the antibody drug conjugate is in the range of 50% to 90%; Preferably, the content of D4 in the antibody drug conjugate is in the range of 50% to 80%; Preferably, the content of D4 in the antibody drug conjugate is in the range of 50% to 70%; Preferably, the content of D4 in the antibody drug conjugate is in the range of 50% to 60%, 60% to 70%, or 55% to 65%.

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