Methods for preparing antibodies with site-specific modifications

Abstract

This application describes a method for preparing an antibody with site-specific modifications. In this application, three of the four interchain disulfide bonds of the antibody are first selectively reduced. Through site-specific modification of the antibody, this application provides many types of ADCs, such as ADCs with D2, ADCs with D1, ADCs with D4, ADCs with D6, ADCs with D3, ADCs with D0+D2, ADCs with D0+D1, bi-payload ADCs with D1+D6, bi-payload ADCs with D1+D2, bi-payload ADCs with D1+D4, bi-payload ADCs with D2+D4, bi-payload ADCs with D6+D2, bi-payload ADCs with D6+D1, bi-payload ADCs with D3+D1, bi-payload ADCs with D3+D2, bi-payload ADCs with D1+D2, bi-payload ADCs with D1+D4, or bi-payload ADCs with D2+D4. Compared with conventional conjugation methods, the homogeneity of antibodies obtained by site-specific modification using the method of the present application can be dramatically improved to 55%, 65%, 70%, 80%, 85% or more, and even 90% or 95%. Meanwhile, the method is simple to operate, does not require antibody or enzyme engineering, and reduces costs.

Description

[Technical Field]

[0001] This application relates to methods for preparing antibodies with site-specific modifications. Specifically, this application relates to a bioconjugation process for preparing ADCs with improved homogeneity. [Background technology]

[0002] The statements in this section provide background information related to the present disclosure and may not necessarily constitute prior art.

[0003] Antibody-drug conjugates (ADCs) are engineered antibodies in which a monoclonal antibody is linked to a small molecule drug via a stable linker. ADCs ideally combine the specificity of antibodies with the potent activity of cytotoxic drugs by delivering potent cytotoxic drugs to antigen-expressing cells and enhancing their targeted cytotoxicity. In contrast to conventional chemotherapy drugs, ADCs target only antigen-expressing cancer cells, sparing healthy cells. (Pettinato, Mark C. (2021) “Introduction to Antibody-Drug Conjugates.” Antibodies (Basel, Switzerland) 10(42):42-52, Joubert N, Beck A, Dumontet C, Denevault-Sabourin C. (2020) “Antibody-Drug Conjugates: The Last Decade.” Pharmaceuticals (Basel). 13(9):245-275.) Antibody-drug conjugates (ADCs) are expected to have a wide range of therapeutic applications, especially in the field of cancer, and are becoming new targeted drugs for disease treatment. Since the approval of Myloturk in 2000, 14 ADCs have been approved by the U.S. Food and Drug Administration (FDA).

[0004] For drug attachment to ADCs, reactive functional groups on both the antibody and the linker payload (i.e., linker drug) are used in the conjugate to form a stable covalent bond. Traditional conjugation, i.e., covalently attaching a drug moiety to an antibody via a linker, typically results in a heterogeneous mixture of molecules in which the drug moiety is attached to multiple sites on the antibody. For example, ADCs have typically been generated using two traditional chemical strategies: lysine conjugation and cysteine conjugation via reduction of interchain disulfide bonds. Cysteine conjugation via reduction of interchain disulfide bonds involves reducing the interchain disulfide bonds in the presence of various reducing agents, followed by nucleophilic reaction of thiol groups. In this conjugation process, ADCs are typically formed by attaching one or more antibody cysteine thiol groups to one or more linker payload moieties, thereby generating a heterogeneous antibody-drug conjugate mixture (e.g., Adcetris) in which the drug moiety is attached to multiple sites on the antibody. For antibody-drug conjugates (ADCs) with a drug-antibody ratio (DAR) of around 4, the heterogeneous mixture typically contains a distribution of antibodies with drug moieties conjugated ranging from 0 to approximately 8 or more. Furthermore, within each subgroup of conjugates with a specific integer ratio of drug moieties to a single antibody, there exists a potentially heterogeneous mixture in which the drug moieties are conjugated to various sites on the antibody. Because this heterogeneous mixture is highly complex, each conjugate may have different pharmacokinetic, toxicity, and efficacy profiles. Meanwhile, their characterization and purification are difficult and expensive. Because traditional nonspecific binding and conjugate distribution are significantly affected by factors such as pH, temperature, concentration, salt concentration, and cosolvents, establishing a robust conjugation process is always challenging.

[0005] Many methods have been developed to improve the homogeneity of ADCs. For example, Genentech's THIOMAB technology was developed to improve the homogeneity of ADCs through antibody engineering by introducing cysteine residues into the primary antibody sequence, enabling site-specific conjugation. This improves product homogeneity ("Cysteine-Based Coupling: Challenges and Solutions". Bioconjug Chem. 2021 Aug 18;32(8):1525-1534.).

[0006] US20210040145 discloses a 14-amino acid peptide, Tub-tag, that can be used at the C-terminus of any POI to catalyze the addition of a variety of different tyrosine derivatives. By utilizing this enzyme, Tub-tag technology reuses tubulin tyrosine ligase to attach functional moieties to the C-terminus of antibodies, resulting in the uniform generation of antibody conjugates bearing DAR 2.

[0007] However, these techniques involve protein engineering or enzyme catalysis, which have drawbacks such as low antibody expression levels, immunogenicity risk, complicated purification, and / or high costs. Meanwhile, there is no effective chemical method for generating ADCs with high D6 selectivity.

[0008] Therefore, there is a need to develop novel bioconjugation processes that can produce highly homogeneous ADCs. Summary of the Invention

[0009] This application describes a method for generating antibodies with site-specific modifications. Site-specific antibody modifications involve selective reduction of three of the four interchain disulfide bonds between the antibody and the target molecule. This allows the application to provide a wide variety of highly homogeneous ADCs, such as ADCs with D2, D1, D4, D6, D3, D1+D6, D2+D4, D1+D2, D1+D4, D0+D2, D0+D1, bi-payload ADCs with D6+D2, D6+D1, bi-payload ADCs with D3+D2, D3+D1, bi-payload ADCs with D1+D2, bi-payload ADCs with D1+D4, or bi-payload ADCs with D2+D4. Compared with conventional conjugation processes, the homogeneity of ADCs can reach up to 55%, 65%, 70%, 80%, 85%, and even 90% or 95%. Furthermore, by increasing the molar ratio of TCEP to antibody, this method can reduce the cost of reduction time. Meanwhile, this method is simple to operate, does not require antibody or enzyme engineering, and also reduces costs. The ADCs with improved homogeneity produced by this method are also optimized for safety and efficacy.

[0010] In one aspect, the present application provides a method for preparing an antibody having a site-specific modification, wherein the site-specific modification is selective reduction of three interchain disulfide bonds in the antibody, the method comprising using tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof in combination with a transition metal ion.

[0011] In a second aspect, the present application provides a method for preparing an antibody having a site-specific modification, wherein the site-specific modification is selective reduction of three interchain disulfide bonds in the antibody, two of which are in the Fab region and one of which is in the hinge region of the antibody, the method comprising using TCEP or a salt thereof and a transition metal ion together.

[0012] In a third aspect, the present application provides a method for preparing an antibody having a site-specific modification, wherein the site-specific modification is selective reduction of three interchain disulfide bonds in the antibody, two of which are in the Fab region and one of which is in the hinge region of the antibody, the method comprising the steps of: (a) Incubating TCEP or its salt with transition metal ions in a buffer system in the presence of an antibody to selectively reduce the interchain disulfide bonds of the antibody to obtain an antibody having reduced thiol groups, at a molar ratio of TCEP to antibody of 3:1-15:1, optionally at a molar ratio of TCEP to antibody of 3:1-6:1.

[0013] In a fourth aspect, the present application provides a method for preparing an antibody with a site-specific modification, the method comprising the method of the present application and further comprising the steps of: (B1) introducing an oxidizing agent to selectively reoxidize the reduced thiol groups resulting from step (a), optionally reoxidizing the reduced thiol groups within the Fab region, and preferably purifying the oxidation product by removing excess oxidizing agent; (C1) Introduce a metal chelator and modifying reagent 1 to react with the remaining thiol groups resulting from step (B1), where modifying reagent 1 is an end-capping reagent, a first linker payload, or a first thio bridging reagent, and optionally, the first thio bridging reagent has a first linker payload or a reactive group.

[0014] In a fifth aspect, the present application provides a method for preparing an antibody with a site-specific modification, the method comprising the method of the present application and further comprising the steps of: (b) introducing a metal chelating agent and modifying reagent 1 to react with the reduced thiol groups obtained in step (a);

[0015] In a sixth aspect, the present application provides an antibody having a site-specific modification prepared by the method of the present application.

[0016] In a seventh aspect, the present application provides a pharmaceutical composition comprising an antibody having a site-specific modification according to the present application and one or more pharmaceutically acceptable carriers.

[0017] In an eighth aspect, the present application provides the use of TCEP or a salt thereof in the preparation of an antibody having a site-specific modification according to the present application.

[0018] In a ninth aspect, the present application provides the use of an antibody having a site-specific modification according to the present application in the manufacture of a therapeutic agent for diagnosing, preventing or treating a disease.

[0019] In a tenth aspect, the present application provides a method for preventing or treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody having a site-specific modification according to the present application. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows HIC-HPLC (hydrophobic interaction chromatography-high performance liquid chromatography) of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 1. [Figure 2] FIG. 2 shows HIC-HPLC of the sacituzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 2. [Figure 3] FIG. 3 shows HIC-HPLC of the belantamab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 3. [Figure 4] Figure 4 AD shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugates of Examples 4-7 (TCEP to antibody molar ratios of 3:1, 3.2:1, 5:1, and 6:1). [Figure 5] Figure 5A-F shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates of Examples 8-13 (TCEP to antibody molar ratios of 8:1, 9:1, 10:1, 11:1, 12:1, and 13:1). [Figure 6]FIG. 6 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 14 (molar ratio of Zn2+ to TCEP is 0.25:1). [Figure 7] FIG. 7 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 15 (molar ratio of Zn2+ to TCEP is 0.5:1). [Figure 8] FIG. 8 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 16 (molar ratio of Zn2+ to TCEP is 1:1). [Figure 9] FIG. 9 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 17 (molar ratio of Zn2+ to TCEP is 2:1). [Figure 10] FIG. 10 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 18 (molar ratio of Zn2+ to TCEP is 3:1). [Figure 11] FIG. 11 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 19 (molar ratio of Zn2+ to TCEP is 4:1). [Figure 12] FIG. 12 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 20 (molar ratio of Zn2+ to TCEP is 7.5:1). [Figure 13] Figure 13 AD shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates of Examples 21-24 (Zn2+ to TCEP molar ratios of 12:1, 27.27:1, 0.11:1, and 0.22:1). [Figure 14] Figure 14 AD shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates of Examples 25-28 (Zn2+ to TCEP molar ratios of 0.44:1, 0.66:1, 0.88:1, and 1.67:1). [Figure 15]Figure 15 AD shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Comparative Example 1-4 (the molar ratio of Zn2+ to TCEP is 0). [Figure 16] Figure 16A-D shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugates of Examples 29 to 32 (different incubation times in step (1)). [Figure 17] Figure 17A and 17B show HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugates of Examples 33 to 36 (different incubation temperatures in step (1)). [Figure 18] FIG. 18 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using Bis-Tris buffer (pH 6.7) of Example 37. [Figure 19] FIG. 19 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using PIPES buffer (pH value 6.7) of Example 38. [Figure 20] FIG. 20 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using the MOPS buffer of Example 39 (pH value 6.7). [Figure 21] FIG. 21 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using the BES buffer of Example 40 (pH value 6.7). [Figure 22] FIG. 22 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using HEPES buffer (pH value 6.7) of Example 41. [Figure 23] FIG. 23 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using DIPSO buffer (pH value 7.4) of Example 42. [Figure 24]FIG. 24 shows the MOBS of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using the MOBS buffer of Example 43 (pH value 7.4). [Figure 25] FIG. 25 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using the MOPSO buffer of Example 44 (pH value 7.4). [Figure 26] FIG. 26 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using TES buffer (pH value 7.4) of Example 45. [Figure 27] FIG. 27 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using ACES buffer (pH value 7.4) of Example 46. [Figure 28] FIG. 28 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using TAPSO buffer (pH value 7.4) of Example 47. [Figure 29] FIG. 29 shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using PB (pH value 6.7) of Comparative Example 5. [Figure 30] FIG. 30 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using the ADA buffer of Comparative Example 6 (pH value 6.7). [Figure 31] FIG. 31 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using BES buffer (pH value 6.4) of Example 48. [Figure 32] FIG. 32 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using BES buffer (pH value 6.7) of Example 49. [Figure 33] FIG. 33 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using the BES buffer of Example 50 (pH value 7.0). [Figure 34] FIG. 34 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using BES buffer (pH value 6.4) of Example 51. [Figure 35] FIG. 35 shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared with 20 mM BES buffer from Example 52. [Figure 36] FIG. 36 shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using 40 mM BES buffer in Example 53. [Figure 37] FIG. 37 shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared with 60 mM BES buffer in Example 54. [Figure 38] FIG. 38 shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared with 80 mM BES buffer from Example 55. [Figure 39] FIG. 39 shows HIC-HPLC of the trastuzumab-[bismaleimide-DBCO]3 conjugate of Example 56. [Figure 40] Figure 40A shows HIC-HPLC of the trastuzumab-[MC-GGFG-DXd]6 conjugate of Example 57. Figure 40B shows HIC-HPLC of the trastuzumab-[MC-GGFG-DXd6[MC-VC-PAB-MMAE]2 conjugate of Example 57. [Figure 41] Figure 41A shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 58. Figure 41B shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]6[maleimide-PEG4-N3-DBCO-Cy3]1 of Example 58. [Figure 42] Figure 42A shows HIC-HPLC of the trastuzumab-[maleimide]6 conjugate of Example 59. Figure 42B shows HIC-HPLC of the trastuzumab-[maleimide]6[MC-VC-PAB-MMAE]2 conjugate of Example 59. [Figure 43]FIG. 43 shows HIC-HPLC of the trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate of Example 60. [Figure 44] FIG. 44 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 61. [Figure 45] FIG. 45 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 62. [Figure 46] FIG. 46 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 63. [Figure 47] FIG. 47 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 64. [Figure 48] FIG. 48 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 65. [Figure 49] FIG. 49 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 66. [Figure 50] FIG. 50 shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 67. [Figure 51] FIG. 51 shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 68. [Figure 52] FIG. 52 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 69. [Figure 53] FIG. 53 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 70. [Figure 54] FIG. 54 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 71. [Figure 55]FIG. 55 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 72. [Figure 56] FIG. 56 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 73. [Figure 57] FIG. 57 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 74. [Figure 58] FIG. 58 shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 75. [Figure 59] Figure 59AH shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates of Examples 76-83 (various parameters for step (1) and / or step (2)). [Figure 60] Figure 60 A-G shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates of Examples 84-90 (with different parameters in step (1) and / or step (2)). [Figure 61] Figure 61A-C shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates of Comparative Examples 7-9 (the concentration of transition metal ions is 0). [Figure 62] Figure 62 AD shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates of Examples 91-94 (different oxidation times and temperatures in step (2)). [Figure 63] Figure 63AH shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates of Examples 95-102 (different buffer systems). [Figure 64] Figure 64AH shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates of Examples 103-110 (different buffer systems). [Figure 65] FIG. 65 shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Comparative Example 10. [Figure 66] Figure 66 shows HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1 conjugate of Example 111. [Figure 67] Figure 67 shows HIC-HPLC of the trastuzumab-[maleimide-PEG-N-DBCO-MMAE][MC-GGFG-DXd] conjugate of Example 112. [Figure 68] Figure 68 shows HIC-HPLC of the trastuzumab-[maleimide-PEG-N-DBCO-Cy][MC-VC-PAB-MMAE] conjugate of Example 113. [Figure 69] Figure 69A shows the HIC-HPLC of the trastuzumab-[maleimide-PEG-N-DBCO-Cy] conjugate of Example 113, and Figure 69B shows the HIC-HPLC of the trastuzumab-[maleimide-PEG-N-DBCO-Cy][MC-VC-PAB-MMAE] conjugate of Examples 114-115. [Figure 70] Figure 70A shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 116. Figure 70B shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]4 conjugate of Example 116. [Figure 71] FIG. 71 shows the HIC-HPLC of the trastuzumab-[MC-GGFG-DXd]2 conjugate of Comparative Example 11. DETAILED DESCRIPTION OF THE INVENTION

[0021] [Detailed explanation] The present disclosure is described in further detail below. This description is not intended to be a detailed catalog of all the different ways in which the present invention may be practiced or all the features that can be added to the present invention. For example, features described with respect to one embodiment may be incorporated into other embodiments. Also, features described with respect to a particular embodiment may be omitted from that embodiment. Moreover, numerous variations and additions to the various embodiments proposed herein will be apparent to those skilled in the art in light of this disclosure without departing from the invention(s) presented herein. Accordingly, the following description is intended to illustrate some specific embodiments of the invention(s) herein, but is not intended to exhaustively identify all permutations, combinations, and variations thereof.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains.In carrying out the testing of this disclosure, methods and materials similar or equivalent to those described herein can be used, but preferred materials and methods are described herein.In describing and claiming this disclosure, the following terms are used:

[0023] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the term "antibody" includes a plurality of antibodies, and the term "transition metal ion" refers to a mixture of transition metal ions. As used in this application, the term "or" means "and / or" unless otherwise indicated.

[0024] Throughout this disclosure, unless the context otherwise requires, the terms "consisting of," "includes," "comprises," "containing," "containing," and "containing" shall be interpreted to mean the inclusion of the stated step or element or group of steps or elements, but not the exclusion of other steps or elements or group of steps or elements. "Consisting of" means including and is limited to everything that follows the term "consisting of." Thus, the term "consisting of" indicates that the listed elements are necessary or mandatory, and that other elements may not be present. "Consisting essentially of" means including all elements that follow the term "consisting of," and is limited to other elements that do not interfere with or facilitate the activity or function specified in the disclosure for the listed elements. Thus, the term "consisting essentially of" means that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending on whether they affect the activity or behavior of the listed elements.

[0025] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% from a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, the term "about" or "approximately" preceding a numerical value indicates a value that is plus or minus a range of 15%, 10%, 5%, or 1%.

[0026] As used herein, the terms "one embodiment," "in one embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places herein are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0027] A mixture of antibody-drug conjugates is produced by a conventional conjugation process or the bioconjugation process of the present disclosure. Generally, antibody molecules belonging to the IgG1 or IgG4 subclass have four interchain disulfide bonds formed by two -SH groups. An antibody molecule can undergo partial or complete reduction of one or more interchain disulfide bonds to form 2n (n is an integer selected from 1, 2, 3, or 4) reactive -SH groups. Depending on the number of drugs conjugated to one antibody molecule, different conjugates containing different numbers of drug molecules are designated D0, D1, D2, D3, D4, D6, and D8. Therefore, the "homogeneity" of an antibody-drug conjugate is used to describe the predominance of one specific type of antibody-drug conjugate (i.e., one type selected from D0, D1, D2, D3, D4, D6, and D8 conjugates) in a given mixture of antibody-drug conjugates.

[0028] Drug loading is expressed as the number of drug moieties per antibody molecule in an ADC molecule. In some antibody-drug conjugates, drug loading may be limited by the number of binding sites on the antibody. For example, when the attachment is a cysteine thiol, in certain exemplary embodiments described herein, drug loading ranges from 0 to 8 drug moieties per antibody. In one embodiment, the average drug loading in an antibody-drug conjugate ranges from 1 to about 8, from about 2 to about 6, or from about 3 to about 5.

[0029] As used herein, the term "D0" refers to an ADC in which the number of drugs bound to a single antibody molecule is approximately zero.

[0030] As used herein, the term "D1" or "ADC with D1" refers to an ADC in which one of the thiol bridging groups with a linker payload re-bridges two thiol groups of one single antibody molecule.

[0031] As used herein, the term "D2" or "ADC having D2" refers to an ADC in which two drug molecules are attached to a single antibody molecule, and the two drug molecules are attached via a linker to the -SH group generated by reduction of the S-S bond between the heavy and light chains, or are attached via a linker to the -SH group generated by reduction of the S-S bond between the heavy and heavy chains.

[0032] As used herein, the term "D3" or "ADC with D3" refers to an ADC in which a thio bridging group with a linker payload re-bridges six thiol groups of one antibody molecule.

[0033] As used herein, the term "D4" or "ADC with D4" refers to an ADC in which four drug molecules are bound to a single antibody molecule, where the four drug molecules may be bound via a linker to four -SH groups generated by reduction of two S-S bonds between the heavy and light chains. Alternatively, the four drug molecules may be bound via a linker to four -SH groups generated by reduction of two S-S bonds between the heavy and light chains. Alternatively, two drug molecules may be bound via a linker to two -SH groups generated by reduction of one S-S bond between the heavy and light chains, and the other two drug molecules may be bound via a linker to two -SH groups generated by reduction of one S-S bond between the heavy and heavy chains.

[0034] As used herein, the term "D6" or "ADC with D6" refers to an ADC in which six drug molecules are attached to one single antibody molecule, and the six drug molecules can be attached to the six -SH groups generated by reduction of the three S-S bonds.

[0035] As used herein, the term "D8" or "ADC with D8" refers to an ADC in which eight drug molecules are attached to one single antibody molecule, and the eight drug molecules can be attached to eight -SH groups generated by reduction of the four S-S bonds.

[0036] As used herein, the term "D6+D1" or "bi-payload ADC with D6+D1" refers to an ADC in which six first linker payloads and one second thio-bridge group group having a second linker payload are attached to one antibody molecule.

[0037] As used herein, the term "D6+D2" or "D6+D2 dual payload ADC" refers to an ADC in which six first linker payloads and two second linker payloads are attached to one antibody molecule.

[0038] As used herein, the term "D3+D1" or "bi-payload ADC with D3+D1" refers to an ADC in which three of the first thio bridging groups with a first linker payload re-bridge six thiol groups, and two of the second linker payloads are attached to one single antibody molecule.

[0039] As used herein, the term "D3+D2" or "bi-payload ADC with D3+D2" refers to an ADC in which three of the first thio bridging groups with a first linker payload re-bridge six thiol groups, and two of the second linker payloads are attached to one single antibody molecule.

[0040] As used herein, the term "D0+D2" or "ADC with D0+D2" refers to an ADC in which one of the first thio bridging groups re-bridges two thiol groups and four of the second linker payloads are attached to one antibody molecule, or an ADC in which two of the end-capping reagents and four of the second linker payloads are attached to one antibody molecule.

[0041] As used herein, the terms "D0+D1" or "ADC with D0+D1" refer to an ADC in which three of the first thio bridging groups re-bridge six thiol groups and one of the second thio bridging groups bearing a linker payload re-bridges two thiol groups on a single antibody molecule, or an ADC in which six of the end-capping reagents react with six thiol groups and one of the second thio bridging groups bearing a linker payload re-bridges two thiol groups on a single antibody molecule.

[0042] As used herein, the term "D1+D6" or "bi-payload ADC with D1+D6" refers to an ADC in which one of the first thio bridging groups with a first linker payload re-bridges two thiol groups and six of the second linker payloads are attached to one antibody molecule, where the first and second linker payloads can be the same or different.

[0043] As used herein, the term "D1+D2" or "bi-payload ADC with D1+D2" refers to an ADC in which one of the first thio bridging groups with a first linker-payload re-bridges two thiol groups and two of the second linker-payloads are attached to one antibody molecule, where the first and second linker-payloads can be the same or different.

[0044] As used herein, the term "D1+D4" or "bi-payload ADC with D1+D4" refers to an ADC in which one of the first thio bridging groups with a first linker-payload re-bridges two thiol groups and four of the second linker-payloads are attached to one antibody molecule, where the first and second linker-payloads can be the same or different.

[0045] As used herein, the term "D2+D4" or "bi-payload ADC with D2+D4" refers to an ADC in which two first linker-payloads and four second linker-payloads are conjugated to one antibody molecule.

[0046] Method for preparing site-specifically modified antibodies In one aspect, the present application provides a method for preparing an antibody having a site-specific modification, wherein the site-specific modification is selective reduction of three interchain disulfide bonds in the antibody, the method comprising using tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof in combination with a transition metal ion.

[0047] In a second aspect, the present application provides a method for preparing an antibody having a site-specific modification, wherein the site-specific modification is selective reduction of three interchain disulfide bonds in the antibody, two of which are in the Fab region and one of which is in the hinge region of the antibody, the method comprising using TCEP or a salt thereof and a transition metal ion together.

[0048] As used herein, the term "disulfide bond" refers to a covalent bond having the structure RSS-R'. Cysteine, an amino acid, contains a thiol group that can form a disulfide bond with, for example, a second thiol group from another cysteine residue. A disulfide bond is formed between the thiol groups of two cysteine residues present in each of two polypeptide chains, thereby forming an interchain crosslink or bond.

[0049] As used herein, the term "hinge region" refers to an antibody heavy chain molecule comprising the portion connecting the CH1 and CH2 domains. The hinge region comprises approximately 25 amino acid residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently.

[0050] As used herein, the term "Fab fragment" refers to the structural region of an antibody capable of binding to an antigen. It consists of an intact light chain (variable and constant regions) and a portion of the heavy chain structure (variable and constant region fragments), with the light and heavy chains linked by disulfide bonds. Fab fragments are obtained by protease digestion of a full-length antibody. Papain degrades human immunoglobulin G into two Fab fragments and one Fc fragment. Pepsin degrades IgG into an F(ab')2 fragment and a pFc' fragment. The F(ab')2 fragment is further reduced to form two Fab' fragments. In some embodiments, an interchain disulfide bond connects two upper heavy chains in the hinge region, or an interchain disulfide bond connects a heavy chain to a light chain in the Fab region.

[0051] In a third aspect, the present application provides a method for preparing an antibody having a site-specific modification, wherein the site-specific modification is selective reduction of three interchain disulfide bonds in the antibody, two of which are in the Fab region and one of which is in the hinge region of the antibody, the method comprising the steps of: (a) Incubating TCEP or its salt with transition metal ions in a buffer system in the presence of an antibody to selectively reduce the interchain disulfide bonds of the antibody to obtain an antibody having reduced thiol groups, at a molar ratio of TCEP to antibody of 3:1-15:1, optionally at a molar ratio of TCEP to antibody of 3:1-6:1.

[0052] In some embodiments, in step (a), three interchain disulfide bonds in the antibody are selectively reduced. The molar ratio of TCEP to antibody is very important for selectively reducing the three interchain disulfide bonds.

[0053] In some embodiments, the salt refers to an acid addition salt or a base addition salt.

[0054] In some embodiments, acid addition salts may be formed using inorganic and organic acids. Inorganic acids from which salts are derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc. Organic acids from which salts are derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfasalicylic acid, etc.

[0055] In some embodiments, base addition salts can be formed using inorganic and organic bases. Inorganic bases from which salts can be derived include those from Groups 1 and 2 of the periodic table. In one embodiment, salts are derived from lithium, sodium, potassium, calcium, magnesium, etc. Organic bases from which salts can be derived include, for example, primary amines, secondary amines, tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Specific organic amines include isopropylamine, benzathine, cholinamide, diethanolamine, diethylamine, lysine, meglumine, piperazine, tromethamine, etc.

[0056] In some embodiments, in step (a), the molar ratio of TCEP to antibody is 3.2:1 to 5:1 or 3.5:1 to 4.4:1.

[0057] The molar ratio of TCEP to antibody is important for selectively reducing the three interchain disulfide bonds in the antibody. In some embodiments, the molar ratio of TCEP to antibody in step (a) is 3.1:1 to 5.5:1, 3.1:1 to 5.0:1, 3:1 to 4.8:1, 3.2:1 to 4.8:1, 3.4:1 to 4.8:1, 3.6:1 to 4.8:1, or 3.8:1 to 4.8:1. In some embodiments, the molar ratio of TCEP to antibody in step (a) is 3:1 to 4.5:1 or 3:1 to 4:1. In some embodiments, the molar ratio of TCEP to antibody in step (a) is 3.2:1 to 4.4:1.

[0058] In some embodiments, in step (a), the molar ratio of TCEP to antibody is 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, or 15:1.

[0059] In some embodiments, in step (a), the incubation temperature is 0°C to 37°C, optionally, in step (a), the incubation temperature is 0°C to 25°C, and more optionally, in step (a), the incubation temperature is 0°C to 15°C. In some embodiments, in step (a), the incubation temperature is 0°C to 10°C, 0°C to 8°C, or 0°C to 6°C. In some embodiments, in step (c), the incubation temperature is 4°C. In some embodiments, in step (a), the incubation temperature is 37°C, 35°C, 33°C, 30°C, 28°C, 24°C, 20°C, 18°C, 15°C, 13°C, 10°C, 8°C, or 4°C.

[0060] The incubation time in step (a) is important for selectively reducing the three interchain disulfide bonds in the antibody. In some embodiments, the incubation time in step (a) is 3 to 24 hours. In some embodiments, the incubation time in step (a) is 12 to 24 hours, optionally, the incubation time in step (a) is 16 to 20 hours, and more optionally, the incubation time in step (a) is 16 to 18 hours.

[0061] In some embodiments, the incubation time in step (a) is 4 to 24 hours, 14 to 24 hours, or 16 to 24 hours. In some embodiments, the incubation time in step (a) is 17 to 18 hours. In some embodiments, the incubation time in step (a) is 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours.

[0062] In some embodiments, in step (a), the incubation temperature is 4° C. and the incubation time is 18 hours.

[0063] In some embodiments, in step (a), the molar ratio of TCEP to antibody is 3:1 to 6:1, and the incubation time is 10 to 24 hours. In some embodiments, in step (a), the molar ratio of TCEP to antibody is 3:1, 3.2:1, 3.5:1, 3.8:1, 4:1, 4.5:1, 5:1, 5.5:1, or 5:8:1, and the incubation time is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.

[0064] In some embodiments, the incubation time in step (a) decreases as the molar ratio of TCEP to antibody increases. In some embodiments, the molar ratio of TCEP to antibody in step (a) is 4:1 to 15:1, and the incubation time is 4 to 12 hours. In some embodiments, the molar ratio of TCEP to antibody in step (a) is 7:1 to 15:1, and the incubation time is 4 to 12 hours. In some embodiments, the molar ratio of TCEP to antibody in step (a) is 8:1 to 13:1, and the incubation time is 4 to 10 hours. In some embodiments, in step (a), the molar ratio of TCEP to antibody is 6.2:1, 6.5:1, 6.8:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, or 15:1, and the incubation time is 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0065] In some embodiments, in step (a), the molar ratio of transition metal ion to TCEP is 0.1:1 to 30:1, optionally the molar ratio of transition metal ion to TCEP is 0.1:1 to 20:1, and further optionally the molar ratio of transition metal ion to TCEP is 0.5:1 to 8:1. In some embodiments, the molar ratio of transition metal ion to TCEP is 0.1:1 to 15:1, 0.1:1 to 10:1, 0.1:1 to 8:1, 0.25:1 to 15:1, 0.25:1 to 12:1, 0.25:1 to 10:1, 0.25:1 to 8:1, 0.25:1 to 7.5:1, 0.25:1 to 7:1, 0.25:1 to 5:1, 0.25:1 to 4:1, or 0.5:1 to 4:1.

[0066] In some embodiments, in step (a), the molar ratio of transition metal ion to TCEP is 0.1:1, 0.2:1, 0.5:1, 0.8:1, 1:1, 2:1, 4:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, or 20:1.

[0067] In some embodiments, the concentration of TCEP is not particularly limited as long as the concentrations of the transition metal ion and the antibody are increased or decreased at equal rates. In some embodiments of the present application, the concentration of the first reducing agent is 0.01 mM to 0.2 mM. In some embodiments of the present application, the concentration of the first reducing agent is 0.02 mM to 0.15 mM. In some embodiments of the present application, the concentration of the first reducing agent is 0.05 mM to 0.1 mM. In some embodiments of the present application, the concentration of the first reducing agent is 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.10 mM, 0.11 mM, 0.12 mM, 0.13 mM, 0.14 mM, 0.15 mM, 0.16 mM, 0.17 mM, 0.18 mM, 0.19 mM, or 0.20 mM.

[0068] In some embodiments, there is no particular limitation on the concentration of the transition metal ion in step (a), as long as the concentrations of TCEP and antibody are increased or decreased in equal proportions.

[0069] In some embodiments of the present application, there is no particular limitation on the concentration of the antibody in step (a), as long as the concentrations of TCEP and transition metal ions are increased or decreased at equal rates.

[0070] In some embodiments, the buffer system is selected from the group consisting of MES buffer, Bis-Tris buffer, PIPES buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, TAPSO buffer, PBS buffer, PB buffer, acetate buffer, BTP buffer, HEPPSO buffer, POPSO buffer, EPPS buffer, or Tris buffer.

[0071] As used herein, the term "MES buffer" refers to 2-(N-morpholino)ethanesulfonic acid buffer.

[0072] As used herein, the term "bis-tris buffer" refers to bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane buffer.

[0073] As used herein, the term "PIPES buffer" refers to piperazine-1,4-bisethanesulfonic acid buffer.

[0074] As used herein, the term "MOPS buffer" refers to 3-morpholinopropanesulfonic acid buffer.

[0075] As used herein, the term "BES buffer" refers to N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer.

[0076] As used herein, the term "HEPES buffer" refers to "4-hydroxyethylpiperazine ethanesulfonic acid buffer."

[0077] As used herein, the term "DIPSO buffer" refers to 3-[bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer.

[0078] As used herein, the term "MOBS buffer" refers to 3-morpholinopropanesulfonic acid buffer.

[0079] As used herein, the term "MOPSO buffer" refers to 3-(N-morpholino)-2-hydroxy-1-propanesulfonic acid buffer.

[0080] As used herein, the term "TES buffer" refers to 2-[tris(hydroxymethyl)methylamino]-1-ethanesulfonic acid buffer.

[0081] As used herein, the term "ACES buffer" refers to N-(carbamoylmethyl)taurine buffer.

[0082] As used herein, the term "TAPSO buffer" refers to 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid buffer.

[0083] As used herein, the term "PBS" refers to "phosphate buffered saline."

[0084] As used herein, the term "ADA buffer" refers to N-(carbamoylmethyl)iminodiacetic acid buffer.

[0085] As used herein, the term "PB buffer" refers to "phosphate buffer."

[0086] As used herein, the term "BTP buffer" refers to a bis-tris-propane buffer.

[0087] As used herein, the term "HEPSO buffer" refers to N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid buffer.

[0088] As used herein, the term "POPSO buffer" refers to piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) buffer.

[0089] As used herein, the term "EPPS buffer" refers to 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid buffer.

[0090] As used herein, the term "Tris buffer" refers to tris(hydroxymethyl)aminomethane buffer.

[0091] In some embodiments, the buffer system is selected from the group consisting of MES buffer, Bis-Tris buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, or TAPSO buffer.

[0092] In some embodiments, the buffer system is a BES buffer.

[0093] In some embodiments, the pH value of the buffer system is between 5.5 and 8.

[0094] In some embodiments, the pH of the buffer system is between 5.8 and 7.4, preferably between 6.7 and 7.4. In some embodiments, the pH of the system buffer is between 6.0 and 7.4 or between 6.4 and 7.4. In some embodiments, the pH of the buffer system is 6.4, 6.7, 7.0, or 7.4.

[0095] In some embodiments, the buffer system is a BES buffer, and the pH value of the BES buffer is 7.0. In some embodiments, the buffer system is a BES buffer, and the pH value of the BES buffer is 6.4. In some embodiments, the buffer system is a BES buffer, and the pH value of the BES buffer is 6.7. In some embodiments, the buffer system is a BES buffer, and the pH value of the BES buffer is 7.4. In some embodiments, the buffer system is an MES buffer, and the pH value of the BES buffer is 7.0.

[0096] In some embodiments, the coherence of the buffer system is between 10 mM and 100 mM.

[0097] In some embodiments, the consonance of the buffer system is between 20 mM and 80 mM, preferably between 20 mM and 40 mM. In some embodiments, the consonance of the buffer system is between 20 mM and 60 mM. In some embodiments, the consonance of the buffer system is 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

[0098] In some embodiments, the transition metal ion is Zn 2+ , Cd 2+ , Ni 2+ , Hg 2+ , Mn 2+ , Co2+ and combinations thereof.

[0099] The term "transition metal ions" refers to elements from groups 4 to 12, justified by their typical chemical properties: a wide range of complex ions in various oxidation states, colored complexes, and catalytic properties as elements or ions (or both). Sc and Y from group 3 are also generally recognized as transition metals.

[0100] In some embodiments, the transition metal ion is Zn 2+ , Cd 2+ , Hg 2+ , Ni 2+ , Co 2+ or a combination thereof.

[0101] In some embodiments, the transition metal ion is Zn 2+ is.

[0102] In some embodiments, the salt of the transition metal ion is not particularly limited, as long as the transition metal ion is soluble in the reaction solution and free transition metal ions are released into the reaction solution. In some embodiments of the present application, the salt of the transition metal ion is chloride, nitrate, sulfate, acetate, iodide, bromide, formate, or tetrafluoroborate.

[0103] In some embodiments, Zn 2+ The salt is ZnCl 2+ , Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, zinc formate, or zinc tetrafluoroborate. In some embodiments, Zn 2+ The salt of is ZnCl2.

[0104] In some embodiments, site-specific modification does not refer to antibody engineering, enzymatic techniques, and glycoengineering.

[0105] In some embodiments, the method includes introducing a metal chelator after step (a).

[0106] In some embodiments, the metal chelator can capture excess transition metal ions and serve to selectively reduce the three interchain disulfide bonds in the antibody. In some embodiments, the specificity of the metal chelator is not important, so long as it can capture excess transition metal ions and does not affect the reduction of antibody disulfide bonds.

[0107] In some embodiments, the metal chelator is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), citric acid (CA), tartaric acid (TA), gluconic acid (GA), or N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA).

[0108] In some embodiments, the metal chelator is selected from the group consisting of EDTA, NTA, or DTPA. In some embodiments, the metal chelator is EDTA.

[0109] In a fourth aspect, the present application provides a method for preparing an ADC having D2 or an ADC having D1, the method comprising the method of the present application and further comprising the steps of: (B1) introducing an oxidizing agent to selectively reoxidize the reduced thiol groups resulting from step (a), optionally reoxidizing the reduced thiol groups within the Fab region, and preferably removing excess oxidizing agent to purify the oxidation product. (C1) Introduce a metal chelator and modifying reagent 1 to react with the remaining thiol groups resulting from step (B1), where modifying reagent 1 is an end-capping reagent, a first linker payload, or a first thio bridging reagent, and optionally, the first thio bridging reagent has a first linker payload or a reactive group.

[0110] As used herein, the term "bears" or "bearing" refers to "to have" or "having."

[0111] In some embodiments, when the first thio bridging reagent has a reactive group, step (C1) comprises the following steps: introducing a first thio bridging reagent having a metal chelator and a reactive group to re-crosslink the reduced thiol groups obtained in step (B1), and then incubating the first linker payload in a buffer system to react with the reactive group of the thio bridging group.

[0112] In some embodiments, in step (a), the molar ratio of TCEP to antibody is 4:1 to 15:1, and the incubation time is 1 to 24 hours. In some embodiments, in step (a), the molar ratio of TCEP to antibody is 4:1 to 15:1, and the incubation time is 1 to 16 hours. In some embodiments, in step (a), the molar ratio of TCEP to antibody is 4:1 to 10:1, and the incubation time is 4 to 8 hours.

[0113] In some embodiments, the concentration of the transition metal ion in step (a) is also important for improving reduction selectivity. In some embodiments, a method for preparing an ADC having D2 comprises the steps of: in step (a), the molar ratio of transition metal ion to antibody is 1:1 to 10:1 or 1:1 to 2:1. In some embodiments, in step (a), the molar ratio of transition metal ion to antibody is 1:1 to 9:1, 1:1 to 8:1, 1:1 to 7:1, 1:1 to 6:1, 1:1 to 5:1:1, 1:1 to 4.5:1, 1:1 to 4:1, 1:1 to 3.5:1, 1:1 to 3:1, or 1:1 to 2.5:1.

[0114] In some embodiments, the oxidizing agent is not particularly limited as long as it is capable of reoxidizing reduced thiol groups, hi some embodiments, the oxidizing agent is dehydroascorbic acid (DHAA).

[0115] In some embodiments, in step (B1), the oxidizing agent selectively reoxidizes the reduced thiol groups resulting from step (a), providing two reduced thiol groups on the antibody.

[0116] In some embodiments, the concentration of the oxidizing agent in step (B1) is important for improving oxidation selectivity. In some embodiments, the molar ratio of oxidizing agent to antibody in step (B1) is 2:1 to 25:1, optionally, the molar ratio of oxidizing agent to antibody in step (B1) is 2:1 to 20:1, and further optionally, the molar ratio of oxidizing agent to antibody in step (B1) is 8:1 to 15:1. In some embodiments, the molar ratio of oxidizing agent to antibody in step (a) is 2.5:1 to 15:1, 3:1 to 15:1, 3.5:1 to 15:1, 4:1 to 15:1, 4.5:1 to 15:1, 5:1 to 15:1, 5.5:1 to 15:1, 6:1 to 15:1, or 7:1 to 15:1.

[0117] In some embodiments, in step (B1), the molar ratio of oxidizing agent to antibody is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, or 25:1.

[0118] In some embodiments, in step (B1), the oxidation temperature is 0°C to 37°C and the oxidation time is 1 hour to 48 hours. In some embodiments, in step (B1), the oxidation temperature is 0°C to 30°C and the oxidation time is 1 hour to 8 hours.

[0119] In some embodiments, in step (B1), the oxidation temperature is 0° C. to 37° C., 0° C. to 25° C., 0° C. to 20° C., 0° C. to 10° C., 0° C. to 4° C., or 4° C. to 10° C. In some embodiments, the oxidation temperature is 0° C., 3° C., 6° C., 8° C., 10° C., 12° C., 15° C., 18° C., 20° C., 22° C., 25° C., 28° C., 30° C., 32° C., 35° C., or 37° C.

[0120] In some embodiments, in step (B1), the oxidation time is 1 to 7 hours, 1 to 6 hours, 1 to 5 hours, 1 to 3 hours, 2 to 5 hours, 2 to 4 hours, or 2 to 3 hours. In some embodiments, the oxidation time is 1 hour, 3 hours, 5 hours, 7 hours, 9 hours, 11 hours, 13 hours, 15 hours, 18 hours, 20 hours, 23 hours, 25 hours, 27 hours, 30 hours, 33 hours, 35 hours, 37 hours, 40 hours, 43 hours, 45 hours, or 48 hours.

[0121] In some embodiments, in step (B1), the oxidation temperature is 25° C. and the oxidation time is 1 hour to 3 hours. In some embodiments, the oxidation temperature is 0° C. to −10° C. and the oxidation time is 5 hours to 8 hours.

[0122] In some embodiments, in step (B1), the oxidation reaction is carried out in the dark.

[0123] In some embodiments, it is important in step (B1) to improve the contents of ADC having D2, ADC having D4, and ADC having D1 by purifying the oxidation product by removing excess oxidizing agent.

[0124] In some embodiments, in step (C1), the reaction temperature with the reduced thiol group is 0° C. to 37° C., 0° C. to 30° C., 5° C. to 25° C., 10° C. to 25° C., or 14° C. to 25° C. In some embodiments, in step (C1), the reaction temperature with the reduced thiol group is 0° C., 0° C., 3° C., 9° C., 13° C., 18° C., 20° C., 23° C., 25° C., 27° C., 29° C., 30° C., 33° C., 35° C., or 37° C.

[0125] In some embodiments, the reaction time with the reduced thiol group in step (C1) is 1 hour to 6 hours, 1 hour to 5 hours, 1 hour to 3 hours, 1 hour to 2 hours, or 1 hour to 1.5 hours. In some embodiments, the reaction time with the reduced thiol group in step (C1) is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

[0126] In some embodiments, in step (C1), the reaction temperature with the reduced thiol group is 0°C to 30°C, and the reaction time with the reduced thiol group is 1 hour to 4 hours. In some embodiments, in step (C1), the reaction temperature with the reduced thiol group is 15°C to 25°C, and the reaction time with the reduced thiol group is 1 hour to 2 hours.

[0127] In some embodiments, in step (C1), the reaction temperature with the reactive group is 10° C. to 37° C., 20° C. to 30° C., 10° C. to 30° C., 15° C. to 30° C., or 25° C. to 30° C. In some embodiments, in steps (b) and (d), the reaction temperature with the reactive group is 10° C., 13° C., 15° C., 17° C., 20° C., 23° C., 25° C., 28° C., 30° C., 33° C., 35° C., or 37° C.

[0128] In some embodiments, the reaction time with the reactive group in step (C1) is 2 to 12 hours, 2 to 10 hours, 4 to 10 hours, 6 to 10 hours, or 8 to 10 hours. In some embodiments, the reaction time with the reactive group in step (C1) is 2 hours, 3 hours, 5 hours, 7 hours, 9 hours, 11 hours, or 12 hours.

[0129] In some embodiments, in step (C1), Modifying Reagent 1 is in excess relative to the amount of antibody.

[0130] In some embodiments, in step (C1), the molar ratio of the first reducing agent to the antibody is 5:1 to 1:1, 2:1 to 1:1, 1.5:1 to 1:1, 1.2:1 to 1:1, or 1.1:1 to 1.1. In some embodiments, in step (C1), the molar ratio of the first Thiobriguet reagent to the antibody is 1.05:1.

[0131] In some embodiments, in step (C1), when the first linker payload reacts with the reactive group in the first thio bridging reagent, the molar ratio of the first linker payload to the antibody is 5:1 to 1:1, 4:1 to 1:1.1, 3:1 to 1:1, or 2:1 to 1:1. In some embodiments, in step (C1), the molar ratio of the first linker payload to the antibody is 5:3.

[0132] In some embodiments, in step (C1), when the first linker payload reacts with a reduced thiol group, the molar ratio of first linker payload to antibody is 2:1 to 10:1, 3:1 to 10:1, 4:1 to 9:1, or 5:1 to 7:1. In some embodiments, when the first linker payload reacts with a reduced thiol group in step (C1), the molar ratio of first linker payload to antibody is 5:1.

[0133] In some embodiments, the method further comprises the steps of: (D1) Optionally, a compound containing at least one thiol group is introduced to consume excess first linker payload. (E1) ADCs bearing D2 or D1 are purified and recovered.

[0134] In some embodiments, the compound that consumes the excess first linker payload is not particularly limited, as long as the compound contains at least one thiol group. In some embodiments of the present application, the compound is cysteine.

[0135] In some embodiments, the oxidation product in step (B1), the product ADC having D2, and / or the product ADC having D1 is purified by a desalting column, size exclusion chromatography, ultrafiltration, dialysis, etc. In some embodiments, the oxidation product in step (B1), the product ADC having D2, and / or the product ADC having D1 is purified by a desalting column.

[0136] In some embodiments, the method of preparing an ADC having D2 comprises steps (a), (B1), and (C1), wherein Modifying Reagent 1 is a first linker payload.

[0137] In some embodiments, the method of preparing an ADC having D2 comprises the steps of: (1) To a solution of trastuzumab (0.01 mM–0.2 mM) in BES buffer (20 mM, pH 7.0), TCEP (4 eq–15 eq) and ZnCl2 (1 eq–2 eq) were added, and the reaction mixture was vortexed and then incubated at 4°C for 4–24 hours. (2) To selectively reoxidize the reduced thiol groups in the Fab region obtained in step (1), DHAA (4 eq to 20 eq) is added, and the mixture is incubated in the dark at 0°C to 37°C for 1 hour to 48 hours. Preferably, excess DHAA is removed to purify the oxidation product. (3) To react with the remaining thiol groups in step (2), EDTA (8 eq–120 eq) and MC-VC-PAB-MMAE (6 eq) were added to 10% v / v DMA and incubated at 0°C–30°C for 1–4 h. (4) The reaction mixture was subjected to purification using a desalting column.

[0138] In some embodiments, the homogeneity of the ADC with D2 is up to 60%, 70%, 75%, or even 80%, 85%, 90%, or 95%.

[0139] In some embodiments, the method of preparing an ADC having D1 comprises steps (a), (B1), and (C1), wherein modifying reagent 1 is a first thio bridging reagent having a first linker payload.

[0140] In some embodiments, the method of preparing an ADC having D1 comprises the steps of: (1) To a solution of trastuzumab (0.01 mM–0.2 mM) in BES buffer (20 mM, pH 7.0), TCEP (4 eq–15 eq) and ZnCl2 (1 eq–2 eq) were added, and the reaction mixture was vortexed and then incubated at 4°C for 4–24 hours. (2) To selectively reoxidize the reduced thiol groups in the Fab region obtained in step (1), DHAA (4 eq to 20 eq) is added at 0°C to 37°C for 1 hour to 48 hours. (3) EDTA (8 eq-120 eq) and the first thio bridging reagent dibromomaleimide-PEG4-N3 (0.013 mM) were introduced to react with the reduced thiol group obtained in step (3). The reaction temperature was 4°C-37°C and the reaction time was 1 hour-6 hours. The product was then recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1. (4) Trastuzumab-[maleimide-PEG4-N3]1 and DBCO-MMAE (0.02 mM) were incubated in BES buffer (20 mM, pH 7.0), with the reaction temperature ranging from 10 to 37°C and the reaction time ranging from 2 to 12 hours. (5) The reaction mixture was subjected to purification using a desalting column.

[0141] In some embodiments, the homogeneity of the ADC with D1 is up to 90%, or even 95%.

[0142] In some embodiments, the present application provides a method for preparing an ADC having D4, an ADC having D2+D3, an ADC having D2+D1, an ADC having D1+D2, an ADC having D1+D4, an ADC having D2+D4, an ADC having D1+D6, and an ADC having D1+D3, the method comprising the steps of: (C2) A metal chelator and a second reducing agent are introduced to selectively reduce the antibody from step (B1), and optionally reduce the interchain disulfide bond in the hinge region of the antibody. or (C2') A second reducing agent is introduced to reduce the interchain disulfide bonds in the product from step (C1), and optionally, a transition metal ion is introduced. (D2) Introducing a modifying reagent 2 to react with the reduced thiol group resulting from step (C2) or step (C2`), and optionally a metal chelator, wherein the modifying reagent 2 is a second linker payload or a second thio bridging reagent, and optionally the second thio bridging reagent has a second linker payload or a reactive group.

[0143] In some embodiments, when the second thio bridging reagent has a reactive group, step (D2) comprises the steps of: introducing the product from step (C2) or step (C2') and a second thio bridging reagent having a reactive group to re-crosslink the reduced thiol group obtained from step (C2) or step (C2'), optionally introducing a metal chelator, and then incubating a second linker payload in a buffer system to react with the reactive group of the second thio bridging group.

[0144] In some embodiments, when transition metal ions are introduced in step (C2'), a metal chelating agent that scavenges excess transition metal ions is introduced in step (D2).

[0145] In some embodiments, the second reducing agent is not particularly limited as long as it can reduce the interchain disulfide bond in the antibody. In some embodiments, the second reducing agent is TCEP, tris(3-hydroxypropyl)phosphine (THPP), or dithiothreitol (DTT). In some embodiments, the second reducing agent is TCEP.

[0146] In some embodiments, in step (C2), the molar ratio of metal chelator to antibody is 2:1 to 120:1. In some embodiments, in step (C2), the molar ratio of metal chelator to antibody is 2:1 to 100:1. In some embodiments, in step (C2), the molar ratio of metal chelator to antibody is 2:1 to 80:1. In some embodiments, in step (C2), the molar ratio of metal chelator to antibody is 5:1 to 60:1. In some embodiments, in step (C2), the molar ratio of metal chelator to antibody is 10:1 to 60:1. In some embodiments, in step (C2), the molar ratio of metal chelator to antibody is 20:1 to 60:1. In some embodiments, in step (C2), the molar ratio of metal chelator to antibody is 30:1 to 60:1. In some embodiments, in step (C2), the molar ratio of metal chelator to antibody is 40:1 to 60:1. In some embodiments, in step (C2), the molar ratio of metal chelator to antibody is 50:1 to 60:1.

[0147] In some embodiments, in step (C2), the molar ratio of the second reducing agent to the antibody is 1:1 to 2:1. In some embodiments, in step (C2), the molar ratio of the second reducing agent to the antibody is 1.2:1 to 1.8:1. In some embodiments, in step (C2), the molar ratio of the second reducing agent to the antibody is 1:1 to 1.6:1. In some embodiments, in step (C2), the molar ratio of the second reducing agent to the antibody is 1:1 to 1.4:1.

[0148] In some embodiments, in step (C2), the reduction temperature is 0°C to 30°C and the reduction time is 1 hour to 8 hours. In some embodiments, in step (C2), the reduction temperature is 0°C to 37°C, 5°C to 25°C, 10°C to 20°C, or 10°C to 15°C.

[0149] In some embodiments, in step (C2), the oxidation time is 1 hour to 8 hours, 1 hour to 7 hours, 1 hour to 6 hours, 1 hour to 5 hours, 2 hours to 5 hours, 2 hours to 4 hours, or 2 hours to 3 hours.

[0150] In some embodiments, in step (C2'), three interchain disulfide bonds in the product prepared from step (C1) are completely reduced without transition metal ions. In some embodiments, one interchain disulfide bond or two interchain disulfide bonds in the product prepared from step (C1) are reduced with transition metal ions.

[0151] In some embodiments, the concentration of the second reducing agent in step (C2') is not particularly limited, as long as the second reducing agent can completely reduce the interchain disulfide bonds of the antibody without the use of transition metal ions. In some embodiments, the molar ratio of the second reducing agent to the antibody in step (C2') is 3:1 to 20:1, 3:1 to 10:1, 4:1 to 10:1, 5:1 to 9:1, 6:1 to 9:1, or 6:1 to 8:1. In some embodiments, the molar ratio of the second reducing agent to the antibody in step (C2') is 20:3.

[0152] In some embodiments, two interchain disulfide bonds are selectively reduced by introducing a transition metal ion. In some embodiments, in step (C2'), the molar ratio of the second reducing agent to the transition metal ion is 1:0.05 to 1:40, and / or the molar ratio of the second reducing agent to the antibody is 2.5:1 to 20:1, and / or the incubation time is 1 hour to 24 hours. In some embodiments, in step (C2'), the molar ratio of the second reducing agent to the transition metal ion is 1:0.05, 1:0.08, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, or 1:20. In some embodiments, in step (C2'), the molar ratio of the second reducing agent to the antibody is 2.5:1, 3:1, 5:1, 7:1, 9:1, 11:1, 13:1, 15:1, 17:1, 19:1, or 20:1. In some embodiments, in step (C2'), the incubation time is 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours. In some embodiments, in step (C2'), the molar ratio of the second reducing agent to the transition metal ion is 1:0.05 to 1:40, and / or the molar ratio of the second reducing agent to the antibody is 3:1 to 15:1, and / or the incubation time is 1 hour to 12 hours. In some embodiments, in step (C2'), the molar ratio of the second reducing agent to the transition metal ion is 1:0.05 to 1:40, and / or the molar ratio of the second reducing agent to the antibody is 2.5:1 to 15:1, and / or the incubation time is 12 hours to 24 hours.

[0153] In some embodiments, one interchain disulfide bond is selectively reduced by introducing a transition metal ion. In some embodiments, in step (C2'), the molar ratio of the second reducing agent to the transition metal ion is 1:0.4 to 1:100, and / or the molar ratio of the second reducing agent to the antibody is 0.8:1 to 2.5:1, and / or the incubation time is 0.5 to 24 hours. In some embodiments, in step (C2'), the molar ratio of the second reducing agent to the transition metal ion is 1:0.5, 1:1, 1:4, 1:8, 1:12, 1:24, 1:30, 1:40, 1:50, 1:50, 1:70, 1:80, 1:90, or 1:100. In some embodiments, in step (C2'), the molar ratio of the second reducing agent to the antibody is 0.8:1, 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1, or 2.5:1. In some embodiments, in step (C2'), the incubation time is 0.2 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours. In some embodiments, in step (c), the molar ratio of the second reducing agent to the transition metal ion is 1:0.5 to 1:100, and / or the molar ratio of the second reducing agent to the antibody is 0.8:1 to 2:1, or the incubation time is 0.5 to 24 hours. In some embodiments, in step (C2`), the molar ratio of the second reducing agent to the transition metal ion is 1:0.5 to 1:100, and / or the molar ratio of the second reducing agent to the antibody is 2:1 to 2.5:1, or the incubation time is 1 hour to 9 hours.

[0154] In some embodiments, the incubation temperature of the second reducing agent in step (C2') is 0°C to 37°C, or 5°C to 30°C. In some embodiments, the incubation temperature of the second reducing agent in step (C2') is 10°C to 30°C, 15°C to 30°C, 20°C to 30°C, or 25°C to 30°C. In some embodiments, the incubation temperature of the second reducing agent in step (C2') is 25°C.

[0155] In some embodiments, the incubation time of the second reducing agent in step (C2') is 0.5 hours to 24 hours, or 5 hours to 20 hours. In some embodiments, the incubation time of the second reducing agent in step (C2') is 6 hours to 18 hours, 8 hours to 18 hours, 8 hours to 15 hours, or 8 hours to 12 hours. In some embodiments, the incubation time of the second reducing agent in step (C2') is 3 hours, 8 hours, 12 hours, or 18 hours.

[0156] In some embodiments, in step (C2`), the molar ratio of metal chelator to antibody is 1:1 to 100:1, 10:1 to 100:1, 20:1 to 100:1, 20:1 to 80:1, 20:1 to 70:1, 30:1 to 60:1, 40:1 to 50:1, 35:1 to 60:1, or 40:1 to 55:1.

[0157] In some embodiments, the reaction temperature and time with the reduced thiol group in step (D2) are the same as those in step (C1). In some embodiments, the reaction temperature and reaction time with the thiol group in step (D2) and step (C1) are independent.

[0158] In some embodiments, the reaction temperature and reaction time with the reactive group in step (D2) are the same as those in step (C1). In some embodiments, the reaction temperature and reaction time with the reactive group in step (D2) and step (C1) are independent.

[0159] In some embodiments, in step (D2), modifying reagent 2 is in excess relative to the amount of antibody.

[0160] In some embodiments, in step (D2), the molar ratio of the second thio bridging reagent to the antibody is 10:1 to 1:1, 5:1 to 1:1, 5:1 to 3:1, 4:1 to 3:1, 4:1 to 3.2:1, or 4:1 to 3.5:1. In some embodiments, in step (D2), the molar ratio of the second thio bridging reagent to the antibody is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4.5:1, 4:1, 3.8:1, 3.5:1, 3.2:1, 2:1, or 1:1.

[0161] In some embodiments, in step (D2), when the second linker payload reacts with a reactive group in the second thio bridging reagent, the molar ratio of the second linker payload to the antibody is 10:1 to 1:1, 10:1 to 3:1, 9:1 to 3:1, 8:1 to 3:1, 7:1 to 3:1, 6:1 to 3:1, 5:1 to 3:1, or 4:1 to 3:1.

[0162] In some embodiments, when the second linker payload reacts with a reduced thiol group in step (D2), the molar ratio of the second linker payload to the antibody is 20:1 to 2:1, 20:1 to 6:1, 18:1 to 8:1, 16:1 to 8:1, 14:1 to 8:1, or 12:1 to 10:1. In some embodiments, when the second linker payload reacts with a reduced thiol group in step (D2), the molar ratio of the second linker payload to the antibody is 35:3.

[0163] In some embodiments, the method for preparing an ADC having D4 also includes purifying the product from step (B1).

[0164] In some embodiments, the product from step (B1) is purified by a desalting column, size exclusion chromatography, ultrafiltration, dialysis, and / or the like. In some embodiments of the present application, the product from step (B1) is purified by a desalting column.

[0165] In some embodiments, the method of preparing an antibody with site-specific modifications also includes the steps of: (E2) Optionally, a compound containing at least one thiol group is introduced to consume excess linker payload. (F2) The site-specifically modified antibody is purified and recovered.

[0166] In some embodiments, the details of step (E2) and step (F2) are similar to those of step (D1) and step (E1).

[0167] In some embodiments, the method for preparing an ADC having D4 comprises step (a), step (B1), step (C2), and step (D2), wherein modifying reagent 2 is a second linker payload.

[0168] In some embodiments, the method of preparing an ADC having D4 comprises the steps of: (1) TCEP (4 eq–15 eq) and ZnCl2 (1 eq–2 eq) were added to a solution of trastuzumab (0.01 mM–0.2 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 14–24 hours. (2) To selectively reoxidize the reduced thiol groups in the Fab region obtained in step (1), DHAA (2 eq-20 eq) is added, and the mixture is incubated at 0°C to 37°C in the dark for 1 hour to 48 hours, followed by purification using a desalting column. (3) In order to selectively reduce the interchain disulfide bonds remaining in the hinge region of the antibody in step (2), EDTA (2 eq-120 eq) and TCEP (1 eq-2 eq) are introduced, the reduction temperature is 0°C to 30°C, and the reduction time is 1 hour to 5 hours. (4) MC-VC-PAB-MMAE (3 eq-6 eq) was introduced into 10% v / v DMA to react with the residual thiol groups obtained in step (3), and the reaction mixture was incubated at 0°C to 30°C for 1 hour to 4 hours. (5) The reaction mixture was subjected to purification using a desalting column.

[0169] In some embodiments, the method for preparing an ADC having D1+D6 comprises steps (a), (B1), (C1), (C2') and (D2), wherein modifying reagent 1 is a first thio bridging reagent and modifying reagent 2 is a second linker payload, and step (C2') does not include a transition metal ion.

[0170] In some embodiments, the method of preparing an ADC having D1+D6 comprises the steps of: (1) ADC and D1 according to the present application and TCEP (0.08 mM) in BES buffer (20 mM, pH 7.0) were introduced, the reaction temperature was 25°C, and the reaction time was 12 hours. (2) MC-GGFG-DXd (0.14 mM) was introduced into the solution from step (1), and the reaction mixture was allowed to stand at 24 °C for 1 hour. Trastuzumab-[maleimide-PEG-N-DBCO-MMAE]1[MC-GGFG-DXd]6 was then recovered using a desalting column.

[0171] In some embodiments, the homogeneity of the ADC with D1+D6 is up to 70%, 75%, or even 80% or 85%.

[0172] In some embodiments, a method for preparing an ADC having D1+D2 includes steps (a), (B1), (C1), (C2'), and (D2), wherein modifying reagent 1 is a first thio-bridging reagent having a first linker payload, modifying reagent 2 is a second linker payload, and a transition metal ion is introduced in step (C2').

[0173] In some embodiments, the method for preparing an ADC using D1+D2 comprises steps (a), (B1), (C1), (C2'), and (D2), wherein modifying reagent 1 is a first thio-bridging reagent having a reactive group that reacts with a first linker payload, modifying reagent 2 is a second linker payload, and a transition metal ion is introduced in step (C2').

[0174] In some embodiments, the content of ADC with D1+D2 is up to 65%, 68%, 70%, or even 71% or 75%.

[0175] In some embodiments, a method for preparing an ADC having D1+D4 includes steps (a), (B1), (C1), (C2'), and (D2), wherein modifying reagent 1 is a first thio bridging reagent having a first linker payload, modifying reagent 2 is a second linker payload, and a transition metal ion is introduced in step (C2').

[0176] In some embodiments, the method for preparing an ADC using D1+D4 comprises steps (a), (B1), (C1), (C2'), and (D2), wherein modifying reagent 1 is a first thio-bridging reagent having a reactive group that reacts with a first linker payload, modifying reagent 2 is a second linker payload, and a transition metal ion is introduced in step (C2').

[0177] In some embodiments, the content of ADC with D1+D4 is up to 70%, 75%, or even 80% or 83%.

[0178] In some embodiments, the method for preparing an ADC having D2+D4 comprises steps (a), (B1), (C1), (C2') and (D2), wherein modifying reagent 1 is a first linker payload and modifying reagent 2 is a second linker payload, and a transition metal ion is introduced in step (C2').

[0179] In some embodiments, the content of ADC with D2+D4 is up to 75%, 80%, 85%, or even 90%.

[0180] In a fifth aspect, the present application provides a method for preparing an antibody with a site-specific modification, the method comprising the method of the present application and further comprising the steps of: (b) introducing a metal chelating agent and modifying reagent 1 to react with the reduced thiol groups obtained in step (a);

[0181] In some embodiments, when the first thio bridging reagent has a reactive group, step (b) comprises the steps of: introducing a first thio bridging reagent having a metal chelator and a reactive group to re-crosslink the reduced thiol groups obtained in step (a), and then incubating the first linker payload in a buffer system to react with the reactive group of the thio bridging group.

[0182] In some embodiments, the method further comprises the step of: purifying and recovering the product from step (b).

[0183] In some embodiments, the method further comprises the steps of: (c) incubating the reaction product of step (b) and a second reducing agent in a buffer system to reduce interchain disulfide bonds in the reaction product of step (b). (d) The incubation product of step (c) and Modification Reagent 2 are introduced and reacted with the reduced thiol group obtained in step (c).

[0184] In some embodiments, when the second thio bridging reagent has a reactive group, step (d) comprises: introducing the product from step (c) and a second thio bridging reagent having a reactive group to re-crosslink the reduced thiol group obtained from step (c), and then incubating a second linker payload in a buffer system to react with the reactive group of the second thio bridging group.

[0185] In some embodiments, the method further comprises the step of: purifying and recovering the product from step (d).

[0186] In some embodiments, the method includes the following steps: introducing a compound containing at least one thiol group to consume the excess first linker payload and the excess second linker payload. In some embodiments, the compound is the same as the compound of (D1).

[0187] In some embodiments, the resulting antibody-drug conjugate is recovered by any suitable purification method, such as a desalting column, size-exclusion chromatography, ultrafiltration, dialysis, ultrafiltration (UF)-diafiltration (DF), etc. Optionally, further ADC enrichment (e.g., D2) may be applied using hydrophobic interaction chromatography (HIC).

[0188] In some embodiments, in step (d), the obtained ADC is purified by a desalting column, size exclusion chromatography, ultrafiltration, dialysis, and / or similar methods. In some embodiments of the present application, in step (d), the obtained ADC is purified by a desalting column.

[0189] In some embodiments, in step (a), the molar ratio of TCEP to antibody is 7:1 to 15:1 and the incubation time is 4 to 12 hours. In some embodiments, in step (a), the molar ratio of TCEP to antibody is 8:1 to 14:1 and the incubation time is 4 to 10 hours.

[0190] In some embodiments, in step (a), TCEP selectively reduces three interchain disulfide bonds in the antibody using a transition metal ion and an appropriate molar ratio of TCEP to antibody, and optionally, in step (c), a second reducing agent reduces the remaining interchain disulfide bond. Antibodies with site-specific modifications, such as ADCs with D6 or ADCs with D3, can be prepared by a method comprising steps (a) and (b). Antibodies with site-specific modifications, such as ADCs with D6+D1, ADCs with D3+D1, ADCs with D6+D2, ADCs with D3+D2, ADCs with D0+D1, or ADCs with D0+D2, can be prepared by a method comprising steps (a), (b), (c), and (d).

[0191] In some embodiments, in steps (b) and (d), the reaction temperature with the reduced thiol group is 4° C. to 37° C., and the reaction time with the reduced thiol group is 0.5 hours to 20 hours.

[0192] In some embodiments, the reaction temperature with the reduced thiol group in steps (b) and (d) is 20°C to 30°C or 20°C to 25°C. In some embodiments, the reaction temperature with the reduced thiol group in steps (b) and (d) is room temperature. In some embodiments, the reaction temperature with the reduced thiol group in steps (b) and (d) is 4°C, 6°C, 8°C, 10°C, 13°C, 17°C, 20°C, 23°C, 27°C, 30°C, 34°C, or 37°C.

[0193] As used herein, the term "room temperature" refers to 23°C ± 2°C, 25°C ± 5°C, or 20°C ± 5°C.

[0194] In some embodiments, the reaction time with the reduced thiol group in steps (b) and (d) is 0.5 to 6 hours, 0.5 to 4 hours, 0.5 to 2 hours, 1 to 2 hours, or 0.5 to 1 hour. In some modifications, the reaction time with the reduced thiol group in steps (b) and (d) is 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 hours.

[0195] In some embodiments, the temperature and time of reaction with the reduced thiol group in step (b) and step (d) are independent.

[0196] In some embodiments, in steps (b) and (d), the temperature for the reaction with the reactive group is 10° C. to 37° C., and the reaction time for the reaction with the reduced thiol group is 2 hours to 12 hours.

[0197] In some embodiments, the reaction temperature with the reactive group in step (b) and step (d) is 10° C. to 30° C., 15° C. to 30° C., or 25° C. to 30° C. In some embodiments, the reaction temperature with the reactive group in step (b) and step (d) is 4° C., 6° C., 8° C., 10° C., 13° C., 17° C., 20° C., 23° C., 27° C., 30° C., 34° C., 35° C., or 37° C.

[0198] In some embodiments, the reaction time with the reactive group in step (b) and step (d) is 2 to 10 hours, 4 to 10 hours, or 8 to 10 hours. In some embodiments, the reaction time with the reactive group in step (b) and step (d) is 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0199] In some embodiments, the temperature and time of reaction with the reactive group in step (b) and step (d) are independent.

[0200] In some embodiments, in step (b), Modification Reagent 1 is in excess relative to the amount of antibody.

[0201] In some embodiments, in step (b), the molar ratio of the first thio bridging reagent to the antibody is 3:1 to 15:1. In some embodiments, in step (b), the molar ratio of the first thio bridging reagent to the antibody is 3:1, 3.3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 13:1.

[0202] In some embodiments, when the first linker payload reacts with a reactive group in the first thio bridging reagent in step (b), the molar ratio of the first linker payload to the antibody is 3:1 to 15:1. In some embodiments, when the first linker payload reacts with a reactive group in the first thio bridging reagent in step (b), the molar ratio of the first linker payload to the antibody is 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 13:1.

[0203] In some embodiments, when the first linker payload reacts with a reduced thiol group in step (b), the molar ratio of the first linker payload to the antibody is 6:1 to 20:1. In some embodiments, when the first linker payload reacts with a reduced thiol group in step (b), the molar ratio of the first linker payload to the antibody is 6:1, 20:3, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1.

[0204] In some embodiments, in step (d), modifying reagent 2 is in excess relative to the amount of antibody.

[0205] In some embodiments, in step (d), the molar ratio of the second thio bridging reagent to the antibody is 1:1 to 3:1. In some embodiments, in step (b), the molar ratio of the second thio bridging reagent to the antibody is 1:1, 1.5:1, 2:1, or 3:1.

[0206] In some embodiments, when the second linker payload reacts with a reactive group in a second thio bridging reagent in step (d), the molar ratio of the second linker payload to the antibody is 1:1 to 8:1. In some embodiments, when the second linker payload reacts with a reactive group in a second thio bridging reagent in step (d), the molar ratio of the second linker payload to the antibody is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1.

[0207] In some embodiments, when the second linker payload reacts with a reduced thiol group in step (d), the molar ratio of the second linker payload to the antibody is 2:1 to 16:1. In some embodiments, when the second linker payload reacts with a reduced thiol group in step (d), the molar ratio of the second linker payload to the antibody is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 10:1, 12:1, 14:1, or 16:1.

[0208] In some embodiments, the second reducing agent in step (c) is not particularly limited as long as it can reduce the interchain disulfide bond in the antibody. In some embodiments, the second reducing agent in step (c) is TCEP, tris(3-hydroxypropyl)phosphine (THPP), or dithiothreitol (DTT). In some embodiments, the second reducing agent is TCEP.

[0209] In some embodiments, the concentration of the second reducing agent in step (c) is not particularly limited, as long as it is capable of reducing interchain disulfide bonds in the antibody. In some embodiments, the molar ratio of the second reducing agent to the antibody is 1:1 to 20:1. In some embodiments, the molar ratio of the second reducing agent to the antibody is 1:1, 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, or 20:1.

[0210] In some embodiments, in step (c), the incubation temperature for the second reducing agent is 0° C. to 37° C., and the incubation time is 0.5 hours to 24 hours.

[0211] In some embodiments, in step (c), the incubation temperature of the second reducing agent is 5° C. to 30° C., 10° C. to 30° C., 15° C. to 30° C., 20° C. to 30° C., or 25° C. to 30° C. In some embodiments, in step (c), the incubation temperature of the second reducing agent is 25° C.

[0212] In some embodiments, in step (c), the incubation time is 1 hour to 20 hours, 5 hours to 20 hours, 6 hours to 18 hours, 8 hours to 18 hours, 8 hours to 15 hours, or 8 hours to 12 hours. In some embodiments, in step (c), the incubation time is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or 12 hours.

[0213] In some embodiments, the method of preparing an ADC having D6 comprises the steps of: (a) A step of incubating TCEP or a salt thereof and a transition metal ion in a buffer system in the presence of an antibody to selectively reduce interchain disulfide bonds in the antibody to obtain an antibody having reduced thiol groups, wherein the molar ratio of TCEP to antibody is 3:1 to 15:1. (b1) A metal chelator and a first linker payload are introduced to react with the reduced thiol groups obtained in step (a).

[0214] In some embodiments, the homogeneity of the ADC with D6 is up to 55%, 65%, 70%, 80%, 85% or even 90%.

[0215] In some embodiments, a method for preparing a bi-payload ADC having D6+D2 comprises the steps of: (c2) Incubating the ADC with D6 and a second reducing agent in a buffer system to reduce the interchain disulfide bonds in the ADC with D6. (d2) introducing the incubation product from step (c2) and a second linker payload to react with the reduced thiol group resulting from step (c2).

[0216] In some embodiments, the homogeneity of the bi-payload ADC with D6+D2 is up to 80%, 85%, or even 90%.

[0217] In some embodiments, the method of preparing a bi-payload ADC having D6+D1 comprises the steps of: (d3) introducing the incubation product from step (c2) and a second thio bridging reagent having a second linker payload to react with the reduced thiol group resulting from step (c2).

[0218] In some embodiments, the method of preparing a bi-payload ADC having D6+D1 comprises the steps of: (d3`) introducing the incubation product from step (c2) and a second thiobridge reagent having a reactive group to re-crosslink the reduced thiol group resulting from step (c2), and then incubating a second linker payload in a buffer system to react with the reactive group of the second thiobridge group.

[0219] In some embodiments, the homogeneity of a bi-payload ADC with D6+D1 is up to 80%, 85%, or even 90%.

[0220] In some embodiments, the method of preparing an ADC having D3 comprises the steps of: (b4) introducing a first thio bridging reagent having a metal chelator and a first linker payload to react with the reduced thiol group obtained in step (a);

[0221] In some embodiments, the method of preparing an ADC having D3 comprises the steps of: (b4`) introducing a first thio bridging reagent having a metal chelator and a reactive group to re-crosslink the reduced thiol group obtained in step (a), and then incubating the first linker payload in a buffer system to react with the reactive group of the thio bridging group;

[0222] In some embodiments, the homogeneity of the ADC with D3 is up to 80%, 82%, or even 86%.

[0223] In some embodiments, a method of preparing a bi-payload ADC having D3+D2 comprises the steps of: (c5) Incubating the ADC with D3 and a second reducing agent in a buffer system to reduce the interchain disulfide bonds in the ADC with D3. (d5) introducing the incubation product from step (c5) and a second linker payload to react with the reduced thiol group resulting from step (c5).

[0224] In some embodiments, a method for preparing a bi-payload ADC having D3+D1 comprises the steps of: (d6) introducing the incubation product from step (c5) and a second thio bridging reagent having a second linker payload to react with the reduced thiol group resulting from step (c5).

[0225] In some embodiments, a method for preparing a bi-payload ADC having D3+D1 comprises the steps of: (d6`) Introducing the incubation product from step (c5) and a second thio bridging reagent having a reactive group to re-crosslink the reduced thiol group resulting from step (c5), and then incubating a second linker payload in a buffer system to react with the reactive group of the second thio bridging group.

[0226] In some embodiments, a method of preparing an ADC having D0+D2 comprises the steps of: (b7) introducing a metal chelating agent and a first thio bridging reagent to react with the reduced thiol groups obtained in step (a); (c7) Incubating the reactive product from (b7) and a second reducing agent in a buffer system to reduce the interchain disulfide bonds in the product from (b7). (d7) introducing the incubation product from step (c7) and a second linker payload to react with the reduced thiol group resulting from step (c7).

[0227] In some embodiments, the homogeneity of the ADC with D0+D2 is up to 60%, 65%, or even 68% or 70%.

[0228] In some embodiments, the method of preparing an ADC having D0+D1 comprises the steps of: (d8) The incubation product from step (c7) and a second thio bridging reagent having a second linker payload are introduced to react with the reduced thiol group obtained in step (c7).

[0229] In some embodiments, the method of preparing an ADC having D0+D1 comprises the steps of: (d8`) Introducing the incubation product from step (c7) and a second thio bridging reagent having a reactive group to re-crosslink the reduced thiol group resulting from step (c7), and then incubating a second linker payload in a buffer system to react with the reactive group of the second thio bridging group.

[0230] In some embodiments, the homogeneity of the ADC with D0+D1 is up to 85%, 87%, or even 90% or 92%.

[0231] In some embodiments, the first thio bridging reagent and the second thio bridging reagent independently comprise at least two substituents that allow for re-crosslinking of thiol groups.

[0232] In some embodiments, but not limited to, the first thio bridging reagent and the second thio bridging reagent are [ka] is selected from the group consisting of:

[0233] In some embodiments, the reactive groups include azide and / or dibenzocyclooctyne (DBCO), respectively.

[0234] In some embodiments, the thio bridging reagent and the reactive group are attached by an alkyl group or polyethylene glycol (PEG).

[0235] In some embodiments, the first thio bridging reagent having a reactive group and the second thio bridging reagent having a reactive group are selected from the group consisting of, but not limited to: [ka] where n is 0-20, 0-18, 0-15, 0-13, 0-10, 0-7, 0-5 or 0-3.

[0236] In some embodiments, the first thio bridging reagent having a reactive group can be different from the second thio bridging reagent having a reactive group, and in some embodiments, the first thio bridging reagent having a reactive group can be the same as the second thio bridging reagent having a reactive group.

[0237] In some embodiments, the first thio bridging reagent having a reactive group and the second thio bridging reagent having a reactive group are dibromomaleimide-PEG4-N3 having the following formula: [ka]

[0238] In some embodiments, the linkers of the first linker-payload and the second linker-payload are selected from any one of which one end can be linked to a reduced thiol group of an antibody or a reactive group of a thio bridging reagent, and the other end can be linked to a payload.

[0239] As used herein, the term "linker" refers to a reactive molecule that contains at least two substituents, one of which can be covalently attached to a drug molecule and the other of which can be covalently attached to a reactive group on an antibody or a thiobridging reagent.

[0240] In some embodiments, when the first linker payload and / or the second linker payload react with a reduced thiol group, the linker of the first linker payload and the linker of the second linker payload, respectively, comprise a cleavable or non-cleavable linker. Cleavable linkers include chemically labile and enzyme-labile. Due to their high plasma stability and the selectivity and efficiency of intracellular cleavage, enzyme-labile linkers are widely selected as potential cleavable linkers for ADCs. In some embodiments, the enzyme-labile linker comprises -maleimidocaproyl-(-MC-), -maleimidocaproyl-peptide moiety-(-MC-peptide moiety-), -p-aminobenzyl alcohol-(-PAB-), or a structure comprising -peptide moiety- or -MC-peptide moiety-PAB-. In some embodiments, the peptide moiety is a dipeptide, tripeptide, tetrapeptide, or pentapeptide.

[0241] In some embodiments, the dipeptide can be, but is not limited to, valine-alanine (VA), valine-citrulline (VC), alanine-asparagine (AD), alanine-phenylalanine (AF), phenylalanine-lysine (FK), alanine-lysine (AK), alanine-valine (AV), valine-lysine (VK), lysine-lysine (KK), phenylalanine-citrulline (FC), leucine-citrulline (LC), isoleucine-citrulline (IC), tryptophan-citrulline (WC), or phenylalanine-alanine (FA).

[0242] In some embodiments, the tripeptide can be, but is not limited to, alanine-alanine-asparagine (AAD), glycine-valine-citrulline (GVC), glycine-glycine-glycine (GGG), phenylalanine-phenylalanine-lysine (FFK), glutamic acid-valine-citrulline (EVC), or glycine-phenylalanine-lysine (GFK).

[0243] In some embodiments, the tetrapeptide can be, but is not limited to, glycine-glycine-phenylalanine-glycine (GGFG).

[0244] In some embodiments, without limitation, when the first linker payload and / or the second linker payload react with a reduced thiol group, the linkers of the first linker payload and the second linker payload can be MC-VA-PAB, MC-VC-PAB, MC-AD-PAB, MC-AF-PAB, MC-FK-PAB, MC-AK-PAB, MC-AV-PAB, MC-VK-PAB, MC-KK-PAB, MC-FC-PAB, MC-LC-PAB, MC-IC-PAB, MC-WC-PAB, or MC-FA-PAB, respectively. In some embodiments, including but not limited to, when the first and / or second linker payloads are reactive with reduced thiol groups, the linker of the first and second linker payloads can be any of MC-AAD-PAB, MC-GVC-PAB, MC-GGG-PAB, MC-FFK-PAB, MC-EVC-PAB, or MC-GFK-PAB, respectively. In some embodiments, including but not limited to, when the first and / or second linker payloads are reactive with reduced thiol groups, the linker of the first and second linker payloads can be MC-GGFG.

[0245] In some embodiments, the linker comprises a maleimide containing drug, an organic chloride containing drug, an organic bromide containing drug, an organic iodide containing drug, and / or a vinylpyrimidine containing drug.

[0246] In some embodiments, when the first linker payload and / or the second linker payload reacts with a reactive group in a thio bridging reagent, the linker of the first linker payload and / or the second linker payload further comprises azide and / or dibenzocyclooctyne (DBCO). In some embodiments, when the linker of the first linker payload and / or the second linker payload comprises azide, the reactive group of the thio bridging group comprises DBCO. In some embodiments, when the linker of the first linker payload and / or the second linker payload comprises DBCO, the reactive group of the thio bridging group comprises azide.

[0247] In some embodiments, when the linker of the first linker payload and / or the second linker payload reacts with a reactive group in a thio bridging reagent, the linker of the first linker payload and the second linker payload, respectively, is selected from the group consisting of: [ka] wherein n is 0 to 20, 0 to 18, 0 to 15, 0 to 13, 0 to 10, 0 to 7, 0 to 5, or 0 to 3, and m is 0 to 20, 0 to 18, 0 to 15, 0 to 13, 0 to 10, 0 to 7, 0 to 5, or 0 to 3. Optionally, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0248] As used herein, the term "end-capping reagent" refers to a compound that does not carry a drug and contains at least one substituent that is capable of covalently binding to an antibody.

[0249] In some embodiments, the end-capping reagent is a cleavable or non-cleavable linker, hi some embodiments, the end-capping reagent is (2-aminoethyl)maleimide.

[0250] In some embodiments, the first thio bridging reagent with the first linker payload and the second thio bridging reagent with the second linker payload have the formula: [ka]

[0251] where Q is selected from the group consisting of: [ka]

[0252] S is selected from a cleavable linker or a non-cleavable linker, including but not limited to, S is selected from the group consisting of: [ka] wherein n is 0 to 20 and m is 0 to 20, and optionally, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0253] T is the payload.

[0254] In some embodiments, the payload is selected from any one of the following, including at least one permuted group that allows a connection from the payload to the linker.

[0255] As used herein, the term "payload" refers to a cytotoxic molecule or molecule of medical interest that includes at least one substituent or moiety that allows for attachment of the payload to a linker structure. The payload can kill cancer cells and / or inhibit the growth, proliferation, or metastasis of cancer cells, thereby reducing, alleviating, or eliminating one or more symptoms of a disease or disorder.

[0256] In some embodiments, the payload is a cytotoxic drug, a fluorescent dye, a cytokine, a nucleic acid, a radionuclide, a kinase inhibitor, or a derivative thereof. In some embodiments, the payload includes, but is not limited to, a topoisomerase inhibitor and a tubulin inhibitor. In some embodiments, the payload is, but is not limited to, an anti-cancer drug, an anti-viral agent, or an anti-bacterial agent.

[0257] In some embodiments, the cancer is carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0258] Representative payloads include monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), monomethyl auristatin EF (MMAF), calicheamicin (CLM), martancillin (DM1), maytansinoids, duocarmycins, anthracyclines, pyrrolobenzodiazepine dimers, amatoxins, quinoline alkaloids, Dxd, doxorubicin hydrochloride, methotrexate, erlotinib, bortezomib, fulvestrant, sunitib, imatinib mesylate, letrozole, finasunate, oxaliplatin, carboplatin, cisplatin, finasunate, fluorouracil, rapamycin, leucovorin, lapatinib, lonafamib, sorafenib, gefitinib, camptothecin, topotecan, bryostatin, and adzealand. and cyclophosphamide, doxorubicin, vincristine, prednisone or prednisolone, mechlorethamine, chlorambucil, other alkylating agents such as ifosfamide, antimetabolites such as azathioprine and mercaptopurine, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, etoposide phosphate, epipodophyllotoxin, actinomycin, daunorubicin, valrubicin, idarubicin, edrecolomab, epirubicin, bleomycin, plicamycin, or mitomycin, and salts thereof.

[0259] In some embodiments, the payload is deruxtecan (DXd), Cy3 (cyanine 3), MMAE, MMAD, or MMAF. In some embodiments of the present application, the payload is MMAE, DXd, or Cy3.

[0260] The linker-payload is a chemical moiety that is synthesized by linking a linker and a payload. Depending on the desired payload and the selected linker, those skilled in the art can select an appropriate method for coupling them. For example, several conventional coupling methods, such as amine coupling, can be used to form the desired linker-payload containing a reactive group for covalently binding to an antibody. In this disclosure, drug-maleimide conjugates (i.e., maleimide-linked drugs) are used as examples of payloads with reactive groups. Maleimide is the most common reactive group that can be coupled to thiol groups in ADC preparation. In addition, organic chlorides, bromides, and iodides are also frequently used.

[0261] The linker payload can be any physically active compound or any compound used in the diagnosis, prevention, or treatment of disease.

[0262] In some embodiments, when the first linker payload and / or the second linker payload react with a reduced thiol group, the first linker payload and / or the second linker payload are MC-GGFG-DXd, MC-VC-PAB-MMAE, MC-VC-PAB-MMAD, MC-VC-PAB-MMAF, respectively.

[0263] In some embodiments, without limitation, the first thio bridging reagent having a first linker payload and the second thio bridging reagent having a second linker payload are each selected from the group consisting of: [ka]

[0264] In some embodiments, the payload of the first thio bridging reagent having the first linker payload and the payload of the second thio bridging reagent having the second linker payload are different. In some embodiments, the linker of the first thio bridging reagent having the first linker payload and the linker of the second thio bridging reagent having the second linker payload can be different. In some embodiments, the linker of the first thio bridging reagent having the first linker payload and the linker of the second thio bridging reagent having the second linker payload can be the same. In some embodiments, the thio bridging reagent of the first thio bridging reagent having the first linker payload and the thio bridging reagent of the second thio bridging reagent having the second linker payload can be different. In some embodiments, the thio bridging reagent of the first thio bridging reagent having the first linker payload and the thio bridging reagent of the second thio bridging reagent having the second linker payload can be the same.

[0265] In some embodiments, the antibody is not particularly limited, and depending on the antigen associated with the disease, one skilled in the art can select an appropriate antibody useful in the bioconjugation process of the present application.

[0266] In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a monospecific antibody, or a multispecific antibody.

[0267] As used herein, the term "antibody" refers to an immunoglobulin that binds to a specific antigen. Naturally occurring, intact antibodies are composed of two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, and each light chain consists of a variable region and a constant region. Heavy chains in vertebrates are classified into one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM.

[0268] As used herein, the term "Fc region" refers to a monomeric, dimeric, or heterodimeric protein having at least an immunoglobulin CH2 and CH3 domain. The CH2 and CH3 domains may form at least a portion of the dimeric region of a protein / molecule (e.g., an antibody).

[0269] In some embodiments, the antibody is a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding portion thereof.

[0270] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody made by a human or human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody-encoding sequences. The definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0271] As used herein, the term "humanized antibody" refers to a chimeric antibody comprising amino acid residues of the heavy chain variable region (HVR) of non-human origin and amino acid residues of the FRs of human origin. In one embodiment, a humanized antibody comprises substantially all or at least one, typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0272] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0273] In some embodiments, antibody refers to an immunoglobulin, a molecule that contains an antigen-binding site that immunospecifically binds to an antigen. In some embodiments of the present application, the antibody class is IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments of the present application, the antibody class is IgG.

[0274] In some embodiments, the class of the antibody is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody is IgG1 or IgG4.

[0275] In some embodiments of the present application, the antibody comprises at least one mutation in the Fc region, in some embodiments, the at least one mutation modulates effector function or reduces or eliminates Fc-g receptor binding.

[0276] In some embodiments, the one or more mutations stabilize the antibody and / or increase half-life. In some examples, the one or more mutations modulate Fc receptor interactions, reducing or eliminating Fc effector functions such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In further examples, the one or more mutations modulate glycosylation.

[0277] In some embodiments, the one or more mutations are located in the Fc region. In some examples, the Fc region comprises mutations at residue positions L234, L235, or a combination thereof. In some examples, the mutations comprise L234 and L235. In some examples, the mutations comprise L234A and L235A. In some examples, the residue positions are relative to IgG1.

[0278] In some embodiments, the Fc region comprises mutations at residue positions L234, L235, D265, N21, K46, L52, or P53, or a combination thereof. In some examples, the mutations comprise L234 and L235 in combination with mutations at residue positions K46, L52, or P53.

[0279] In some embodiments, the Fc region comprises mutations at L234, L235, and K46. In some examples, the Fc region comprises mutations at L234, L235, and L52. In some examples, the Fc region comprises mutations at L234, L235, and P53. In some examples, the Fc region comprises mutations at D265 and N21. In some examples, residue positions are relative to IgG1.

[0280] In some examples, the Fc region comprises L234A, L235A, D265A, N21G, K46G, L52R, or P53G, or a combination thereof. In some examples, the Fc region comprises L234A and L235A in combination with K46G, L52R, or P53G. In some examples, the Fc region comprises L234A, L235A, and K46G. In some examples, the Fc region comprises L234A, L235A, and L52R. In some examples, the Fc region comprises L234A, L235A, and P53G. In some examples, the Fc region comprises D265A and N21G. In some examples, residue positions are relative to IgG1.

[0281] In some embodiments, the Fc region comprises mutations at residue positions L233, L234, D264, N20, K45, L51, or P52. In some examples, the Fc region comprises mutations at L233 and L234. In some examples, the Fc region comprises mutations at L233 and L234 in combination with mutations at residue positions K45, L51, or P52. In some examples, the Fc region comprises mutations at L233, L234, and K45. In some examples, the Fc region comprises mutations at L233, L234, and L51. In some examples, the Fc region comprises mutations at L233, L234, and K45. In some examples, the Fc region comprises mutations at L233, L234, and P52. In some examples, the Fc region comprises mutations at D264 and N20. In some examples, positions equivalent to residues L233, L234, D264, N20, K45, L51, or P52 in an IgG1, IgG2, IgG3, or IgG4 framework are contemplated.

[0282] In some embodiments, the Fc region comprises L233A, L234A, D264A, N20G, K45G, L51R, or P52G. In some examples, the Fc region comprises L233A and L234A. In some examples, the Fc region comprises L233A and L234A in combination with K45G, L51R, or P52G. In some examples, the Fc region comprises L233A, L234A, and K45G. In some examples, the Fc region comprises L233A, L234A, and L51R. In some examples, the Fc region comprises L233A, L234A, and K45G. In some examples, the Fc region comprises L233A, L234A, and P52G. In some examples, the Fc region comprises D264A and N20G.

[0283] In some embodiments, the human IgG constant region is selected from the group consisting of, for example, Natsume et al., 2008 Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4):2571-5; Moore et al., 2010 mAbs, 2(2):181-189; Lazar et al., 2006 PNAS, 103(11):4005-4010, Shields et al., 2001 JBC, 276(9):6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18):8882-8890; Stavenhagen et al., 2008 Advan.Enzyme Regul., 48:152-164; Alegre et al., 1992 J Immunol, 148:3461-3468; Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11. Amino acid modifications are made to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0284] In some embodiments, the IgG1, IgG2, IgG3, or IgG4 antibody is a human or humanized antibody. IgG1, IgG2, IgG3, or IgG4 information can be obtained from NCBI or UniProt (https: / / www.uniprot.org / ).

[0285] In some embodiments, the antibody is a bispecific antibody. In some embodiments of the present application, the antibody is an IgG1-like bispecific antibody.

[0286] In some embodiments, one skilled in the art can select an appropriate method for preparing bispecific antibodies. In some embodiments of the present application, bispecific antibodies can be synthesized using a variety of techniques, including knobs-into-hole technology (Ridgway JBB, Presta LG, Paul C. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization[J]. Protein Engineering(7):617(2023-08-11).), format chain exchange (FORCE) technology, common light chain format technology (De Nardis C, Hendriks LJA, Poirier E, et al. A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1[J]. Journal of Biological Chemistry, 2017:jbc.M117.793497.), controlled Fab arm exchange technology (Yanakieva De, Pekar L, Evers A, et al. Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities[J]. MABS, 2022, 14(1), e2018960), CrossMAb technology (Klein C, Schaefer W, Regula J T. The use of CrossMAb technology for the generation of bi- and multispecific antibodies[J].MABS, 2016, 8(6), P1010-P1020.), or a combination thereof.

[0287] As used herein, the term "knobs-into-holes" is used in its broadest sense and encompasses a variety of situations, such as the CH1 domain of one heavy chain having a knob mutation and the CH1 domain of the other heavy chain having a hole mutation, the CH2 domain of one heavy chain having a knob mutation and the CH2 domain of the other heavy chain having a hole mutation, and / or the CH3 domain of one heavy chain having a knob mutation and the CH3 domain of the other heavy chain having a hole mutation. For example, "knobs-into-holes" can generally refer to modifications within the interface between two antibody heavy chains in the CH3 domain. i) In the CH3 domain of one heavy chain (first CH3 domain), an amino acid residue is replaced with another amino acid residue having a large side chain, thereby forming a protrusion ("knob") at the interface of the first CH3 domain. ii) In the CH3 domain of the other heavy chain (the second CH3 domain), an amino acid residue is replaced with another amino acid residue having a smaller side chain, thereby forming a cavity ("hole") within the interface of the second CH3 domain into which the knob ("knob") of the first CH3 domain can be positioned.

[0288] In some embodiments, the antibody is selected from any one of a cytotoxic antibody, an inhibitor of cell proliferation, a modulator of cell activation and interaction, a modulator of the human immune system, a neutralizing antigen, an antibody immunologically specific for a viral antigen, or an antibody immunologically specific for a microbial antigen.

[0289] In some embodiments of the present application, the antibody may be a target-specific antibody, including, but not limited to, an anti-HER2 antibody, an anti-FAP antibody, an anti-OX-40 antibody, an anti-41BB antibody, an anti-angiogenic factor 2 antibody, an anti-IL-4Rα antibody, an anti-BCMA antibody, an anti-Blys antibody, an anti-BTNO2 antibody, an anti-C5 antibody, an anti-CD122 antibody, an anti-CD13 antibody, an anti-CD133 antibody, an anti-CD137 antibody, an anti-CD138 antibody, an anti-CD16a antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD2 2 antibody, anti-CD27 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD38 antibody, anti-CD40 antibody, anti-CD47 antibody, anti-CD8 antibody, anti-CD79 antibody, anti-CEA antibody, anti-CGP R / CGRPR antibody, anti-CSPGs antibody, anti-CTLA4 antibody, anti-CTLA-4 domain antibody, anti-DLL-4 antibody, anti-EGFR antibody, anti-EpCAM antibody, anti-factor IXa antibody, anti-factor X antibody, anti-GITR antibody , anti-GP130 antibody, anti-Her3 antibody, anti-HSG antibody, anti-ICOS antibody, anti-IGF1 antibody, anti-IGF1 / 2 antibody, anti-IGF-1R antibody, anti-IGF2 antibody, anti-IGFR antibody, anti-IL-1 antibody, anti-IL-12 antibody body, anti-IL-12p40 antibody, anti-IL-13 antibody, anti-IL-17A antibody, anti-IL-1β antibody, anti-IL-23 antibody, anti-IL-5 antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-Lag-3 antibody, anti-LAG3 antibody, Anti-MAG antibody, anti-Met antibody, anti-NgR antibody, anti-NogoA antibody, anti-OMGp antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-PDGFR antibody, anti-PDL-1 antibody, anti-PSMA antibody, anti-RGMA antibody, anti-RGMB The antibody may be an anti-SARS-CoV-2 antibody, an anti-Te38 antibody, an anti-TIM-3 antibody, an anti-TNF antibody, an anti-TNFα antibody, an anti-TROP-2 antibody, an anti-TWEAK antibody, an anti-VEGF antibody, or an anti-VEGFR antibody.

[0290] In some embodiments, the antibodies are target specific and target HER2 (human epidermal growth factor receptor 2), TROP2 (TACSTD2, tumor-associated calcium signaling factor 2), BCMA (TNFRSF17, TNF receptor superfamily member 17).

[0291] In some embodiments of the present application, the antibody can be trastuzumab, sacituzumab, belantamab, risankizumab, eptanumab, teprotumab, polatuzumab, tafasitamab, rovelizumab, romosozumab, dostallimb, enfortum, or ublituximab.

[0292] In some embodiments, the antibody is trastuzumab, sacituzumab, or belantamab.

[0293] In some embodiments, the antibodies are commercially available or are produced by any method known to one of skill in the art.

[0294] In some embodiments, the method of preparing an ADC having D6 comprises the steps of: (1) To a solution of trastuzumab (0.01 mM to 0.2 mM) in BES buffer (20 mM, pH 7.0), TCEP (4 eq to 15 eq) and ZnCl2 (Zn / TCEP molar ratio 0.1:1 to 30:1) were added, and the reaction mixture was mixed by vortexing. The reaction mixture was then incubated at 4°C for 4 to 18 hours. (2) EDTA (0.6 mM) and MC-VC-PAB-MMAE (0.08 mM) in DMA were introduced and reacted at room temperature for 1 hour. (3) Optionally, a cysteine is introduced to consume excess MC-VC-PAB-MMAE. (4) The reaction mixture was subjected to purification using a desalting column.

[0295] In some embodiments, a method for preparing a bi-payload ADC having D6+D2 comprises the steps of: (1) ZnCl2 (Zn / TCEP molar ratios from 0.1:1 to 30:1) and the first reducing agent TCEP (4 eq-15 eq) were added to a solution of the monoclonal antibody trastuzumab (0.01 mM-0.2 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4 °C for 18 h. (2) EDTA-2Na (0.6 mM) and MC-GGFG-DXd (0.1 mM) in DMA were added, and the reaction was continued at room temperature for 1 hour. (3) The resulting product and the second reducing agent TCEP (0.02 mM) were incubated at room temperature for 2 hours. The second linker payload MC-VC-PAB-MMAE (0.05 mM) was added, and the reaction was continued at room temperature for 2 hours. (4) Purify the product using a desalting column.

[0296] In some embodiments, the method of preparing a bi-payload ADC having D6+D1 comprises the steps of: (1) ZnCl2 (Zn / TCEP molar ratios from 0.1:1 to 30:1) and the first reducing agent TCEP (4 eq-15 eq) were added to a solution of the monoclonal antibody trastuzumab (0.01 mM-0.2 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4 °C for 18 h. (2) EDTA-2Na (0.6 mM) and MC-GGFG-DXd (0.1 mM) in DMA were added, and the reaction was continued at room temperature for 1 hour. (3) The resulting product and the second reducing agent TCEP (0.02 mM) were incubated at room temperature for 2 hours. A thiobridge reagent with the reactive group dibromomaleimide-PEG4-N3 (0.012 mM) was added, and the reaction was continued at room temperature for 2 hours. Then, the second linker payload DBCO-Cy3 (0.05 mM) was added, and the reaction was continued at room temperature for 4 hours. (4) Purify the product using a desalting column.

[0297] In some embodiments, the method of preparing an ADC having D3 comprises the steps of: (1) ZnCl2 (Zn / TCEP molar ratios from 0.1:1 to 30:1) and the first reducing agent TCEP (4 eq-15 eq) were added to a solution of the monoclonal antibody trastuzumab (0.01 mM-0.2 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4 °C for 18 h. (2) To react with the reduced thiol group obtained in step (1), EDTA (0.6 mM) and bismaleimide-DBCO (0.045 mM) were reacted at room temperature for 3 hours, and the product was then recovered using a desalting column to obtain trastuzumab-[bismaleimide-DBCO]3. (3) Trastuzumab-[bismaleimide-DBCO]3 and N3-Cy3 (0.06 mM) are incubated in BES buffer (20 mM, pH 6.7), and the reaction temperature is 25°C for 6 hours. Trastuzumab-[bismaleimide-DBCO-N3-Cy3]3 is then recovered using a desalting column.

[0298] In some embodiments, a method of preparing an ADC having D0+D2 comprises the steps of: (1) ZnCl2 (Zn / TCEP molar ratios from 0.1:1 to 30:1) and the first reducing agent TCEP (4 eq-15 eq) were added to a solution of the monoclonal antibody trastuzumab (0.01 mM-0.2 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4 °C for 18 h. (2) To react with the reduced thiol group obtained in step (1), EDTA (0.6 mM) and (2-aminoethyl)maleimide (0.04 mM) were added at room temperature for 3 hours, and the product was then recovered using a desalting column to obtain trastuzumab-maleimide. (3) Trastuzumab-maleimide and the reducing agent TCEP (0.02 mM) were incubated at room temperature for 2 hours, and the second linker payload MC-VC-PAB-MMAE (0.05 mM) was added, and the reaction was continued at room temperature for 2 hours. (4) Purify the product using a desalting column.

[0299] In some embodiments, the method of preparing an ADC having D0+D1 comprises the steps of: (1) ZnCl2 (Zn / TCEP molar ratios from 0.1:1 to 30:1) and the first reducing agent TCEP (4 eq-15 eq) were added to a solution of the monoclonal antibody trastuzumab (0.01 mM-0.2 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4 °C for 18 h. (2) To react with the reduced thiol group obtained in step (1), EDTA (0.6 mM) and (2-aminoethyl)maleimide (0.04 mM) were added at room temperature for 3 hours, and the product was then recovered using a desalting column to obtain trastuzumab-maleimide. (3) Trastuzumab-maleimide and the second reducing agent TCEP (0.02 mM) were incubated at room temperature for 2 hours. The second thio-bridge reagent with the reactive group dibromomaleimide-PEG4-N3 (0.013 mM) was reacted with the reduced thiol group at 24°C for 3 hours. (4) The resulting product and DBCO-Cy3 (0.02 mM) were incubated in MES (20 mM, pH 6.7) at 25 °C for 8 hours, and trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1 was recovered using a desalting column.

[0300] Various analytical methods can be used to measure the yield and isomeric mixture of ADCs. In some embodiments of the present application, the analytical method is HIC-HPLC. HIC-HPLC can separate ADCs of antibodies loaded with various numbers of drugs. Drug loading levels can be determined, for example, based on the ratio of absorbance at 250 nm to 280 nm. For example, the drug absorbs at 250 nm and the antibody absorbs at 280 nm. Thus, the 250 / 280 ratio increases with drug loading. Using the bioconjugation process described herein, it has been observed that antibodies with an even number of drugs are generally conjugated to the antibody as a conjugate. This is because disulfide reduction generates an even number of free cysteine thiols. Compared to ADCs produced by conventional conjugation processes, the present ADCs exhibit improved homogeneity.

[0301] Site-specifically modified antibodies In a sixth aspect, the present application provides an antibody having a site-specific modification prepared by the method of the present application.

[0302] In some embodiments, the sequence of the antibody with the site-directed modification is wild-type.

[0303] As used herein, the term "wild-type antibody" refers to a naturally occurring antibody that is free of mutations.

[0304] In some embodiments, the glycosylation of antibodies with site-directed modifications is unaltered.

[0305] In some embodiments, antibodies with site-specific modifications do not refer to antibody engineering, enzymatic techniques, and glycoengineering.

[0306] In some embodiments, an antibody having a site-specific modification is conjugated with Modifying Reagent 1 to form an ADC with D6, D1, D3, D2, D4, or D6. In some embodiments, an antibody having a site-specific modification is conjugated with Modifying Reagent 1 and Modifying Reagent 2 to form a bi-payload ADC with D6+D2, a bi-payload ADC with D6+D1, a bi-payload ADC with D3+D1, a bi-payload ADC with D3+D2, an ADC with D0+D2, an ADC with D0+D1, a bi-payload ADC with D2+D6, a bi-payload ADC with D2+D3, a bi-payload ADC with D1+D6, a bi-payload ADC with D1+D3, an ADC with D0+D6, an ADC with D0+D3, a bi-payload ADC with D2+D4, a bi-payload ADC with D2+D2, a bi-payload ADC with D1+D4, or a bi-payload ADC with D1+D2.

[0307] In some embodiments, an antibody with a site-specific modification is an ADC with D2, an ADC with D4, an ADC with D1, an ADC with D6, an ADC with D3, an ADC with D1+D6, an ADC with D6+D2, an ADC with D6+D1, an ADC with D3+D1, an ADC with D3+D2, an ADC with D0+D6, an ADC with D0+D2, an ADC with D1+D2, an ADC with D1+D4, or an ADC with D2+D4.

[0308] In some embodiments, antibodies with site-specific modifications include trastuzumab-[MC-VC-PAB-MMAE]6, sacituzumab-[MC-VC-PAB-MMAE]6, belantamab-[MC-VC-PAB-MMAE]6, trastuzumab-[MC-VC-PAB-MMAE]2, trastuzumab-[bismaleimide-DBCO]3, trastuzumab-[MC-GGFG-DXd]6[MC-VC-PAB-MMAE]2, trastuzumab-[MC-VC-PAB-MMAE]6[maleimide -PEG4-N3-DBCO-Cy3]1, trastuzumab-[maleimide]6[MC-VC-PAB-MMAE]2, trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1, trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1, trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1[MC-GGFG-DXd]6 or trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]4.

[0309] Pharmaceutical compositions containing site-specifically modified antibodies In a seventh aspect, the present application provides a pharmaceutical composition comprising an antibody having a site-specific modification according to the present application and one or more pharmaceutically acceptable carriers.

[0310] The pharmaceutical compositions provided herein can be formulated by any method known in the art, for example, the pharmaceutical compositions provided herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniformity of dosage).

[0311] Pharmaceutical compositions are formulated to be compatible with the intended route of administration (eg, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0312] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonicity agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, ingredients known in the art, or various combinations thereof.

[0313] Suitable ingredients include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars or cyclodextrins. Suitable antioxidants include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, inclusion of one or more antioxidants, such as methionine, in a composition comprising an antibody or antigen-binding fragment thereof and a conjugate provided herein reduces oxidation of the antibody or antigen-binding fragment thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Thus, in one embodiment, a pharmaceutical composition is provided comprising one or more antibodies or antigen-binding fragments thereof disclosed herein and one or more antioxidants, such as methionine.

[0314] In some embodiments, the pharmaceutical composition may be a liquid solution, suspension, or emulsion. In some aspects, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or a solid form suitable for forming a liquid solution, suspension, or emulsion. Injectable preparations include sterile and / or non-caseating solutions ready for injection, sterile dry soluble preparations such as lyophilized powders ready for combination with a solvent immediately before use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble preparations ready for combination with a vehicle immediately before use, and sterile and / or non-caseating emulsions. The solution may be aqueous or non-aqueous.

[0315] In some embodiments, the pharmaceutical composition is used in combination with other therapeutic agents. The other therapeutic agents are not particularly limited as long as they can reduce the side effects of the pharmaceutical composition or enhance the efficacy of the pharmaceutical composition. Examples of other therapeutic agents include anti-cancer agents, anti-autoimmune disease agents, anti-emetic agents, and anti-allergic agents.

[0316] In some embodiments, anticancer drugs include erlotinib, bortezomib, fulvestrant, sunitinib, imatinib, mesylate, letrozole, platins such as finasunate, oxaliplatin, carboplatin, and cisplatin, finasunate, fluorouracil, rapamycin, leucovorin, lapatinib, lonafamib, sorafenib, gefitinib, capmutotecin, topotecan, bryostatin, adzelesin, anthracyclines, carzelesin, bizelesin, dolastatins, auristatins, duocarmycins, eleutherobin, taxols such as paclitaxel or docetaxel, cyclosporin, doxorubicin, cyclosporin ... Other alkylating agents such as rubicin, vincristine, prednisone or prednisolone, mechlorethamine, chlorambucil and ifosfamide, antimetabolites such as azathioprine or mercaptopurine, other microtubule inhibitors (vinca alkaloids such as vincristine, vinblastine, vinorelbine and vindesine and taxanes), podophyllotoxins (etoposide, teniposide, etoposide phosphate, epipodophyllotoxin), topoisomerase inhibitors, actinomycin, daunorubicin, barbicin, idarubicin, epirubicin, bleomycin, plicamycin, mitomycin and other cytotoxins.

[0317] In some embodiments, anti-autoimmune disease agents may include, but are not limited to, ibuprofen, loxoprofen, naproxen, diclofenac, indomethacin, meloxicam, lornoxicam, nabumetone, celecoxib, paracetamol, glucocorticoids, azathioprine, cyclophosphamide, and the like.

[0318] In some embodiments, patients may experience nausea during and after administration of the ADC of the present application. Therefore, antiemetic drugs can be administered to prevent nausea (upper respiratory tract inflammation) and vomiting. Antiemetic drugs include, but are not limited to, aprepitant, ondansetron, granisetron hydrochloride, lorazepam, dexamethasone, prochlorperazine, casopitant, etc.

[0319] In some embodiments, patients may experience allergic reactions during and after administration of the ADC of the present application. Therefore, to minimize the risk of allergic reactions, anti-allergic agents can be administered. Anti-allergic agents include dexamethasone, beclomethasone, hydrocortisone, prednisolone, methylprednisolone, hydroxyzine, cyproheptadine, bronchodilators, terbutaline, etc.

[0320] Use of TCEP or its salts In an eighth aspect, the present application provides use of TCEP or a salt thereof in the preparation of an antibody with site-specific modifications.

[0321] In some embodiments, the site-specifically modified antibody is modified by selectively reducing the interchain S-S bonds, hi some embodiments, the site-specifically modified antibody is modified by selectively reducing three interchain S-S bonds.

[0322] In some embodiments, TCEP or a salt thereof and a transition metal ion are used together.

[0323] In some embodiments, TCEP and transition metal ions are used together to selectively reduce three of the four interchain disulfide bonds of an antibody by using a specific molar ratio of TCEP to antibody, a specific incubation time of TCEP, and introducing a metal chelator after the incubation reaction in step (a).

[0324] Use of site-specifically modified antibodies In a ninth aspect, the present application provides the use of an antibody having a site-specific modification according to the present application in the manufacture of a therapeutic agent for the diagnosis, prevention, or treatment of a disease.

[0325] In a tenth aspect, the present application provides a method for preventing or treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody having a site-specific modification according to the present application.

[0326] As used herein, the term "treating any disease" refers to alleviating or ameliorating the disease (i.e., delaying or preventing the onset of the disease or at least one of its clinical symptoms), or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease (including those not discernible to the patient). In the case of cancer, "treating" refers to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or a combination thereof. In the case of tumors, "treating" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, delaying the development of tumors, or a combination thereof.

[0327] As used herein, the term "prevention of any disease" refers to the prophylactic treatment of the disease or the delay in the onset or progression of the disease.

[0328] As used herein, the term "subject" refers to a mammal, a primate (e.g., a human, male or female), a dog, a rabbit, a guinea pig, a pig, a rat, or a mouse. In one embodiment, the subject is a primate. In yet another embodiment, the subject is a human.

[0329] As used herein, the term "therapeutically effective amount" refers to the amount of a site-specifically modified antibody of the present application that elicits a biological or medical response in a subject, e.g., improves symptoms, alleviates a condition, slows or delays the progression of a disease, or prevents a disease. The therapeutically effective amount will vary depending on the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition. In some embodiments of the present application, the therapeutically effective amount is based on a variety of factors, including the type of disease, age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular antibody used. In some embodiments of the present application, the therapeutically effective amount can vary widely but can be routinely determined using standard methods. In some embodiments of the present application, the therapeutically effective amount can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values.

[0330] In some embodiments, the disease is a tumor or cancer, hi some embodiments, the disease is an autoimmune disease, or the like.

[0331] In some embodiments, cancer includes, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia or lymphoid malignancies, etc. More specific examples of cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0332] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the present application described herein are obvious and may be made using suitable equivalents without departing from the scope of the present disclosure or the embodiments disclosed herein. Now, having described the present disclosure in detail, the same will be more clearly understood by reference to the following examples. Furthermore, unless otherwise specified, the reagents and solvents described herein are readily available from commercial suppliers. [Example]

[0333] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0334] Reagents and manufacturers Trastuzumab is commercially available from Roche. Sacituzumab and belantamab are commercially available from MedChemExpress. MC-VC-PAB-MMAE is commercially available from Levena biopharma. DMA (dimethylacetamide) is commercially available from Aldrich Sigma. TCEP is commercially available from Bidepharm. EDTA is commercially available from Aladdin. DHAA is commercially available from Aladdin. (2-aminoethyl)maleimide is commercially available from Bidepharma. Buffers are commercially available from Macklin.

[0335] Homogeneity Assay: Analysis was performed using HIC-HPLC (Agilent 1200) with a TSK Gel Butyl NPR column (2.5 μm, 4.6 mm × 35 mm) (procured from Tosoh Biosciences) at a flow rate of 0.5 mL / min at 25 °C. Solvent A was 50 mM K2HPO4·3H2O and 1.5 M (NH4)2SO4. Solvent B was 75% v / v 21.3 mM KH2PO4, 28.6 mM K2HPO4, and 25% v / v isopropanol. The sampling temperature was 4 °C. The washing procedure was as follows: [Table 1]

[0336] Example 1: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate by the process of the present application To a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0) were added TCEP (0.048 mM) and ZnCl2 (0.024 mM), and the reaction mixture was vortex mixed and then incubated at 4°C for 18 hours. EDTA (0.6 mM) and MC-VC-PAB-MMAE (0.08 mM) in DMA were introduced and reacted at room temperature for 1 hour. The reaction mixture was purified using a desalting column (Thermo, model number: 40K, 0.5 mL, REF: 87766, Lot: SJ251704).

[0337] Example 2: Preparation of sacituzumab-[MC-VC-PAB-MMAE]6 conjugate by the process of the present application The method of Example 2 is similar to that of Example 1, except that the antibody is sacituzumab, the buffer system is MES buffer, and Zn 2+ The molar ratio of TCEP to TCEP is 0.87:1.

[0338] Example 3: Preparation of Belantamab-[MC-VC-PAB-MMAE]6 Conjugate by the Process of the Present Application The method of Example 3 is similar to that of Example 1, except that the antibody is belantamab, the buffer system is MES buffer, and Zn2+ The molar ratio of TCEP to TCEP is 0.87:1.

[0339] The results of the homogeneity assay were as follows: [Table 2]

[0340] As shown in Table 1 and Figures 1 to 3, MC-VC-PAB-MMAE was successfully conjugated to trastuzumab, sacituzumab, and belantamab, and the D6 content in Examples 1 to 3 was generally up to 80%, e.g., 90%, 85%, and 82%. These results clearly demonstrate that the homogeneity of the D6-containing ADCs prepared by the method of the present application was significantly improved.

[0341] Example 4-13: Preparation of antibody-[MC-VC-PAB-MMAE]6 conjugate (varying molar ratios of TCEP to antibody and / or reduction time in step (1)) The methods of Examples 4 to 13 are the same as those of Example 1, except for the concentrations of ZnCl2 and TCEP in step (1) and / or the reduction time in step (1). In Examples 5 and 6, the antibody is belantamab. The concentrations of ZnCl2 and TCEP, and the reduction time in step (1) are as follows: [Table 3]

[0342] Comparative Example 11: Preparation of antibody-[MC-VC-PAB-MMAE]2 conjugate (ADC with D2) (1) To a solution of trastuzumab (0.012 mM, MES buffer, pH 6.7, 20 mM), ZnCl2 (0.24 mM) and TCEP (0.02 mM) were added, and the reaction mixture was allowed to stand at 4°C for 4 hours. (2) Zn 2+ EDTA (0.6 mM) was added to capture (3) MC-VC-PAB-MMAE (0.06 mM) in DMA was introduced, and the reaction was continued at 24 °C for 30 min. (4) To remove excess MC-VC-PAB-MMAE, cysteine (purchased from Aladdin, 0.08 mM) was added. (5) The reaction mixture was purified using a desalting column.

[0343] The homogeneity assay results for Examples E4-E13 and Comparative Example 11 were as follows: [Table 4]

[0344] As shown in Table 2, compared to Comparative Example 11, when the TCEP to antibody molar ratio was 3:1 to 13:1, the D6 content reached a maximum of 55%, 80%, or even 85% or 87%. When the TCEP to antibody molar ratio was 8:1 to 13:1, the reduction time in step (1) was shortened to 6 hours. When the TCEP to antibody molar ratio was 8:1 to 12:1, the reduction time in step (1) was 6 hours, and the D6 content reached a maximum of 87% or 89%. These results indicated that the TCEP / antibody molar ratio plays an important role in determining the D6 content and selective reduction.

[0345] Examples 14-28: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (Zn 2+ and TCEP molar ratios are different, and / or the reduction time in step (1) is different) The methods of Examples 14-28 were similar to those of Example 1, with the only differences being the concentrations of ZnCl2 and / or TCEP and / or the reduction time in step (1) as shown in Table 3.

[0346] [Table 5]

[0347] Comparative Example 1-4: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (Zn 2+and TCEP molar ratios are different) The method of Comparative Example 1 is similar to that of Example 1, Comparative Example 2 is similar to that of Example 8, Comparative Example 3 is similar to that of Example 10, and Comparative Example 4 is similar to that of Example 12, except that the concentration of ZnCl in step (1) is 0.

[0348] The results of the homogeneity assay were as follows: [Table 6]

[0349] As shown in Table 4, Zn 2+ As the molar ratio of Zn to TCEP increased from 0.11:1 to 27.27:1, the D6 content increased to 75%, then 80%, 85%, and 90%. 2+ When the concentration of was 0, the content of D6 was as low as 10%, indicating that metal transition ions are very important for improving the reduction selectivity and homogeneity in the conjugate with D6.

[0350] Examples 29-32: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates (varying reduction times in step (1)) The methods of Examples 29-32 were similar to those of Example 1, except for the reduction time in step (1), which is shown in Table 5. On the other hand, in Examples 29-32, Zn 2+ The molar ratio of TCEP to trastuzumab was 0.22:1, and the molar ratio of TCEP to trastuzumab was 9:1.

[0351] The results of the homogeneity assay were as follows: [Table 7]

[0352] As shown in Table 5, when the molar ratio of TCEP to antibody was 9:1 and the reduction time in step (1) was 4 to 10 hours, the D6 content reached a maximum of 86% and 90%. This indicates that increasing the molar ratio of TCEP to antibody in the method presented here can reduce the cost of reduction time.

[0353] Examples 33-36: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates (varying reduction temperatures in step (1)) The methods of Examples 33-36 are similar to those of Example 1, with the exception of the reduction temperature, the amount of Zn in step (1), and the like. 2+ and TCEP, and / or the molar ratio of TCEP to antibody, as shown below.

[0354] [Table 8]

[0355] The results of the homogeneity assay were as follows: [Table 9]

[0356] As shown in Table 6, when the temperature of the reducing agent in step (1) was between 0°C and 25°C, the D6 content reached a maximum of 80%, 85%, or even 88%.

[0357] Examples 37-47: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates using different buffer systems The method for Examples 37-47 was the same as Example 1, except that the BES buffer in Example 1 was replaced with a different buffer in Examples 37-47.

[0358] [Table 10]

[0359] Comparative Example 5-6: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate using different buffer systems The method of Comparative Examples 5-6 is similar to that of Example 1, except that the BES buffer of Example 1 is replaced with a different buffer in Comparative Examples 5-6.

[0360] [Table 11]

[0361] The results of the homogeneity assay were as follows: [Table 12]

[0362] As shown in Table 9 and Figures 18-30, the type of buffer system was shown to affect the content of D6 by affecting the reduction kinetics and selectivity. The buffer systems of Examples 37-47 are useful for increasing the content of D6.

[0363] Examples 48-51: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates at different pH values The methods of Examples 48-51 are similar to those of Example 1, with the difference being the pH value of the buffer system.

[0364] [Table 13]

[0365] The results of the homogeneity assay were as follows: [Table 14]

[0366] As shown in Table 11 and Figures 31-34, the pH values of the buffer systems of Examples 48-51 were found to be useful in increasing the content of D6.

[0367] Examples 52-55: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates in different concentrations in buffer systems The methods of Examples 52-55 are similar to those of Example 1, with the difference being the concentration of the buffer system.

[0368] [Table 15]

[0369] The results of the homogeneity assay were as follows: [Table 16]

[0370] As shown in Table 13 and Figures 35-38, the concentrations of the buffer systems in Examples 51-54 were shown to be useful in increasing the content of D6.

[0371] Example 56: Preparation of trastuzumab-[bismaleimide-DBCO]3 1. Synthesis of bismaleimide-DBCO (thio bridging reagent) [ka]

[0372] Intermediate 2: To a mixture of 1 (6.5 g, 50.0 mmol, 1.0 eq) in DMF (100 mL) was added DBU (30.7 g, 0.2 mol, 4.0 eq). The mixture was stirred at 80 °C for 1 h, and then 2-bromobenzyl acetate (25.4 g, 0.11 mol, 2.2 eq) was added. The resulting mixture was stirred at 80 °C for 16 h. The mixture was poured into ice water (600 mL) and extracted with EtOAc (200 mL*3). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, and filtered. The filtrate was concentrated and purified by flash column chromatography (EtOAc / petroleum ether = 0-40%) to give product 2 (4.5 g, 21.0%) as a white solid.

[0373] Intermediate 3: To a mixture of intermediate 2 (4.5 g, 10.58 mmol, 1.0 eq) in DMF (50 mL) was added DBU (3.22 g, 21.16 mmol, 2.0 eq). The mixture was stirred at 80 °C for 0.5 h, and tert-butyl N-(2-bromoethyl)carbamate (3.56 g, 15.87 mmol, 1.5 eq) was added. The resulting mixture was stirred at 80 °C for 6 h. TLC showed that compound 1 was completely consumed. The mixture was poured into ice water (300 mL) and extracted with EtOAc (100 mL*3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, and filtered. The filtrate was concentrated and purified by flash column chromatography (EtOAc / petroleum ether = 0-40%) to give product 3 (4.8 g, 79.8%) as a white solid.

[0374] Intermediate 4: To a mixture of intermediate 3 (4.8 g, 8.44 mmol, 1.0 eq) in THF (50 mL) was added Pd / C (500 mg, 10% Pd / C, wet with approximately 55% water). The mixture was degassed and purged with hydrogen three times. The resulting mixture was stirred under a hydrogen atmosphere at room temperature for 2 hours. TLC showed that intermediate 3 was completely consumed. The mixture was filtered through a Celite pad, and the filtrate was concentrated to give product 4 (3.0 g, 91.5%) as a white solid.

[0375] Intermediate 5: To a mixture of intermediate 4 (50 mg, 1.00 mmol, 1.0 eq) in DMF (5 mL), HATU (0.92 g, 2.4 mmol, 2.4 eq) and DIPEA (0.65 g, 5.0 mmol, 5.0 eq) were added. The mixture was stirred at room temperature for 0.5 h, and 4a (0.51 g, 2.0 mmol, 2.0 eq) was added. The resulting mixture was stirred at room temperature for 2 h. LCMS showed that intermediate 4 was completely consumed. The mixture was neutralized with hydrochloric acid (0.5 M, 2 mL) and purified on a RP column (water / MeCN = 10-70%). The eluate was lyophilized to give product 5 (0.51 g, 80.3%) as a white solid.

[0376] Intermediate 6: To a solution of intermediate 5 (100 mg, 0.16 mmol, 1.0 eq) in DCM (2 mL) was added TFA (100 μL). The mixture was stirred at room temperature for 1 h. LCMS showed that intermediate 5 was completely consumed. The mixture was concentrated, and the residue was taken up with water (10 mL) and lyophilized to give product 6 (100 mg, 97.8%) as a white solid.

[0377] Compound 7 (bismaleimide-DBCO): To a mixture of intermediate 6 (50 mg, 93.9 μmol, 1.0 eq) in DCM (2 mL), 6a (37.8 mg, 93.9 μmol, 1.0 eq) was added, followed by DIPEA (17 μL, 93.9 μmol, 1.0 eq). The mixture was stirred at room temperature for 1 h. LCMS showed that intermediate 6 was completely consumed. The mixture was concentrated, and the residue was purified by preparative HPLC (water / MeCN = 10-50%). The eluate was lyophilized to give product 7 (28.8 mg, 3.41%) as a white solid. MS [M+H] + =820.19, C 40 H 37 N9O 11 The calculated exact mass is 819.26. 1 H NMR (400 MHz, DMSO-d6):δ 8.17 (t, J = 6.0 Hz, 2H), 7.85 (d, J = 6.4 Hz, 1H), 7.64 (dd, J = 18.4, 7.3 Hz, 2H), 7.47 (d, J = 10.3 Hz, 3H), 7.38 - 7.24 (m, 3H), 6.96 (s, 4H), 5.02 (d, J = 14.0 Hz, 1H), 4.22 (s, 4H), 3.68 (t, J = 6.6 Hz, 2H), 3.65-3.58 (m, 4H), 3.43 (t, J = 6.2 Hz, 3H), 3.24-3.20 (m, 3H), 3.16-3.10 (m, 5H).

[0378] 2. Preparation of trastuzumab-[bismaleimide-DBCO] (1) To a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), TCEP (0.048 mM) and ZnCl2 (0.024 mM) were added, and the reaction mixture was vortexed and then incubated at 4°C for 18 hours. (2) To react with the reduced thiol group obtained in step (1), EDTA (0.6 mM) and bismaleimide-DBCO (0.045 mM) were reacted at room temperature for 1 hour, and the product was then recovered using a desalting column to obtain trastuzumab-[bismaleimide-DBCO]3.

[0379] The results of the homogeneity assay were as follows: [Table 17]

[0380] As shown in the table, the content of trastuzumab-[bismaleimide-DBCO]3 reached approximately 86%, indicating that the process of this method is beneficial for site-specific modification of antibodies with D3 and improving homogeneity.

[0381] Example 57: Preparation of trastuzumab-[MC-GGFG-DXd6[MC-VC-PAB-MMAE]2 (ADC with D6+D2) (1) To a solution of the monoclonal antibody trastuzumab (0.012 mM) in BES buffer (pH 7.0, 20 mM), ZnCl2 (0.048 mM) and the reducing agent TCEP (0.048 mM) were added, and the reaction mixture was allowed to stand at 4°C for 18 hours. (2) EDTA-2Na (0.6 mM) and MC-GGFG-DXd (0.1 mM) were introduced into the DMA, and the reaction was continued at room temperature for 1 hour. The product was then purified using a desalting column. (3) The resulting product and the second reducing agent TCEP (0.02 mM) were incubated at room temperature for 3 hours. The second linker payload MC-VC-PAB-MMAE (0.05 mM) was added, and the reaction was continued at room temperature for 2 hours. (4) Purify the product using a desalting column.

[0382] Here, an average of about six drug molecules MC-GGFG-DXd were bound to trastuzumab, and an average of about two drug molecules MC-VC-PAB-MMAE were bound to trastuzumab.

[0383] The results of the homogeneity assay were as follows: [Table 18]

[0384] As shown in the table above, the content of ADC with D6+D2 was approximately 87.10%, which indicates that the process of this method is beneficial for site-specifically modifying antibodies with D6+D2 and improving homogeneity.

[0385] Example 58: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6[maleimide-PEG4-N3-DBCO-Cy3]1 (ADC with D6+D1) 1. Synthesis of dibromomaleimide-PEG4-N3 [ka]

[0386] To a solution of 3,4-dibromomaleimide (127 mg, 0.5 mmol, 1 eq) and N-methylmorpholine (0.22 mL, 2 mmol, 4 eq) in THF (3.5 mL) was added chloromethyl chloroformate (0.18 mL, 2 mmol, 4 eq) and the mixture was stirred at room temperature for 20 min. DCM (10 mL) was then added, the organic phase was washed with HO, dried over MgSO, and the solvent was removed in vacuo to give the title product 1 (139 mg, 0.4 mmol, 80%).

[0387] A solution of azido-PEG4-amine (105 mg, 0.4 mmol, 1 eq, Xi'an Confluore Biological Technology Co., Ltd) in dichloromethane (2 mL) was added to a stirred solution of product 1 (139 mg, 0.4 mmol, 1 eq) in dichloromethane (2 mL).

[0388] After 30 min, dichloromethane (6 mL) was added, and the solution was washed with 0.68 M acetate buffer pH 5 (10 mL), water (1 mL), and dried over MgSO. After concentration in vacuo, purification by column chromatography (100% EtOAc as mobile phase) afforded dibromomaleimide-PEG-N3 (title product 2) as a pale yellow oil (150 mg, 0.3 mmol, 75%).

[0389] 2. Preparation of trastuzumab-[MC-VC-PAB-MMAE]6[maleimide-PEG4-N3-DBCO-Cy3]1 (1) ZnCl2 (0.0408 mM) and TCEP (0.048 mM) were added to a solution of the monoclonal antibody trastuzumab (0.012 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4 °C for 16 h. (2) EDTA-2Na (0.6 mM) and MC-VC-PAB-MMAE (0.1 mM) were introduced into the DMA, and the reaction was continued at room temperature for 1 hour. The product was then recovered using a desalting column. (3) The resulting product and the second reducing agent TCEP (0.09 mM) were incubated at room temperature for 20 hours. A second thio-bridge reagent with the reactive group dibromomaleimide-PEG4-N3 (0.012 mM) was added, and the reaction was continued at room temperature for 2 hours. Then, the second linker payload DBCO-Cy3 (0.05 mM) was added, and the reaction was continued at room temperature for 4 hours. (4) Purify the product using a desalting column.

[0390] Here, an average of about six drug molecules, MC-VC-PAB-MMAE, were conjugated to trastuzumab, and an average of about one drug molecule, maleimide-PEG4-N3-DBCO-Cy3, was conjugated to trastuzumab.

[0391] The results of the homogeneity assay were as follows: [Table 19]

[0392] As shown in the table above, the content of ADCs having D6+D1 can reach approximately 80%, 85%, or even 90%, indicating that the process of this method is useful for site-specifically modifying antibodies having D6+D1 and improving homogeneity.

[0393] Example 59: Preparation of trastuzumab-[maleimide]6[MC-VC-PAB-MMAE]2 (ADC with D0+D2) (1) To a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), TCEP (0.048 mM) and ZnCl2 (0.024 mM) were added, and the reaction mixture was vortexed and then incubated at 4°C for 18 hours. (2) To react with the reduced thiol group obtained in step (1), EDTA (0.6 mM) and (2-aminoethyl)maleimide (0.1 mM) were added at room temperature for 1 hour, and the product was then recovered using a desalting column to obtain trastuzumab-[maleimide] 6. (3) Trastuzumab-[maleimide] 6 and the reducing agent TCEP (0.02 mM) were incubated at room temperature for 18 hours, and the second linker payload MC-VC-PAB-MMAE (0.05 mM) was added, and the reaction was continued at room temperature for 2 hours. (4) Purify the product using a desalting column.

[0394] The results of the homogeneity assay were as follows: [Table 20]

[0395] As shown in the table above, the content of ADC with D0+D2 was approximately 70.92%, which indicates that the process of this method is beneficial for site-specifically modifying antibodies with D0+D2 and improving homogeneity.

[0396] Example 60: Preparation of trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1 (ADC with D0+D1) (1) To a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), TCEP (0.048 mM) and ZnCl2 (0.024 mM) were added, and the reaction mixture was vortexed and then incubated at 4°C for 18 hours. (2) To react with the reduced thiol group obtained in step (1), EDTA (0.6 mM) and (2-aminoethyl)maleimide (0.1 mM) were added at room temperature for 1 hour, and the product was then recovered using a desalting column to obtain trastuzumab-[maleimide] 6. (3) Trastuzumab-[maleimide]2 and the second reducing agent TCEP (0.02 mM) were incubated at room temperature for 18 hours. The second thio-bridge reagent with the reactive group dibromomaleimide-PEG4-N3 (0.013 mM) was reacted with the reduced thiol group at 24 °C for 3 hours. (4) The resulting product and DBCO-Cy3 (0.02 mM) were incubated in MES (20 mM, pH 6.7) at 25°C for 8 hours, and trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3] was recovered using a desalting column.

[0397] The results of the homogeneity assay were as follows: [Table 21]

[0398] As shown in the table above, the content of ADC with D0+D1 was approximately 92.96%, which indicates that the process of this method is beneficial for site-specifically modifying antibodies with D0+D1 and improving homogeneity.

[0399] Example 61: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate by the process of the present application (1) To a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), TCEP (0.048 mM) and ZnCl2 (0.012 mM) were added, and the reaction mixture was vortexed and then incubated at 4°C for 18 hours. (2) To selectively reoxidize the reduced thiol groups in the Fab region obtained in step (1), DHAA (0.096 mM) was added at 25°C for 1 hour in the dark. (3) To react with the remaining thiol groups in step (2), EDTA (0.6 mM) and MC-VC-PAB-MMAE (0.048 mM) were introduced into the DMA and incubated at room temperature for 1 h. (4) The reaction mixture was purified using a desalting column (Thermo, model number: 40K, 0.5 mL, REF: 87766, Lot: SJ251704).

[0400] The results of the homogeneity assay were as follows: [Table 22]

[0401] As shown in Table 12 and Figure 44, MC-VC-PAB-MMAE was successfully conjugated to trastuzumab, showing that the content of D2 in Example 61 was 92%. These results clearly demonstrate that the homogeneity of the D2-containing ADC prepared by the present method is significantly improved.

[0402] Examples 62-90: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates by the process of the present application The methods of Examples 62-90 are similar to those of Example 61, with the only differences being the parameters of steps (1) and (2). The different parameters are shown in Table 15. Meanwhile, the oxidation time of step (2) is 2 hours in Examples 76-90, and the molar ratio of ZnCl2 to antibody is 2:1 in Example 90.

[0403] [Table 23]

[0404] [Table 24]

[0405] Comparative Examples 7-9: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate (Zn 2+ and TCEP molar ratios are different) The method of Comparative Example 7 is similar to that of Example 80, Comparative Example 8 is similar to that of Example 83, and Comparative Example 9 is similar to that of Example 86, except that the concentration of ZnCl2 in step (1) is 0.

[0406] [Table 25]

[0407] [Table 26]

[0408] As shown in Examples 76-90, when the molar ratio of TCEP to antibody was 4:1 to 10:1, the D6 content was up to 80%, 85%, and 90%, respectively. Meanwhile, the reduction time in step (1) was shortened to 1 hour, reducing the reduction time cost.

[0409] As shown in Tables 17 and 18, the molar ratio of DHAA to antibody played an important role in determining the D2 content and selective oxidation. When the DHAA to antibody molar ratio was between 4:1 and 24:1, the D2 content reached up to 60%, 70%, 80%, 85%, and even 90% or 95%.

[0410] In addition, Zn 2+ The molar ratio of Zn to antibody also affects the content and selective oxidation of D2. As shown in Comparative Examples 7 to 9, 2+ When the concentration of Zn is 0, the D2 content is low at 7.87%. 2+ A molar ratio of 1:1 or 2:1 between D2 and antibody is beneficial in improving the homogeneity of the ADC with D2.

[0411] As shown in Examples 65 and 67, or Examples 68 and 70, by purifying the oxidation product, the content of ADC with D2 increased from 89% to 95%, or from 88% to 94%. These results demonstrate the importance of improving the content of ADC with D2 by purifying the oxidation product before step (3).

[0412] Examples 91-94: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates (varying oxidation temperature and / or time in step (2)) The methods of Examples 91-94 were similar to those of Example 61, except for the oxidation temperature and / or time in step (2), as shown in Table 19.

[0413] The results of the homogeneity assay were as follows: [Table 27]

[0414] As shown in Table 19, when the oxidation temperature in step (2) was 4°C to 37°C and the oxidation time was 1 hour to 48 hours, the D6 content reached a maximum of 70% and 90%.

[0415] Examples 95-110 and Comparative Example 10: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates using different buffer systems The methods of Examples 95-110 and Comparative Example 10 are similar to those of Example 61, except that the BES buffer in Example 61 is replaced with a different buffer in Examples 95-110 and Comparative Example 10. Meanwhile, the oxidation time in step (2) is 2 hours in Examples 95-110 and Comparative Example 10.

[0416] The results of the homogeneity assay were as follows: [Table 28]

[0417] As shown in Table 20, the type and pH of the buffer system influence the reduction rate and selectivity, thereby affecting the D2 content. The buffer systems of Examples 95-110 are useful for increasing the D2 content, and the pH value of the buffer system is 5.8-7.4.

[0418] Example 111: Preparation of trastuzumab-[maleimide-PEG-N-DBCO-MMAE] (ADC with D1) (1) To a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), TCEP (0.048 mM) and ZnCl2 (0.012 mM) were added, and the reaction mixture was vortexed and then incubated at 4°C for 18 hours. (2) To selectively reoxidize the reduced thiol groups in the Fab region obtained in step (1), DHAA (0.066 mM) is added at 25°C for 2 hours. (3) To react with the reduced thiol group obtained in step (3), EDTA (0.6 mM) and the first thio-bridge reagent, dibromomaleimide-PEG4-N3 (0.013 mM), were added, the reaction temperature was set to 25°C, and the reaction time was set to 1 hour. The product was recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1. (4) Trastuzumab-[maleimide-PEG4-N3]1 and DBCO-MMAE (0.02 mM) were incubated in BES buffer (20 mM, pH 7.0), the reaction temperature was 25°C, and the reaction time was 8 hours. (5) The reaction mixture was purified using a desalting column and AKTA with HIC chromatography.

[0419] Purification of AKTA:ADC by HIC chromatography was performed using an AKTA Explorer with a polar MC30-HIC butyl column (4.2 mL, 800 Å, 30 μm) (obtained from Sepax Technologies). Solvent A was 50 mM PB and 1 M (NH4)2SO4. Solvent B was 50 mM PB and 20% v / v isopropanol. Solvent C was 50 mM PB and 2 M (NH4)2SO4. The ADC sample was mixed with solvent C in a 1:1 volumetric ratio, filtered, and then loaded at a flow rate of 2 mL / min. The target components were washed out with solvent A / solvent B between 65% / 35% v / v and 0% / 100% v / v at a flow rate of 3 mL / min at 25 °C. The collected product solution was concentrated and exchanged into His buffer (20 mM, pH 5.5) by ultracentrifugation.

[0420] The results of the homogeneity assay were as follows: [Table 29]

[0421] As shown in the above table, the content of ADC with D1 reached approximately 97.58%, indicating that the process of this method is useful for site-specifically modifying antibodies with D1 and improving homogeneity.

[0422] Example 112: Preparation of trastuzumab-[maleimide-PEG-N-DBCO-MMAE][MC-GGFG-DXd] (ADC with D1+D6) (1) Trastuzumab-[maleimide-PEG-N-DBCO-MMAE]1 prepared from Example 111 (0.008 mM) and TCEP (0.08 mM) was introduced into BES buffer (20 mM, pH 7.0), the reaction temperature was 37°C, and the reaction time was 16 hours. (2) MC-GGFG-DXd (0.14 mM) was introduced into the solution from step (1), and the reaction mixture was allowed to stand at 24 °C for 1 hour. Trastuzumab-[maleimide-PEG-N-DBCO-MMAE]1[MC-GGFG-DXd]6 was then recovered using a desalting column.

[0423] The results of the homogeneity assay were as follows: [Table 30]

[0424] As shown in the above table, the content of ADC with D1+D6 was approximately 82.42%, which indicates that the process of this method is beneficial for site-specifically modifying antibodies with D1+D6 and improving homogeneity.

[0425] Example 113: Preparation of trastuzumab-[maleimide-PEG-N-DBCO-Cy][MC-VC-PAB-MMAE] (ADC with D+D) (1) To a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), TCEP (0.048 mM) and ZnCl2 (0.012 mM) were added, and the reaction mixture was vortexed and then incubated at 4°C for 18 hours. (2) To selectively reoxidize the reduced thiol groups in the Fab region obtained in step (1), DHAA (0.096 mM) is added at 25°C for 2 hours. (3) To react with the reduced thiol group obtained in step (3), EDTA (0.6 mM) and the first thio-bridge reagent, dibromomaleimide-PEG4-N3 (0.013 mM), were added, the reaction temperature was set to 25°C, and the reaction time was set to 1 hour. The product was recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1. (4) Trastuzumab-[maleimide-PEG4-N3]1 (0.013 mM) was incubated in BES buffer (20 mM, pH 7.0) at 25°C for 1 hour, followed by DBCO-Cy3 (0.02 mM) in BES buffer (20 mM, pH 7.0) at 25°C for 8 hours. (5) The reaction mixture was subjected to purification using a desalting column. (6) ZnCl2 (0.8 mM), the second reducing agent TCEP (0.011 mM) / ), and the product of step (5) (0.008 mM) were incubated in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4 °C for 4 hours. (7) EDTA (3 mM) was introduced to introduce Zn 2+ , and MC-VC-PAB-MMAE (0.048 mM) was reacted with the reduced thiol group obtained in step (6), the reaction temperature was 25°C, and the reaction time was 2 hours. (8) The reaction mixture was purified using a desalting column.

[0426] The results of the homogeneity assay were as follows: [Table 31]

[0427] In step (6), one of the interchain disulfide bonds of the ADC having D1 was reduced. As shown in the table above, the content of the ADC having D1+D2 was approximately 70%, which indicates that the process of this method is useful for site-specifically modifying the antibody having D1+D2 and improving the homogeneity.

[0428] Examples 114-115: Preparation of trastuzumab-[maleimide-PEG-N-DBCO-Cy][MC-VC-PAB-MMAE] (ADC with D1+D4) (1) To a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), TCEP (0.048 mM) and ZnCl2 (0.024 mM) were added, and the reaction mixture was vortexed and then incubated at 4°C for 18 hours. (2) To selectively reoxidize the reduced thiol groups in the Fab region obtained in step (1), DHAA (0.12 mM) is added at 25°C for 2 hours. (3) To react with the reduced thiol group obtained in step (2), EDTA (0.6 mM) and a mixture of the first thiobridging reagents, dibromomaleimide-PEG4-N3 (0.028 mM) and DBCO-Cy3 (0.033 mM), were introduced. The reaction temperature was set at 25°C for 2 hours, and the product was recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1. (4) ZnCl (Example 113: 0.024 mM, Example 114: 0.36 mM), the second reducing agent TCEP (0.024 mM), and the product of step (3) (0.008 mM) were incubated in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4° C. for 16 hours. (5) Zn was introduced by adding EDTA (3 mM). 2+ , and MC-VC-PAB-MMAE (0.048 mM) was reacted with the reduced thiol group obtained in step (4), the reaction temperature was 25°C, and the reaction time was 2 hours. (6) The reaction mixture was purified using a desalting column.

[0429] The results of the homogeneity assay were as follows: [Table 32]

[0430] In step (4), two of the interchain disulfide bonds of the ADC having D1 were reduced. As shown in the table above, the content of the ADC having D1+D4 was approximately 80%, which indicates that the process of this method is useful for site-specifically modifying the antibody having D1+D4 and improving the homogeneity.

[0431] Example 116: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]4 (ADC with D2+D4) (1) To a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), TCEP (0.0672 mM) and ZnCl2 (0.024 mM) were added, and the reaction mixture was vortexed and then incubated at 4°C for 16 hours. (2) To selectively reoxidize the reduced thiol groups in the Fab region obtained in step (1), DHAA (0.12 mM) is added at 25°C for 2 hours. (3) To react with the remaining thiol groups in step (2), EDTA (0.6 mM) and MC-VC-PAB-MMAE (0.048 mM) were introduced into the DMA and incubated at room temperature for 1 h. (4) The reaction mixture was subjected to purification using a desalting column. (5) ZnCl2 (0.552 mM), the second reducing agent TCEP (1.2 mM), and the product of step (4) were incubated in BES buffer (pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4 °C for 2.5 hours. (6) EDTA (3 mM) was introduced to introduce Zn 2+ To capture the thiol group and react it with the reduced thiol group obtained in step (5), MC-GGFG-DXd (0.048 mM) was added, and the reaction temperature was 25°C and the reaction time was 2 hours. (7) The reaction mixture was purified using a desalting column.

[0432] The results of the homogeneity assay were as follows: [Table 33]

[0433] As shown in the table above, the content of ADCs with D2(DXd)+D4(MMAE) is generally 90% or up to 90%, indicating that the process of this method is useful for site-specifically modifying antibodies with D2+D4 and improving homogeneity.

[0434] In summary, the method of the present application provides a wide variety of ADCs with high homogeneity without the need for genetic engineering of antibodies and enzymes. For example, the homogeneity of ADCs bearing D6 is 55%, 65%, 70%, 80%, 85%, or even 90% or higher, and the homogeneity of ADCs bearing D2 is 60%, 70%, 75%, or even 80%, 85%, 90%, or 95%. Meanwhile, the method of the present application is compatible with current thiol-reactive linker-drug technology, minimizes conformational changes, and preserves intact Fc function. It also simplifies the process and reduces costs.

[0435] While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents may occur that are presently unforeseen or conceivable to applicant or those skilled in the art. Accordingly, the appended claims, as filed and as they may be amended, are intended to embrace all such alternatives, modifications, variations, improvements, and substantial equivalents. [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to PCT Application No. PCT / CN2022 / 113992 filed on August 22, 2022, PCT Application No. PCT / CN2022 / 119955 filed on September 20, 2022, PCT Application No. PCT / CN2022 / 131519 filed on November 11, 2022, and PCT Application No. PCT / CN2023 / 073070 filed on January 19, 2023. The contents of the prior PCT applications are considered part of this disclosure and are incorporated herein in their entireties.

Claims

1. A method for preparing an antibody having a site-specific modification, wherein the site-specific modification is achieved by selectively reducing three interchain disulfide bonds in the antibody, the method comprising the combined use of tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof and a transition metal ion.

2. A method for preparing an antibody having a site-specific modification, wherein the site-specific modification selectively reduces three interchain disulfide bonds in the antibody, two of which are in the Fab region of the antibody and one of which is in the hinge region of the antibody, the method comprising using TCEP or a salt thereof in combination with a transition metal ion.

3. A method for preparing an antibody having a site-specific modification, characterized in that the site-specific modification selectively reduces three interchain disulfide bonds in the antibody, two of which are in the Fab region of the antibody and one of which is in the hinge region of the antibody, the method comprising the steps of: (a) incubating TCEP or a salt thereof with a transition metal ion in the presence of an antibody in a buffer system to selectively reduce interchain disulfide bonds of the antibody to obtain an antibody having a reduced thiol group, wherein the molar ratio of TCEP to antibody is 3:1 to 15:1, and optionally the molar ratio of TCEP to antibody is 3:1 to 6:

1.

4. 4. The method of claim 3, further comprising introducing a metal chelating agent after step (a).

5. The method according to claim 3, wherein in step (a), the molar ratio of TCEP to antibody is 3.2:1 to 5:1 or 3.5:1 to 4.4:

1.

6. 4. The method of claim 3, wherein in step (a), the incubation temperature is between 0°C and 37°C, optionally, in step (a), the incubation temperature is between 0°C and 25°C, more optionally, in step (a), the incubation temperature is between 0°C and 15°C.

7. 4. The method of claim 3, wherein in step (a), the incubation time is between 3 hours and 24 hours, optionally, in step (a), the incubation time is between 12 hours and 24 hours, more optionally, in step (a), the incubation time is between 16 hours and 20 hours, and most optionally, in step (a), the incubation time is between 16 hours and 18 hours.

8. The method according to claim 5, wherein in step (a), the molar ratio of TCEP to antibody is 3:1 to 6:1, and the incubation time is 10 to 24 hours.

9. 4. The method of claim 3, wherein in step (a), the molar ratio of the transition metal ion to TCEP is from 0.1:1 to 30:1, optionally the molar ratio of the transition metal ion to TCEP is from 0.1:1 to 20:1, and further optionally the molar ratio of the transition metal ion to TCEP is from 0.5:1 to 8:

1.

10. 4. The method of claim 3, wherein the buffer system is selected from the group consisting of MES buffer, Bis-Tris buffer, PIPES buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, TAPSO buffer, PBS, PB, acetate buffer, BTP buffer, HEPPSO buffer, POPSO buffer, EPPS buffer, or Tris buffer; optionally, the buffer system is selected from the group consisting of MES buffer, Bis-Tris buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, or TAPSO buffer.

11. 4. The method according to claim 3, wherein the pH value of the buffer system is between 5.5 and 8, preferably between 5.8 and 7.4, more preferably between 6.7 and 7.

4.

12. 4. The method of claim 3, wherein the concentration of the buffer system is between 10 mM and 100 mM, between 20 mM and 80 mM, or between 20 mM and 40 mM.

13. 4. The method of claim 3, wherein the transition metal ion is Zn ２＋ , Cd ２＋ , Hg ２＋ , Ni ２＋ , Co ２＋ or a combination thereof, and optionally the transition metal ion is selected from the group consisting of Zn ２＋ The method according to claim 1, wherein

14. A method for preparing an antibody having site-specific modifications, comprising the method according to claims 3 to 13, further comprising the steps of: (B1) introducing an oxidizing agent to selectively reoxidize the reduced thiol groups resulting from step (a), optionally reoxidizing the reduced thiol groups within the Fab region of the antibody, and preferably purifying the oxidation product by removing excess oxidizing agent; (C1) introducing a metal chelator and modifying reagent 1 to react with the remaining thiol groups resulting from step (B1), wherein modifying reagent 1 is an end-capping reagent, a first linker payload, or a first thio bridging reagent, and optionally, the first thio bridging reagent has a first linker payload or a reactive group.

15. 15. The method of claim 14, comprising the steps of: (C2) introducing a metal chelator and a second reducing agent to selectively reduce the antibody from step (B1), and optionally reduce the interchain disulfide bond in the hinge region of the antibody; or (C2') introducing a second reducing agent to reduce interchain disulfide bonds in the product from step (C1) and, optionally, introducing a transition metal ion; (D2) introducing modifying reagent 2, and optionally a metal chelator, for reaction with the reduced thiol group obtained in step (C2) or step (C2'), wherein modifying reagent 2 is a second linker payload or a second thio bridging reagent, and optionally the second thio bridging reagent has a second linker payload or a reactive group.

16. 16. The method of claim 14 or 15, wherein the first thio bridging reagent and the second thio bridging reagent independently comprise at least two substituents that allow for re-crosslinking of thiol groups.

17. 17. The method of claim 16, wherein the first thio bridging reagent and the second thio bridging reagent are each independently: 【Chemical 1】 A method characterized in that the method is selected from the group consisting of:

18. 16. The method of claim 14 or 15, wherein the reactive groups independently comprise azide and / or dibenzocyclooctyne (DBCO).

19. 15. The method of claim 14, wherein in step (a), the molar ratio of TCEP to antibody is 4:1 to 15:1 and the incubation time is 1 hour to 16 hours, and optionally, in step (a), the molar ratio of TCEP to antibody is 4:1 to 10:1 and the incubation time is 4 hours to 8 hours.

20. The method according to claim 14, wherein in step (a), the molar ratio of transition metal ion to antibody is 1:1 to 10:1 or 1:1 to 2:

1.

21. 15. The method of claim 14, wherein in step (B1), the molar ratio of oxidizing agent to antibody is from 2:1 to 25:1, optionally the molar ratio of oxidizing agent to antibody is from 2:1 to 20:1, and further optionally the molar ratio of oxidizing agent to antibody is from 8:1 to 15:

1.

22. The method according to claim 14, wherein in step (B1), the oxidation temperature is 0°C to 37°C and the oxidation time is 1 hour to 48 hours, and optionally the oxidation temperature is 0°C to 30°C and the oxidation time is 1 hour to 8 hours.

23. 15. The method according to claim 14, wherein in step (B1), the oxidation reaction is carried out in the dark.

24. The method according to claim 15, wherein in step (C2), the molar ratio of the second reducing agent to the antibody is 1:1 to 2:

1.

25. The method according to claim 15, wherein in step (C2), the molar ratio of the metal chelator to the antibody is 2:1 to 120:

1.

26. 16. The method according to claim 15, wherein in step (C2), the reduction temperature is 0°C to 30°C and the reduction time is 1 hour to 8 hours.

27. The method according to claim 15, wherein in step (C2'), the molar ratio of the second reducing agent to the transition metal ion is 1:0.05 to 1:40, and / or the molar ratio of the second reducing agent to the antibody is 2.5:1 to 20:1, and / or the incubation time is 1 hour to 24 hours.

28. The method according to claim 15, wherein in step (C2'), the molar ratio of the second reducing agent to the transition metal ion is 1:0.4 to 1:100, and / or the molar ratio of the second reducing agent to the antibody is 0.8:1 to 2.5:1, and / or the incubation time is 0.5 hours to 24 hours.

29. A method for preparing an antibody having site-specific modifications, comprising the method according to claims 3 to 13, further comprising the steps of: (b) introducing a metal chelating agent and modifying reagent 1 to react with the reduced thiol groups obtained in step (a);

30. 30. The method of claim 29, further comprising the steps of: (c) incubating the reaction product from (b) and a second reducing agent in a buffer system to reduce interchain disulfide bonds in the reaction product from (b); (d) introducing the incubation product of step (c) and modifying reagent 2 to react with the reduced thiol group obtained in step (c);

31. 30. The method of claim 29, wherein in step (a), the molar ratio of TCEP to antibody is 7:1 to 15:1 and the incubation time is 4 to 12 hours, and optionally, in step (a), the molar ratio of TCEP to antibody is 8:1 to 14:1 and the incubation time is 4 to 10 hours.

32. 30. The method of claim 14 or 29, wherein when the first thio bridging reagent has a reactive group, step (C1) and / or step (b) comprises the steps of: introducing a first thio bridging reagent having a metal chelator and a reactive group to re-crosslink the reduced thiol groups obtained in step (B1) or step (a), and then incubating the first linker payload in a buffer system to react with the reactive group of the first thio bridging group.

33. 31. The method of claim 15 or 30, wherein when the second thio bridging reagent has a reactive group, step (D2) and / or step (d) comprises the steps of: introducing the product from step (C2) or step (C2') or step (c) and a second thio bridging reagent having a reactive group to re-crosslink the reduced thiol groups obtained from step (C2) or step (C2') or step (c), and then incubating a second linker payload in a buffer system to react with the reactive group of the second thio bridging group.

34. The method according to any one of claims 1 to 3, 14 and 29, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a monospecific antibody or a multispecific antibody.

35. 35. The method of claim 34, wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding portion thereof, and optionally, the antibody is IgG1 or IgG4.

36. The method according to any one of claims 14 to 15 and 29 to 30, wherein the linkers of the first linker-payload and the second linker-payload are selected from any one of those having one end that can be linked to a reduced thiol group of an antibody or a reactive group of a thiobridging reagent and the other end that can be linked to a payload.

37. The method according to any one of claims 14 to 15 and claims 29 to 30, wherein the payload is selected from any one of the following, comprising at least one substituent that allows attachment of the payload to a linker.

38. An antibody having a site-specific modification prepared by the method of any one of claims 1 to 37.

39. 39. The antibody having a site-specific modification of claim 38, wherein the antibody having a site-specific modification is an ADC having D2, an ADC having D4, an ADC having D1, an ADC having D6, an ADC having D3, an ADC having D1+D6, an ADC having D6+D2, an ADC having D6+D1, an ADC having D3+D1, an ADC having D3+D2, an ADC having D0+D6, an ADC having D0+D2, an ADC having D1+D2, an ADC having D1+D4, or an ADC having D2+D4.

40. A pharmaceutical composition comprising an antibody having a site-specific modification according to any one of claims 38 to 39 and one or more pharmaceutically acceptable carriers.

41. Use of TCEP or a salt thereof in the preparation of an antibody having a site-specific modification according to any one of claims 38 to 39.

42. 42. The use according to claim 41, characterized in that TCEP or a salt thereof is used in combination with a transition metal ion.

43. Use of an antibody having a site-specific modification according to any one of claims 38 to 39 in the manufacture of a therapeutic agent for the diagnosis, prevention or treatment of a disease.

44. A method for preventing or treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody having a site-specific modification described in any one of claims 38 to 39.

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