Preparation method of antibody with site specific modification

Abstract

The invention provides a preparation method of an antibody with site specific modification. First, three of the four interchain disulfide bonds of the antibody are selectively reduced. By site-specific modification of an antibody, the invention provides a variety of ADCs, such as a D2 type ADC, a D1 type ADC, a D4 type ADC, a D6 type ADC, a D3 type ADC, a D0 + D2 type ADC, a D0 + D1 type ADC, a D1 + D6 type dual payload ADC, a D1 + D2 type dual payload ADC, a D1 + D4 type dual payload ADC, a D2 + D4 type dual payload ADC, a D6 + D1 type dual payload ADC, a D3 + D1 type dual payload ADC, a D1 + D2 type dual payload ADC, a D1 + D2 type dual payload ADC, a D1 + D2 type dual payload ADC, a D1 + D4 type dual payload ADC, a D1 + D4 type dual payload ADC, or a D2 + D4 type dual payload ADC. Compared with a conventional coupling method, the homogeneity of the antibody with site specific modification can be remarkably improved, and the homogeneity is larger than 55%, 65%, 70%, 80% and 85% and even reaches 90% or 95%. Meanwhile, the method is easy to operate and low in cost, and antibody engineering and enzyme engineering are not needed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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 the present application and are incorporated herein in their entirety. Technical Field

[0003] The present invention relates to a method for preparing an antibody with site-specific modification. Specifically, the present invention relates to a bioconjugation process for preparing ADC with improved homogeneity. Background Art

[0004] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0005] Antibody-drug conjugates (ADCs) are modified antibodies in which a monoclonal antibody is linked to a small molecule drug using a stable linker. By delivering potent cytotoxic drugs to cells expressing antigens, ADCs ideally combine the specificity of antibodies with the high potency of cytotoxic drugs, thereby enhancing their targeted cytotoxic activity. Compared with traditional chemotherapy drugs, antibody-drug conjugates only target cancer cells that express antigens, so healthy cells are less affected (Pettinato, Mark C. (2021) "Introduction to Antibody-Drug Conjugates." Antibodies (mAb) (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.). ADCs have a wide range of potential therapeutic applications in multiple disease areas, especially in cancer, becoming a new type of targeted drug for disease treatment. Since the approval of Mylotarg in 2000, 14 ADC drugs have been approved by the U.S. Food and Drug Administration.

[0006] For drug attachment of ADCs, functional groups that are highly reactive to both the antibody and the linker-payload (i.e., linker-drug) are used for coupling to form stable covalent bonds. Conventional coupling methods (i.e., covalently binding the drug moiety to the antibody via a linker) typically result in uneven molecular attachment, with the drug moiety attached to several sites on the antibody. For example, ADCs are generally produced by two conventional chemical strategies: lysine-based coupling and coupling based on interchain disulfide bond reduction of cysteine. For coupling based on interchain disulfide bond reduction of cysteine, it includes a step of reducing the interchain disulfide bond under various reducing agent environments, followed by a nucleophilic reaction of the thiol group. In this coupling process, ADCs are typically formed by coupling one or more antibody cysteine ​​thiol groups to one or more linker-payload moieties, thereby producing a heterogeneous antibody-drug conjugate mixture (e.g., Adcetris), in which the drug moiety is attached to several sites on the antibody. For ADCs with a drug-antibody ratio (DAR) of approximately 4, the heterogeneous mixture typically contains a distribution of antibodies with 0 to about 8 or more drug moieties attached. Furthermore, within each subset of conjugates with a specific integer ratio of drug moiety to a single antibody, there is a potentially heterogeneous mixture of drug moieties attached to various sites on the antibody. This heterogeneous mixture is highly complex, and each conjugated product may exhibit distinct pharmacokinetic, toxicity, and efficacy profiles. Furthermore, characterizing and purifying these conjugates is difficult and costly. Conventional nonspecific conjugation and conjugate distribution are significantly affected by factors such as pH, temperature, concentration, salt concentration, and cosolvents, making the establishment of a robust conjugation process challenging.

[0007] Many methods have been developed to improve the uniformity of ADCs. For example, Genentech's THIOMAB technology was developed based on improving the uniformity of ADCs through antibody engineering. By introducing cysteine ​​into the primary sequence of the antibody, site-directed coupling is achieved to improve the uniformity of the product ("Cysteine-Based Coupling: Challenges and Solutions". Bioconjug Chem. 2021 Aug 18;32(8):1525-1534.).

[0008] US20210040145 discloses a 14-amino acid peptide Tub-tag that is applied to the C-terminus of any POI and catalyzes the addition of various tyrosine derivatives. Tub-tag technology utilizes this enzyme to repurpose tubulin-tyrosine ligase to attach to the functional portion of the antibody C-terminus, thereby homogeneously generating DAR2-type antibody conjugates.

[0009] However, these technologies involve protein engineering and / or enzyme catalysis, and therefore have several drawbacks, such as low antibody expression levels, risk of immunogenicity, complex purification processes, and / or high costs. Furthermore, there are no effective chemical methods for generating ADCs with high D6 selectivity.

[0010] Therefore, there is a need to develop a novel bioconjugation process that can generate ADCs with improved high homogeneity. Summary of the Invention

[0011] The present invention has developed a method for preparing an antibody with site-specific modification. Regarding the site-specific modification of the antibody, three of the four interchain disulfide bonds of the antibody are first selectively reduced. In this way, the present invention provides a variety of ADCs with high homogeneity, such as D2-type ADCs, D1-type ADCs, D4-type ADCs, D6-type ADCs, D3-type ADCs, D1+D6-type ADCs, D2+D4-type ADCs, D1+D2-type dual payload ADCs, D1+D4-type dual payload ADCs, D0+D2-type dual payload ADCs, D0+D1-type dual payload ADCs, D6+D2-type dual payload ADCs, D6+D1-type dual payload ADCs, D3+D2-type dual payload ADCs, D3+D1-type dual payload ADCs, D1+D2-type dual payload ADCs, D1+D4-type dual payload ADCs, or D2+D4-type dual payload ADCs. Compared to conventional conjugation processes, ADC homogeneity can reach as high as 55%, 65%, 70%, 80%, 85%, and even 90% or 95%. Furthermore, increasing the molar ratio of TCEP to antibody reduces the time and cost of reduction. Furthermore, the method is simple to operate and reduces costs, eliminating the need for antibody and enzyme engineering. The ADCs with improved homogeneity produced by the method of the present invention also have optimized safety and efficacy.

[0012] On the one hand, the present invention provides a method for preparing an antibody with site-specific modification, characterized in that the site-specific modification is that three interchain disulfide bonds in the antibody are selectively reduced, and the method comprises using tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof and a transition metal ion together.

[0013] In a second aspect, the present invention provides a method for preparing an antibody with site-specific modification, characterized in that the site-specific modification is that three interchain disulfide bonds in the antibody are selectively reduced, two of the three interchain disulfide bonds are located in the Fab region and one interchain disulfide bond is located in the hinge region of the antibody, and the method comprises using TCEP or a salt thereof and a transition metal ion together.

[0014] In a third aspect, the present invention provides a method for preparing an antibody with a site-specific modification, characterized in that the site-specific modification is that three interchain disulfide bonds in the antibody are selectively reduced, two of the three interchain disulfide bonds are located in the Fab region and one interchain disulfide bond is located in the hinge region of the antibody, the method comprising the following steps:

[0015] (a) 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 carrying reduced thiol groups, wherein the molar ratio of TCEP to the antibody is 3:1 to 15:1, and optionally, the molar ratio of TCEP to the antibody is 3:1 to 6:1.

[0016] In a fourth aspect, the present invention provides a method for preparing an antibody having a site-specific modification, characterized in that the method comprises the method of the present invention and further comprises the following steps:

[0017] (B1) introducing an oxidizing agent to selectively reoxidize the reduced thiol groups from step (a), optionally reoxidizing the reduced thiol groups in the Fab region, preferably removing excess oxidizing agent to purify the oxidized product;

[0018] (C1) introducing the metal chelator and modifying reagent 1 to react with the reduced thiol group from step (B1), wherein the modifying reagent 1 is a capping reagent, a first linker-payload or a first sulfide bridging reagent, optionally, the first sulfide bridging reagent carries the first linker-payload or a reactive group.

[0019] In a fifth aspect, the present invention provides a method for preparing an antibody having a site-specific modification, characterized in that the method comprises the method of the present invention and further comprises the following steps:

[0020] (b) introducing the metal chelator and the modifying reagent 1 to react with the reduced thiol group from step (a).

[0021] In a sixth aspect, the present invention provides an antibody with site-specific modification prepared by the method of the present invention.

[0022] In a seventh aspect, the present invention provides a pharmaceutical composition comprising an antibody with site-specific modification according to the present invention and one or more pharmaceutically acceptable carriers.

[0023] In an eighth aspect, the present invention provides use of TCEP or a salt thereof in preparing an antibody having site-specific modification according to the present invention.

[0024] In a ninth aspect, the present invention provides use of an antibody having site-specific modification according to the present invention in the production of a therapeutic agent for diagnosing, preventing or treating a disease.

[0025] In a tenth aspect, the present invention 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 invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 HIC-HPLC (hydrophobic interaction chromatography-high performance liquid chromatography) of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 1 is shown.

[0027] Figure 2 HIC-HPLC of the gosartuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 2 is shown.

[0028] Figure 3 HIC-HPLC of the mabelantuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 3 is shown.

[0029] Figure 4 A to Figure 4 D shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugates of Examples 4 to 7 (TCEP to antibody molar ratios of 3:1, 3.2:1, 5:1, 6:1).

[0030] Figure 5 A to Figure 5 F shows HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates of Examples 8 to 13 (TCEP to antibody molar ratios of 8:1, 9:1, 10:1, 11:1, 12:1, 13:1).

[0031] Figure 6 The trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (Zn 2+ The molar ratio of TCEP to TCEP was 0.25:1) using HIC-HPLC.

[0032] Figure 7 The trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (Zn 2+ The molar ratio of TCEP to TCEP was 0.5:1) using HIC-HPLC.

[0033] Figure 8The trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (Zn 2+ The molar ratio of TCEP to TCEP was 1:1) by HIC-HPLC.

[0034] Figure 9 The trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (Zn 2+ The molar ratio of TCEP to TCEP was 2:1) using HIC-HPLC.

[0035] Figure 10 The trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (Zn 2+ The molar ratio of TCEP to TCEP was 3:1) using HIC-HPLC.

[0036] Figure 11 The trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (Zn 2+ The molar ratio of TCEP to TCEP was 4:1) by HIC-HPLC.

[0037] Figure 12 The trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (Zn 2+ The molar ratio of TCEP to TCEP was 7.5:1) by HIC-HPLC.

[0038] Figure 13 A to Figure 13 D shows the HIC-HPLC (Zn 2+ The molar ratios of TCEP were 12:1, 27.27:1, 0.11:1, and 0.22:1).

[0039] Figure 14 A to Figure 14 D shows the HIC-HPLC (Zn 2+ The molar ratio of TCEP was 0.44:1, 0.66:1, 0.88:1, and 1.67:1).

[0040] Figure 15 A to Figure 15 D shows the HIC-HPLC (Zn 2+ The molar ratio of TCEP to TCEP is 0).

[0041] Figure 16 A to Figure 16 D shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugates of Examples 29 to 32 (with different incubation times in step (1)).

[0042] Figure 17 A to Figure 17 D shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugates of Examples 33 to 36 (the incubation temperature in step (1) was different).

[0043] Figure 18 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using Bis-Tris buffer (pH 6.7) according to Example 37 is shown.

[0044] Figure 19 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using PIPES buffer (pH 6.7) according to Example 38 is shown.

[0045] Figure 20 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using MOPS buffer (pH 6.7) according to Example 39 is shown.

[0046] Figure 21 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using BES buffer (pH 6.7) of Example 40 is shown.

[0047] Figure 22 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using HEPES buffer (pH 6.7) of Example 41 is shown.

[0048] Figure 23 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using DIPSO buffer (pH 7.4) of Example 42 is shown.

[0049] Figure 24 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using MOBS buffer (pH 7.4) of Example 43 is shown.

[0050] Figure 25HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using MOPSO buffer (pH 7.4) in Example 44 is shown.

[0051] Figure 26 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using TES buffer (pH 7.4) of Example 45 is shown.

[0052] Figure 27 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using ACES buffer (pH 7.4) of Example 46 is shown.

[0053] Figure 28 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using TAPSO buffer (pH 7.4) of Example 47 is shown.

[0054] Figure 29 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using PB (pH 6.7) of Comparative Example 5 is shown.

[0055] Figure 30 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using ADA buffer (pH 6.7) in Comparative Example 6 is shown.

[0056] Figure 31 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using BES buffer (pH 6.4) in Example 48 is shown.

[0057] Figure 32 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using BES buffer (pH 6.7) of Example 49 is shown.

[0058] Figure 33 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using BES buffer (pH 7.0) of Example 50 is shown.

[0059] Figure 34 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared using BES buffer (pH 6.4) in Example 51 is shown.

[0060] Figure 35 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 52 prepared using 20 mM BES buffer is shown.

[0061] Figure 36 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 53 prepared using 40 mM BES buffer is shown.

[0062] Figure 37 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 54 prepared using 60 mM BES buffer is shown.

[0063] Figure 38 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 55 prepared using 80 mM BES buffer is shown.

[0064] Figure 39 HIC-HPLC of the trastuzumab-[bismaleimide-DBCO]3 conjugate of Example 56 is shown.

[0065] Figure 40 A shows the HIC-HPLC of the trastuzumab-[MC-GGFG-DXd]6 conjugate of Example 57; Figure 40 B shows HIC-HPLC of the trastuzumab-[MC-GGFG-DXd]6[MC-VC-PAB-MMAE]2 conjugate of Example 57.

[0066] Figure 41 A shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 58; Figure 41 B shows HIC-HPLC of Trastuzumab-[MC-VC-PAB-MMAE]6[Maleimide-PEG4-N3-DBCO-Cy3]1 of Example 58.

[0067] Figure 42 A shows the HIC-HPLC of the trastuzumab-[maleimide]6 conjugate of Example 59; Figure 42 B shows HIC-HPLC of the trastuzumab-[maleimide]6[MC-VC-PAB-MMAE]2 conjugate of Example 59.

[0068] Figure 43HIC-HPLC of the trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate of Example 60 is shown.

[0069] Figure 44 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 61 is shown.

[0070] Figure 45 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 62 is shown.

[0071] Figure 46 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 63 is shown.

[0072] Figure 47 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 64 is shown.

[0073] Figure 48 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 65 is shown.

[0074] Figure 49 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 66 is shown.

[0075] Figure 50 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 67 is shown.

[0076] Figure 51 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 68 is shown.

[0077] Figure 52 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 69 is shown.

[0078] Figure 53 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 70 is shown.

[0079] Figure 54 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 71 is shown.

[0080] Figure 55 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 72 is shown.

[0081] Figure 56 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 73 is shown.

[0082] Figure 57 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 74 is shown.

[0083] Figure 58 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 75 is shown.

[0084] Figure 59 A to Figure 59 H shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates of Examples 76 to 83 (the parameters of step (1) and / or step (2) were different).

[0085] Figure 60 A to Figure 60 G shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates of Examples 84 to 90 (parameters of step (1) and / or step (2) were different).

[0086] Figure 61 A to Figure 61 C shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE] 2 conjugates of Comparative Examples 7 to 9 (the concentration of transition metal ions is 0).

[0087] Figure 62 A to Figure 62 D shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates of Examples 91 to 94 (oxidation time and oxidation temperature in step (2) were different).

[0088] Figure 63 A to Figure 63 H shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates of Examples 95 to 102 (different buffer systems).

[0089] Figure 64 A to Figure 63H shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates of Examples 103 to 110 (different buffer systems).

[0090] Figure 65 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Comparative Example 10 is shown.

[0091] Figure 66 HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1 conjugate of Example 111 is shown.

[0092] Figure 67 HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1[MC-GGFG-DXd]6 conjugate of Example 112 is shown.

[0093] Figure 68 HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2 conjugate of Example 113 is shown.

[0094] Figure 69 A shows the HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate of Example 113; Figure 69 B to Figure 69 C shows HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 conjugates of Examples 114 to 115.

[0095] Figure 70 A shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 116; Figure 70 B shows HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]4 conjugate of Example 116.

[0096] Figure 71 HIC-HPLC of the trastuzumab-[MC-GGFG-DXd]2 conjugate of Comparative Example 11 is shown. DETAILED DESCRIPTION

[0097] The present invention is explained in more detail below. This description is not an exhaustive list of all the different ways in which the present invention may be implemented, nor is it an exhaustive list of all the features that may be incorporated into the present invention. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be deleted from that embodiment. In addition, many variations and additions to the various embodiments presented herein will be apparent to those skilled in the art without departing from the present invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention and is not intended to be exhaustive of all permutations, combinations, and variations thereof.

[0098] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the detection practice of the present invention, preferred materials and methods are described herein. In describing and advocating the present invention, the following terms will be used.

[0099] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, the singular forms "a," "an," and "the" in this specification and the appended claims include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to an "antibody" includes a plurality of antibodies; a reference to a "transition metal ion" includes a mixture of transition metal ions, and so on. In the present invention, unless otherwise indicated, "or" as used means "and / or."

[0100] Throughout this document, unless the context requires otherwise, the words "comprises," "comprising," and "containing" should be understood to imply the inclusion of the stated steps, elements, or groups of steps or elements, but not the exclusion of any other steps, elements, or groups of steps or elements. "Consisting of" means including, but not limited to, what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to only those other elements that do not interfere with or have an effect on the specific activity or operation of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, while other elements are optional and may or may not be present, depending on whether they affect the activity or operation of the listed elements.

[0101] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that differs from a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length by at most 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In certain embodiments, the term "about" or "approximately" preceding a numerical value indicates a range of plus or minus 15%, 10%, 5%, or 1% of that value.

[0102] As used herein, the terms "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "further embodiments," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Therefore, appearances of the aforementioned phrases in various places throughout this specification are not necessarily referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0103] By conventional coupling process or bioconjugate process of the present invention, a mixture of antibody drug conjugates is generated. Generally speaking, an antibody molecule belonging to IgG1 or IgG4 subclass has 4 interchain SS bonds, and each interchain disulfide bond is formed by two-SH groups. The antibody molecule can be partially or completely reduced to form 2n (n is an integer selected from 1, 2, 3 or 4) reactive-SH groups. According to the number of drugs coupled to a single antibody molecule, the different conjugates containing different numbers of drug molecules are named D0, D1, D2, D3, D4, D6 and D8. Therefore, the "homogeneity" of antibody drug conjugates is used to describe the dominant properties of a particular type of antibody drug conjugate (i.e., a type selected from D0, D1, D2, D3, D4, D6 and D8 conjugates) in a given antibody drug conjugate mixture.

[0104] Drug loading is represented by the number of drug moieties per antibody in the ADC molecule. For some antibody-drug conjugates, drug loading may be limited by the number of attachment sites on the antibody. For example, when the attachment is a cysteine ​​thiol, in certain illustrative examples described herein, drug loading may be 0 to 8 drug moieties per antibody. In certain embodiments, the average drug loading of the antibody-drug conjugate ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5.

[0105] The term "D0" herein refers to an ADC in which the amount of drug conjugated to a single antibody molecule is zero.

[0106] The term "D1" or "D1-type ADC" herein refers to an ADC in which one of the first sulfide-bridging groups of the linker-payload rebridges two thiol groups of a single antibody molecule.

[0107] The term "D2" or "D2-type ADC" herein refers to an ADC in which two drug molecules are coupled to a single antibody molecule, wherein the two drug molecules can be coupled to the -SH group generated by reducing the SS bond between the heavy chain and the light chain via a linker, or can be coupled to the -SH group generated by reducing the SS bond between the heavy chain and the heavy chain via a linker.

[0108] The term "D3" or "D3-type ADC" herein refers to an ADC that carries three of the first sulfide-bridging groups of the linker-payload to rebridge the six thiol groups of a single antibody molecule.

[0109] The term "D4" or "D4-type ADC" herein refers to an ADC in which four drug molecules are coupled to a single antibody molecule, wherein the four drug molecules can be coupled to four -SH groups generated by reducing two SS bonds between the heavy chain and the light chain via a linker, or the four drug molecules can be coupled to four -SH groups generated by reducing two SS bonds between the heavy chain and the heavy chain via a linker, or two drug molecules can be coupled to two -SH groups generated by reducing one SS bond between the heavy chain and the light chain via a linker, and the other two drug molecules can be coupled to two -SH groups generated by reducing one SS bond between the heavy chain and the heavy chain via a linker.

[0110] The term "D6" or "D6-type ADC" herein refers to an ADC in which six drug molecules are coupled to a single antibody molecule, wherein the six drug molecules can be coupled to six -SH groups generated by reducing three SS bonds.

[0111] The term "D8" or "D8-type ADC" herein refers to an ADC in which eight drug molecules are coupled to a single antibody molecule, wherein the eight drug molecules can be coupled to eight -SH groups generated by reducing four SS bonds.

[0112] The term "D6+D1" or "D6+D1-type dual-payload ADC" herein refers to an ADC in which six of the first linker-payloads and one of the second sulfide bridge groups carrying the second linker-payload are coupled to a single antibody molecule.

[0113] The term "D6+D2" or "D6+D2-type dual-payload ADC" herein refers to an ADC in which six of the first linker-payloads and two of the second linker-payloads are coupled to a single antibody molecule.

[0114] The term "D3+D1" or "D3+D1 type dual-payload ADC" herein refers to an ADC in which three of the first sulfide bridging groups carrying the first linker-payload and one of the second sulfide bridging groups carrying the second linker-payload rebridge the eight thiol groups of a single antibody molecule.

[0115] The term "D3+D2" or "D3+D2-type dual-payload ADC" herein refers to an ADC in which three of the first sulfide-bridging groups carrying the first linker-payload rebridge six thiol groups and two of the second linker-payload are coupled to a single antibody molecule.

[0116] The term "D0+D2" or "D0+D2 type ADC" herein refers to an ADC in which one, two, or three of the first sulfide bridging groups rebridge six thiol groups and two of the second linker-payloads are coupled to a single antibody molecule, or refers to an ADC in which two, four, or six of the capping agents and two of the second linker-payloads are coupled to a single antibody molecule.

[0117] The term "D0+D1" or "D0+D1 type ADC" herein refers to an ADC in which three of the first sulfide bridging groups rebridge six thiol groups and one of the second sulfide bridging groups carrying a linker-payload rebridges two thiol groups of a single antibody molecule, or refers to an ADC in which six of the capping reagents react with six thiol groups and one of the second thiol groups carrying a linker-payload rebridges two thiol groups of a single antibody molecule.

[0118] The term "D1+D6" or "D1+D6 type dual-payload ADC" herein refers to an ADC in which one of the first sulfide-bridging groups of a first linker-payload rebridges two thiol groups and six of the second linker-payloads are coupled to a single antibody molecule, wherein the first linker-payload and the second linker-payload can be the same or different.

[0119] The term "D1+D2" or "D1+D2 type dual-payload ADC" herein refers to an ADC in which one of the first sulfide-bridging groups of a first linker-payload rebridges two thiol groups and two of the second linker-payloads are coupled to a single antibody molecule, wherein the first linker-payload and the second linker-payload can be the same or different.

[0120] The term "D1+D4" or "D1+D4 type dual-payload ADC" herein refers to an ADC in which one of the first sulfide-bridging groups of a first linker-payload rebridges two thiol groups and four of the second linker-payloads are coupled to a single antibody molecule, wherein the first linker-payload and the second linker-payload can be the same or different.

[0121] The term "D2+D4" or "D2+D4-type dual-payload ADC" herein refers to an ADC in which two of the first linker-payloads and four of the second linker-payloads are coupled to a single antibody molecule.

[0122] Method for preparing antibodies with site-specific modifications

[0123] On the one hand, the present invention provides a method for preparing an antibody with site-specific modification, characterized in that the site-specific modification is that three interchain disulfide bonds in the antibody are selectively reduced, and the method comprises using tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof and a transition metal ion together.

[0124] In a second aspect, the present invention provides a method for preparing an antibody with site-specific modification, characterized in that the site-specific modification is that three interchain disulfide bonds in the antibody are selectively reduced, two of the three interchain disulfide bonds are located in the Fab region and one interchain disulfide bond is located in the hinge region of the antibody, and the method comprises using TCEP or a salt thereof and a transition metal ion together.

[0125] The term "disulfide bond" as used herein refers to a covalent bond having an RSS-R' structure. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond with a second thiol group (e.g., a thiol group from another cysteine ​​residue). A disulfide bond can form between the thiol groups of two cysteine ​​residues, each located on two polypeptide chains, thereby forming an interchain bridge or bond.

[0126] The term "hinge region" herein refers to the portion of an antibody comprising a heavy chain molecule connecting the CH1 domain and the CH2 domain. The hinge region comprises approximately 25 amino acid residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently.

[0127] The term "Fab fragment" herein refers to the region of the antibody structure that is capable of binding to an antigen. It consists of a complete light chain (variable region and constant region) and a partial heavy chain structure (variable region and constant region fragments), and the light chain and heavy chain are connected by disulfide bonds. Fab fragments can be obtained by protease digestion of full-length antibodies. Under the action of papain, human immunoglobulin G can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into F(ab')2 fragments and pFc' fragments. The F(ab')2 fragment can be further reduced to form two Fab' fragments. In some embodiments, the interchain disulfide bond connects two of the upper heavy chains in the hinge region, or the interchain disulfide bond connects the heavy chain to the light chain in the Fab region.

[0128] In a third aspect, the present invention provides a method for preparing an antibody with a site-specific modification, characterized in that the site-specific modification is that three interchain disulfide bonds in the antibody are selectively reduced, two of the three interchain disulfide bonds are located in the Fab region and one interchain disulfide bond is located in the hinge region of the antibody, the method comprising the following steps:

[0129] (a) 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 carrying reduced thiol groups, wherein the molar ratio of TCEP to the antibody is 3:1 to 15:1, and optionally, the molar ratio of TCEP to the antibody is 3:1 to 6:1;

[0130] In some embodiments, in step (a), three interchain disulfide bonds within the antibody are selectively reduced. The molar ratio of TCEP to the transition metal ion is important for selectively reducing three interchain disulfide bonds.

[0131] In some embodiments, the salt refers to an acid addition salt or a base addition salt.

[0132] In some embodiments, the acid addition salt can be formed with inorganic acids and organic acids. Examples of inorganic acids from which salts can be derived include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like. Examples of organic acids from which salts can be derived include 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, sulfosalicylic acid, and the like.

[0133] In certain embodiments, the base addition salt can be formed with inorganic bases and organic bases. The inorganic base from which the salt can be derived includes Groups 1 to 2 of the periodic table. In certain embodiments, the salt is derived from lithium, sodium, potassium, calcium, magnesium, etc. The organic base from which the salt can be derived includes, for example, primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, etc. Some organic amines include isopropylamine, benzylethylenediamine, choline, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0134] 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.

[0135] The molar ratio of TCEP to antibody is important for selectively reducing the three interchain disulfide bonds within the antibody. In some embodiments, in step (a), the molar ratio of TCEP to antibody 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, in step (a), the molar ratio of TCEP to antibody is 3:1 to 4.5:1 or 3:1 to 4:1. In some embodiments of the present invention, in step (a), the molar ratio of TCEP to antibody is 3.2:1 to 4.4:1.

[0136] In some embodiments, in step (a), the molar ratio of TCEP to the 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.

[0137] 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, 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 (a), 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.

[0138] The incubation time in step (a) is important for selectively reducing the three interchain disulfide bonds within the antibody. In some embodiments, in step (a), the incubation time is 3 to 24 hours. In some embodiments, in step (a), the incubation time is 12 to 24 hours, alternatively, in step (a), the incubation time is 16 to 20 hours, and more alternatively, in step (a), the incubation time is 16 to 18 hours.

[0139] In some embodiments, in step (a), the incubation time is 4 to 24 hours, 14 to 24 hours, or 16 to 24 hours. In some embodiments, in step (a), the incubation time is 17 to 18 hours. In some embodiments, in step (a), the incubation time is 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours.

[0140] In some embodiments, in step (a), the incubation temperature is 4° C. and the incubation time is 18 h.

[0141] 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.

[0142] In some embodiments, in step (a), the incubation time is shortened as the molar ratio of TCEP to antibody increases. In some embodiments, in step (a), the molar ratio of TCEP to antibody is 4:1 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 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 13:1, and the incubation time is 4 to 10 hours. In some embodiments, the molar ratio of antibody to TCEP 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.

[0143] In some embodiments, in step (a), the molar ratio of transition metal ion to TCEP is 0.1:1 to 30:1, alternatively, the molar ratio of transition metal ion to TCEP is 0.1:1 to 20:1, more alternatively, 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.

[0144] 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.

[0145] In some embodiments, there is no specific limitation on the concentration of TCEP, as long as the concentrations of the transition metal ion and the antibody are increased or decreased in equal proportions. In some embodiments of the present invention, the concentration of the first reducing agent is 0.01 mM to 0.2 mM. In some embodiments of the present invention, the concentration of the first reducing agent is 0.02 mM to 0.15 mM. In some embodiments of the present invention, the concentration of the first reducing agent is 0.05 mM to 0.1 mM. In some embodiments of the present invention, 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.

[0146] In some embodiments, there is no specific limitation on the concentration of the transition metal ion in step (a), as long as the concentrations of TCEP and the antibody are increased or decreased in equal proportions.

[0147] In some embodiments of the present invention, there is no specific limitation on the concentration of the antibody in step (a), as long as the concentrations of TCEP and the transition metal ion are increased or decreased in equal proportions.

[0148] In some embodiments, the first buffer system and the second buffer system are independently 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.

[0149] The term "MES buffer" herein refers to 2-(N-morpholino)ethanesulfonic acid buffer.

[0150] The term "Bis-Tris buffer" herein refers to a bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane buffer.

[0151] The term "PIPES buffer" herein refers to piperazine-1,4-bisethanesulfonic acid buffer.

[0152] The term "MOPS buffer" herein refers to 3-morpholinepropanesulfonic acid buffer.

[0153] The term "BES buffer" herein refers to N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer.

[0154] The term "HEPES buffer" herein refers to 4-hydroxyethylpiperazineethanesulfonic acid buffer.

[0155] The term "DIPSO buffer" herein refers to 3-[bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer.

[0156] The term "MOBS buffer" herein refers to 3-morpholinepropanesulfonic acid buffer.

[0157] The term "MOPSO buffer" herein refers to 3-(N-morpholino)-2-hydroxy-1-propanesulfonic acid buffer.

[0158] The term "TES buffer" herein refers to 2-[tris(hydroxymethyl)methylamino]-1-ethanesulfonic acid buffer.

[0159] The term "ACES buffer" herein refers to N-(carbamoylmethyl)taurine buffer.

[0160] The term "TAPSO buffer" herein refers to 3-[N-tris-(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid buffer.

[0161] The term "PBS" herein refers to phosphate buffered saline.

[0162] The term "ADA buffer" herein refers to N-(carbamoylmethyl)iminodiacetic acid buffer.

[0163] The term "PB buffer" herein refers to phosphate buffered saline.

[0164] The term "BTP buffer" herein refers to Bis-tris propane buffer.

[0165] The term "Heppso buffer" herein refers to N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid buffer.

[0166] The term "POPSO buffer" herein refers to piperazine-N,N'-bis(2-hydroxy-propanesulfonic acid) buffer.

[0167] The term "EPPS buffer" herein refers to 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid buffer.

[0168] The term "Tris buffer" herein refers to tris(hydroxymethyl)aminomethane buffer.

[0169] 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.

[0170] In some embodiments, the buffer system is BES buffer.

[0171] In some embodiments, the pH of the buffer system is 5.5 to 8.

[0172] In some embodiments, the pH of the buffer system is 5.8 to 7.4, preferably, the pH of the buffer system is 6.7 to 7.4. In some embodiments, the pH of the buffer system is 6.0 to 7.4 or 6.4 to 7.4. In some embodiments, the pH of the buffer system is 6.4, 6.7, 7.0 or 7.4.

[0173] 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 a MES buffer, and the pH value of the BES buffer is 7.0.

[0174] In some embodiments, the concentration of the buffer system is 10 mM to 100 mM.

[0175] In some embodiments, the concentration of the buffer system is 20 mM to 80 mM, preferably, the concentration of the buffer system is 20 mM to 40 mM. In some embodiments, the concentration of the buffer system is 20 mM to 60 mM. In some embodiments, the concentration 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.

[0176] In some embodiments, the transition metal ion is selected from the group consisting of: Zn 2+ 、Cd 2+ 、Ni 2+ 、Hg 2+ 、Mn 2+ 、Co 2+ and their combinations.

[0177] The term "transition metal ion" refers to elements from Groups 4 to 12, defined by their typical chemical properties, i.e., coordinated 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 considered transition metals.

[0178] In some embodiments, the transition metal ion is selected from the group consisting of: Zn 2+ 、Cd 2+ 、Hg 2+ 、Ni 2+ 、Co 2+ or a combination thereof.

[0179] In some embodiments, the transition metal ion is Zn 2+ .

[0180] In some embodiments, there is no specific limitation on the salt of the transition metal ion, as long as the transition metal ion is soluble in the reaction solution so that free transition metal ions can be released in the reaction solution. In some embodiments of the present invention, the salt of the transition metal ion is a chloride, nitrate, sulfate, acetate, iodide, bromide, formates, or tetrafluoroborate.

[0181] In some embodiments, Zn 2+ The salt is ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, zinc formate or zinc tetrafluoroborate. In some embodiments, Zn 2+ The salt is ZnCl2.

[0182] In some embodiments, the site-specific modification does not involve antibody engineering, enzyme technology, and glycan modification.

[0183] In some embodiments, the method comprises introducing a metal chelator after step (a).

[0184] In some embodiments, the metal chelator can capture excess transition metal ions, which helps to selectively reduce the three interchain disulfide bonds in the antibody. In some embodiments, there is no specific limitation on the metal chelator, as long as the metal chelator can capture excess transition metal ions and does not affect the reduction of disulfide bonds in the antibody.

[0185] 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).

[0186] 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.

[0187] In a fourth aspect, the present invention provides a method for preparing a D2-type ADC or a D1-type ADC, characterized in that the method comprises the method of the present invention and further comprises the following steps:

[0188] (B1) introducing an oxidizing agent to selectively reoxidize the reduced thiol groups from step (a), optionally reoxidizing the reduced thiol groups in the Fab region, preferably removing excess oxidizing agent to purify the oxidized product;

[0189] (C1) introducing the metal chelator and modifying reagent 1 to react with the reduced thiol group from step (B1), wherein the modifying reagent 1 is a capping reagent, a first linker-payload or a first sulfide bridging reagent, optionally, the first sulfide bridging reagent carries the first linker-payload or reactive group.

[0190] The term "carry" herein means to have or possess.

[0191] In some embodiments, when the first sulfide-bridging reagent carries a reactive group, step (C1) comprises the following steps:

[0192] A metal chelator and a first sulfide bridging reagent carrying a reactive group are introduced to rebridge the reduced thiol group from step (B1), and then the first linker-payload is incubated in a buffer system to react with the reactive group of the first sulfide bridging group.

[0193] 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.

[0194] In some embodiments, in step (a), the concentration of the transition metal ion is also important for improving reduction selectivity. In some embodiments, the method for preparing the D2-type ADC comprises the following steps: in step (a), the molar ratio of the transition metal ion to the antibody is 1:1 to 10:1 or 1:1 to 2:1. In some embodiments, in step (a), the molar ratio of the transition metal ion to the 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.

[0195] In some embodiments, the oxidant is not particularly limited, as long as the oxidant can reoxidize the reduced thiol group. In some embodiments, the oxidant is dehydroascorbic acid (DHAA).

[0196] In some embodiments, in step (B1), the oxidizing agent selectively reoxidizes the reduced thiol group from step (a), thereby providing the antibody with two reduced thiol groups.

[0197] In some embodiments, in step (B1), the concentration of the oxidant is important for improving the oxidation selectivity. In some embodiments, in step (B1), the molar ratio of the oxidant to the antibody is 2: 1 to 25: 1, alternatively, in step (B1), the molar ratio of the oxidant to the antibody is 2: 1 to 20: 1, more alternatively, the molar ratio of the oxidant to the antibody is 8: 1 to 15: 1. In some embodiments, in step (B1), the molar ratio of the oxidant to the antibody 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.

[0198] In some embodiments, in step (B1), the molar ratio of the oxidizing agent to the 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.

[0199] 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.

[0200] 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.

[0201] In some embodiments, in step (B1), the oxidation time is 1 h to 7 h, 1 h to 6 h, 1 h to 5 h, 1 h to 3 h, 2 h to 5 h, 2 h to 4 h, or 2 h to 3 h. In some embodiments, the oxidation time is 1 h, 3 h, 5 h, 7 h, 9 h, 11 h, 13 h, 15 h, 18 h, 20 h, 23 h, 25 h, 27 h, 30 h, 33 h, 35 h, 37 h, 40 h, 43 h, 45 h, or 48 h.

[0202] In some embodiments, in step (B1), the oxidation temperature is 25° C., and the oxidation time is 1 to 3 hours. In some embodiments, the oxidation temperature is 0° C.-10° C., and the oxidation time is 5 to 8 hours.

[0203] In some embodiments, in step (B1), the oxidation reaction is carried out in the dark.

[0204] In some embodiments, in step (B1), to increase the content of D2-type ADC, D4-type ADC, and D1-type ADC, it is important to remove excess oxidant to purify the oxidation product.

[0205] 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.

[0206] In some embodiments, in step (C1), the reaction time with the reduced thiol group is 1 h to 6 h, 1 h to 5 h, 1 h to 3 h, 1 h to 2 h, or 1 h to 1.5 h. In some embodiments, in step (C1), the reaction time with the reduced thiol group is 1 h, 2 h, 3 h, 4 h, 5 h, or 6 h.

[0207] 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.

[0208] 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.

[0209] In some embodiments, in step (C1), the reaction time with the reactive group is 2 h to 12 h, 2 h to 10 h, 4 h to 10 h, 6 h to 10 h, or 8 h to 10 h. In some embodiments, in step (C1), the reaction time with the reactive group is 2 h, 3 h, 5 h, 7 h, 9 h, 11 h, or 12 h.

[0210] In some embodiments, in step (C1), the modifying reagent 1 is in excess based on the amount of the antibody.

[0211] In some embodiments, in step (C1), the molar ratio of the first sulfide bridging reagent 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 sulfide bridging reagent to the antibody is 1.05:1.

[0212] In some embodiments, in step (C1), when the first linker-payload reacts with the reactive group in the first sulfide 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.

[0213] In some embodiments, in step (C1), when the first linker-payload reacts with the reduced thiol group, the molar ratio of the first linker-payload to the 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, in step (C1), when the first linker-payload reacts with the reduced thiol group, the molar ratio of the first linker-payload to the antibody is 5: 1.

[0214] In some embodiments, the method further comprises the following steps:

[0215] (D1) optionally, introducing a compound containing at least one thiol group to consume excess first linker-payload;

[0216] (E1) Purifying and recovering the D2-type ADC or the D1-type ADC.

[0217] In some embodiments, there is no particular limitation on the compound that consumes the excess linker-payload, as long as the compound contains at least one thiol group. In some embodiments of the present invention, the compound is cysteine.

[0218] In some embodiments, the oxidation product, the resulting D2-type ADC, and / or the resulting D1-type ADC in step (B1) are purified by desalting columns, size exclusion chromatography, ultrafiltration, dialysis, and / or similar methods. In some embodiments, the oxidation product, the resulting D2-type ADC, and / or the resulting D1-type ADC in step (B1) are purified by desalting columns.

[0219] In some embodiments, the preparation method of the D2-type ADC comprises steps (a), (B1) and (C1), wherein the modification reagent 1 is a first linker-payload.

[0220] In some embodiments, the preparation method of the D2-type ADC comprises the following steps:

[0221] (1) TCEP (4 to 15 equivalents) and ZnCl2 (1 to 2 equivalents) were added to a solution of trastuzumab (0.01 to 0.2 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 4 to 24 hours;

[0222] (2) adding DHAA (4 to 20 equivalents) to selectively reoxidize the reduced thiol groups in the Fab region from step (1), and incubating the mixture at 0° C. to 37° C. in the dark for 1 to 48 hours. Preferably, excess DHAA is removed to purify the oxidized product;

[0223] (3) EDTA (8 to 120 equivalents) and MC-VC-PAB-MMAE (6 equivalents) were introduced in 10% v / v DMA to react with the remaining thiol groups from step (2), and the reaction was incubated at 0°C to 30°C for 1 to 4 hours;

[0224] (4) Purify the reaction mixture using a desalting column.

[0225] In some embodiments, the homogeneity of the D2-type ADC reaches 60%, 70%, 75%, or even 80%, 85%, 90% or 95%.

[0226] In some embodiments, the preparation method of the D1-type ADC comprises steps (a), (B1) and (C1), wherein the modification reagent 1 is a first sulfide bridge reagent carrying a first linker-payload.

[0227] In some embodiments, the preparation method of the D1-type ADC comprises the following steps:

[0228] (1) TCEP (4 to 15 equivalents) and ZnCl2 (1 to 2 equivalents) were added to a solution of trastuzumab (0.01 to 0.2 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 4 to 24 hours;

[0229] (2) adding DHAA (4 to 20 equivalents) to selectively reoxidize the reduced thiol groups in the Fab region from step (1) at 0°C to 37°C for 1 to 48 hours;

[0230] (3) introducing EDTA (8 equivalents, 120 equivalents) and the first sulfur bridge reagent dibromomaleimide-PEG4-N3 (0.013 mM) to react with the reduced thiol group from step (3), the reaction temperature is 4° C. to 37° C. and the reaction time is 1 h to 6 h, and then the product is recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1;

[0231] (4) In BES buffer (20 mM, pH 7.0), trastuzumab-[maleimide-PEG4-N3]1 and DBCO-MMAE (0.02 mM) were incubated at a temperature of 10°C to 37°C and a reaction time of 2 h to 12 h.

[0232] (5) Purify the reaction mixture using a desalting column.

[0233] In some embodiments, the uniformity of the D1-type ADC is as high as 90%, or even 95%.

[0234] In some embodiments, the present invention provides a method for preparing a D4 ADC, a D2+D3 ADC, a D2+D1 ADC, a D1+D2 ADC, a D1+D4 ADC, a D2+D4 ADC, a D1+D6 ADC, and a D1+D3 ADC, the method comprising the following steps:

[0235] (C2) introducing the metal chelator and a second reducing agent to selectively reduce the antibody from step (B1), optionally, to reduce the interchain disulfide bonds in the hinge region of the antibody;

[0236] or

[0237] (C2′) introducing the second reducing agent to reduce the interchain disulfide bonds in the product from step (C1), and optionally, introducing the transition metal ion;

[0238] (D2) introducing a modifying reagent 2 to react with the reduced thiol group from step (C2) or step (C2'), optionally introducing the metal chelator, wherein the modifying reagent 2 is a second linker-payload or a second sulfide bridging reagent, optionally, the second sulfide bridging reagent carries the second linker-payload or reactive group.

[0239] In some embodiments, when the second sulfide-bridging reagent carries the reactive group, step (D2) comprises the following steps:

[0240] The product from step (C2) or step (C2') is introduced with the second sulfide bridging reagent carrying the reactive group to re-bridge the reduced thiol group from step (C2) or step (C2'), optionally, the metal chelator is introduced, and then, in the buffer system, the second linker-payload is incubated to react with the reactive group of the second sulfide bridging group.

[0241] In some embodiments, when the transition metal ion is introduced in step (C2'), the metal chelator is introduced in step (D2) to capture excess transition metal ions.

[0242] In some embodiments, there is no specific limitation on the second reducing agent, as long as the second reducing agent can reduce the interchain disulfide bonds 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.

[0243] In some embodiments, in step (C2), the molar ratio of the metal chelator to the antibody is 2:1 to 120:1. In some embodiments, in step (C2), the molar ratio of the metal chelator to the antibody is 2:1 to 100:1. In some embodiments, in step (C2), the molar ratio of the metal chelator to the antibody is 2:1 to 80:1. In some embodiments, in step (C2), the molar ratio of the metal chelator to the antibody is 5:1 to 60:1. In some embodiments, in step (C2), the molar ratio of the metal chelator to the antibody is 10:1 to 60:1. In some embodiments, in step (C2), the molar ratio of the metal chelator to the antibody is 20:1 to 60:1. In some embodiments, in step (C2), the molar ratio of the metal chelator to the antibody is 30:1 to 60:1. In some embodiments, in step (C2), the molar ratio of the metal chelator to the antibody is 40:1 to 60:1. In some embodiments, in step (C2), the molar ratio of the metal chelator to the antibody is 50:1 to 60:1.

[0244] 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.

[0245] 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.

[0246] In some embodiments, in step (C2), the reduction time is 1 h to 8 h, 1 h to 7 h, 1 h to 6 h, 1 h to 5 h, 2 h to 5 h, 2 h to 4 h, or 2 h to 3 h.

[0247] In some embodiments, in step (C2'), three of the interchain disulfide bonds in the product prepared by step (C1) are completely reduced in the absence of transition metal ions. In some embodiments, one or two interchain disulfide bonds in the product prepared by step (C1) are reduced using transition metal ions.

[0248] In some embodiments, in step (C2'), in the absence of transition metal ions, the concentration of the second reducing agent is not specifically limited, as long as the second reducing agent can completely reduce the interchain disulfide bonds in the antibody, but is not limited thereto. In some embodiments, in step (C2'), the molar ratio of the second reducing agent to the antibody 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, in step (C2'), the molar ratio of the second reducing agent to the antibody is 20:3.

[0249] In some embodiments, the transition metal ion is introduced to selectively reduce two interchain disulfide bonds in the interchain disulfide bonds. 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 antibody to the second reducing agent 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 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 antibody to the second reducing agent is 3: 1 to 15: 1, and 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 antibody to the second reducing agent is 2.5:1 to 15:1, and the incubation time is 12h to 24h.

[0250] In some embodiments, the transition metal ion is introduced to selectively reduce one of the interchain disulfide bonds. 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 antibody to the second reducing agent is 0.8:1 to 2.5:1, and / or the incubation time is 0.5h to 24h. 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, 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, 2.5:1. In some embodiments, in step (C2'), the incubation time is 0.2h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22 or 24h. 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 antibody to the second reducing agent is 0.8:1 to 2:1, and the incubation time is 0.5h to 24h. 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, and the incubation time is 1 h to 9 h.

[0251] In some embodiments, in step (C2'), the incubation temperature of the second reducing agent is 0° C. to 37° C., or 5° C. to 30° C. In some embodiments, in step (C2'), the incubation temperature of the second reducing agent 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, in step (C2'), the incubation temperature of the second reducing agent is 25° C.

[0252] In some embodiments, in step (C2'), the incubation time of the second reducing agent is 0.5h to 24h, or 5h to 20h. In some embodiments, in step (C2'), the incubation time of the second reducing agent is 6h to 18h, 8h to 18h, 8h to 15h, or 8h to 12h. In some embodiments, in step (C2'), the incubation time of the second reducing agent is 3h, 8h, 12h, or 18h.

[0253] In some embodiments, in step (C2'), the molar ratio of the metal chelator to the 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, 40:1 to 55:1.

[0254] In some embodiments, the reaction temperature and reaction time of the reduced thiol group in step (D2) are the same as those in step (C1). In some embodiments, the reaction temperature and reaction time of the reduced thiol group in step (D2) are independent of those in step (C1).

[0255] 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) are independent of those in step (C1).

[0256] In some embodiments, in step (D2), the modifying agent 2 is in excess based on the amount of the antibody.

[0257] In some embodiments, in step (D2), the molar ratio of the second sulfide 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 sulfide 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.

[0258] In some embodiments, in step (D2), when the second linker-payload reacts with the reactive group in the second sulfide 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.

[0259] In some embodiments, in step (D2), when the second linker-payload reacts with the reduced thiol group, 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, 12: 1 to 10: 1. In some embodiments, in step (D2), when the first linker-payload reacts with the reduced thiol group, the molar ratio of the second linker-payload to the antibody is 35:3.

[0260] In some embodiments, the method for preparing the D4-type ADC comprises: purifying the product from step (B1).

[0261] In some embodiments, the product from step (B1) is purified by desalting column, size exclusion chromatography, ultrafiltration, dialysis and / or similar methods.In some embodiments of the present invention, the product from step (B1) is purified by desalting column.

[0262] In some embodiments, the method for preparing the ADC with site-specific modification further comprises the following steps:

[0263] (E2) optionally, introducing a compound containing at least one thiol group to consume excess linker-payload;

[0264] (F2) Purify and recover the antibody with site-specific modification.

[0265] In some embodiments, details of step (E2) and step (F2) are similar to those of step (D1) and step (E1).

[0266] In some embodiments, the preparation method of the D4-type ADC comprises step (a), step (B1), step (C2) and step (D2), wherein the modification reagent 2 is a second linker-payload.

[0267] In some embodiments, the preparation method of the D4-type ADC comprises the following steps:

[0268] (1) TCEP (4 to 15 equivalents) and ZnCl2 (1 to 2 equivalents) were added to a solution of trastuzumab (0.01 to 0.2 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 4 to 24 hours;

[0269] (2) adding DHAA (2 equivalents to 20 equivalents) to selectively reoxidize the reduced thiol groups in the Fab region from step (1), incubating the mixture at 0° C. to 37° C. in the dark for 1 h to 48 h, and then purifying the mixture using a desalting column;

[0270] (3) introducing EDTA (2 equivalents to 120 equivalents) and TCEP (1 equivalent to 2 equivalents) to selectively reduce the remaining interchain disulfide bonds in the hinge region of the antibody from step (2), at a reduction temperature of 0° C. to 30° C. and a reduction time of 1 h to 5 h;

[0271] (4) introducing MC-VC-PAB-MMAE (3 to 6 equivalents) in 10% v / v DMA to react with the remaining thiol groups from step (3), and incubating the reaction at 0°C to 30°C for 1 to 4 hours;

[0272] (5) Purify the reaction mixture using a desalting column.

[0273] In some embodiments, the preparation method of the D1+D6 type ADC comprises step (a), step (B1), step (C1), step (C2') and step (D2), wherein the modification reagent 1 is a first sulfide bridge reagent, the modification reagent 2 is a second linker-payload, and there is no transition metal ion in step (C2').

[0274] In some embodiments, the preparation method of the D1+D6 ADC comprises the following steps:

[0275] (1) In BES buffer (20 mM, pH 7.0), the D1-type ADC according to the present invention and TCEP (0.08 mM) were introduced at a reaction temperature of 25° C. and a reaction time of 12 h;

[0276] (2) MC-GGFG-DXd (0.14 mM) was introduced into the solution from step (1), and the reaction mixture was placed at 24°C for 1 h, and then trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1[MC-GGFG-DXd]6 was recovered using a desalting column.

[0277] In some embodiments, the uniformity of the D1+D6 ADC is as high as 70%, 75%, or even 80% or 85%.

[0278] In some embodiments, the preparation method of the D1+D2 type ADC comprises steps (a), (B1), (C1), (C2') and (D2), wherein the modification reagent 1 is a first sulfur bridge reagent carrying a first linker-payload, the modification reagent 2 is a second linker-payload, and a transition metal ion is introduced in step (C2').

[0279] In some embodiments, the preparation method of the D1+D2 type ADC comprises steps (a), (B1), (C1), (C2') and (D2), wherein the modification reagent 1 is a first sulfide bridge reagent carrying a reactive group that reacts with the first linker-payload, the modification reagent 2 is a second linker-payload, and a transition metal ion is introduced in step (C2').

[0280] In some embodiments, the D1+D2 ADC content is as high as 65%, 68%, 70%, or even 71% or 75%.

[0281] In some embodiments, the preparation method of the D1+D4 type ADC comprises step (a), step (B1), step (C1), step (C2') and step (D2), wherein the modification reagent 1 is a first sulfur bridge reagent carrying a first linker-payload, the modification reagent 2 is a second linker-payload, and a transition metal ion is introduced in step (C2').

[0282] In some embodiments, the preparation method of the D1+D4 type ADC comprises step (a), step (B1), step (C1), step (C2') and step (D2), wherein the modification reagent 1 is a first sulfide bridge reagent carrying a reactive group that reacts with the first linker-payload, the modification reagent 2 is a second linker-payload, and a transition metal ion is introduced in step (C2').

[0283] In some embodiments, the content of the D1+D4 ADC is as high as 70%, 75%, or even 80% or 83%.

[0284] In some embodiments, the preparation method of the D2+D4 type ADC comprises step (a), step (B1), step (C1), step (C2') and step (D2), wherein the modification reagent 1 is a first linker-payload, the modification reagent 2 is a second linker-payload, and a transition metal ion is introduced in step (C2').

[0285] In some embodiments, the content of the D2+D4 ADC is as high as 75%, 80%, 85%, or even 90%.

[0286] In a fifth aspect, the present invention provides a method for preparing an antibody having a site-specific modification, characterized in that the method comprises the method of the present invention and further comprises the following steps:

[0287] (b) introducing the metal chelator and the modifying reagent 1 to react with the reduced thiol group from step (a).

[0288] In some embodiments, when the first sulfide-bridging reagent carries the reactive group, the step (b) comprises the following steps:

[0289] A metal chelator and the first sulfide bridging reagent carrying the reactive group are introduced to re-bridge the reduced thiol group from step (a), and then the first linker-payload is incubated in the buffer system to react with the reactive group of the first sulfide bridging group.

[0290] In some embodiments, the method further comprises the steps of purifying and recovering the product from step (b).

[0291] In some embodiments, the method further comprises the following steps:

[0292] (c) incubating the reaction product from (b) and the second reducing agent in the buffer system to reduce the interchain disulfide bonds in the reaction product from (b);

[0293] (d) introducing the incubation product from step (c) and the modification reagent 2 to react with the reduced thiol group from step (c).

[0294] In some embodiments, when the second sulfide-bridging reagent carries the reactive group, the step (d) comprises the following steps:

[0295] The product from step (c) and the second sulfide bridging reagent carrying a reactive group are introduced to re-bridge the reduced thiol group from step (c), and then the second linker-payload is incubated in the buffer system to react with the reactive group of the second sulfide bridging group.

[0296] In some embodiments, the method further comprises the steps of purifying and recovering the product from step (d).

[0297] In some embodiments, the method further comprises the step of introducing a compound containing at least one thiol group to consume excess first linker-payload and excess second linker-payload. In some embodiments, the compound is the same as the compound in (D1).

[0298] In some embodiments, the resulting antibody drug conjugate is recovered by any suitable purification method, such as using a desalting column, size exclusion chromatography, ultrafiltration, dialysis, ultrafiltration (UF)-diafiltration (DF), etc. If desired, in some cases, further ADC enrichment (e.g., D2) can be applied using hydrophobic interaction chromatography (HIC).

[0299] In some embodiments, in step (d), the ADC is purified by a desalting column, size exclusion chromatography, ultrafiltration, dialysis, and / or similar methods. In some embodiments of the present invention, in step (d), the ADC is purified by a desalting column.

[0300] 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.

[0301] In some embodiments, in step (a), TCEP selectively reduces three interchain disulfide bonds within the antibody using transition metal ions and at an appropriate molar ratio of TCEP to antibody, and optionally, in step (c), the second reducing agent reduces the remaining one interchain disulfide bond. The method comprising steps (a) and (b) can prepare antibodies with site-specific modifications, such as D6-type ADCs or D3-type ADCs. The method comprising steps (a), (b), (c), and (d) can prepare antibodies with site-specific modifications, such as D6+D1-type ADCs, D3+D1-type ADCs, D6+D2-type ADCs, D3+D2-type ADCs, D0+D1-type ADCs, or D0+D2-type ADCs.

[0302] In some embodiments, in step (b) and step (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 h to 20 h.

[0303] In some embodiments, in step (b) and step (d), the reaction temperature with the reduced thiol group is 20°C to 30°C or 20°C to 25°C. In some embodiments, in step (b) and step (d), the reaction temperature with the reduced thiol group is room temperature. In some embodiments, in step (b) and step (d), the reaction temperature with the reduced thiol group 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.

[0304] As used herein, the term "room temperature" refers to 23°C±2°C, 25°C±5°C or 20°C±5°C.

[0305] In some embodiments, in step (b) and step (d), the reaction time with the reduced thiol group is 0.5 h to 6 h, 0.5 h to 4 h, 0.5 h to 2 h, 1 h to 2 h, or 0.5 h to 1 h. In some embodiments, in step (b) and step (d), the reaction time with the reduced thiol group is 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, or 20 h.

[0306] In some embodiments, in step (b) and step (d), the reaction temperature and reaction time for reducing the thiol groups are independent.

[0307] In some embodiments, in step (b) and step (d), the reaction temperature with the reactive group is 10° C. to 37° C., and the reaction time with the reduced thiol group is 2 h to 12 h.

[0308] In some embodiments, in step (b) and step (d), the reaction temperature with the reactive group is 10° C. to 30° C., 15° C. to 30° C., or 25° C. to 30° C. In some embodiments, in step (b) and step (d), the reaction temperature with the reactive group 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.

[0309] In some embodiments, in step (b) and step (d), the reaction time with the reactive group is 2 h to 10 h, 4 h to 10 h, 8 h to 10 h. In some embodiments, in step (b) and step (d), the reaction time with the reactive group is 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h.

[0310] In some embodiments, in step (b) and step (d), the reaction temperature and reaction time with the reactive group are independent.

[0311] In some embodiments, in step (b), the modifying reagent 1 is in excess based on the amount of the antibody.

[0312] In some embodiments, in step (b), the molar ratio of the first sulfide bridging reagent to the antibody is 3: 1 to 15: 1. In some embodiments, in step (b), the molar ratio of the first sulfide 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.

[0313] In some embodiments, in step (b), when the first linker-payload reacts with the reactive group in the first sulfide bridging reagent, the molar ratio of the first linker-payload to the antibody is 3: 1 to 15: 1. In some embodiments, in step (b), when the first linker-payload reacts with the reactive group in the first sulfide bridging reagent, 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.

[0314] In some embodiments, in step (b), when the first linker-payload reacts with the reduced thiol group, the molar ratio of the first linker-payload to the antibody is 6: 1 to 20: 1. In some embodiments, in step (b), when the first linker-payload reacts with the reduced thiol group, 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.

[0315] In some embodiments, in step (d), the modifying agent 2 is in excess based on the amount of the antibody.

[0316] In some embodiments, in step (d), the molar ratio of the second sulfide bridging reagent to the antibody is 1:1 to 3:1. In some embodiments, in step (b), the molar ratio of the second sulfide bridging reagent to the antibody is 1:1, 1.5:1, 2:1, or 3:1.

[0317] In some embodiments, in step (d), when the second linker-payload reacts with the reactive group in the second sulfide bridging reagent, the molar ratio of the second linker-payload to the antibody is 1: 1 to 8: 1. In some embodiments, in step (d), when the second linker-payload reacts with the reactive group in the second sulfide bridging reagent, 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.

[0318] In some embodiments, in step (d), when the second linker-payload reacts with the reduced thiol group, the molar ratio of the second linker-payload to the antibody is 2: 1 to 16: 1. In some embodiments, in step (d), when the second linker-payload reacts with the reduced thiol group, 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.

[0319] In some embodiments, in step (c), there is no specific limitation on the second reducing agent, as long as the second reducing agent can reduce the interchain disulfide bonds in the antibody. In some embodiments, in step (c), the second reducing agent is TCEP, tris(3-hydroxypropyl)phosphine (THPP), or dithiothreitol (DTT). In some embodiments, the second reducing agent is TCEP.

[0320] In some embodiments, in step (c), there is no specific limitation on the concentration of the second reducing agent, as long as the second reducing agent is capable of reducing interchain disulfide bonds within 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.

[0321] In some embodiments, in step (c), the incubation temperature of the second reducing agent is 0° C. to 37° C., and the incubation time is 0.5 h to 24 h.

[0322] In some embodiments of the present invention, 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.

[0323] In some embodiments, in step (c), the incubation time is 1 h to 20 h, 5 h to 20 h, 6 h to 18 h, 8 h to 18 h, 8 h to 15 h or 8 h to 12 h. In some embodiments, in step (c), the incubation time is 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h or 12 h.

[0324] In some embodiments, the preparation method of the D6-type ADC comprises the following steps:

[0325] (a) Incubating TCEP or a salt thereof with a transition metal ion in a buffer system in the presence of an antibody to selectively reduce interchain disulfide bonds within the antibody to obtain an antibody carrying reduced thiol groups, wherein the molar ratio of TCEP to antibody is 3:1 to 15:1.

[0326] (b1) introducing the metal chelator and the first linker-payload to react with the reduced thiol group from step (a).

[0327] In some embodiments, the uniformity of the D6-type ADC is as high as 55%, 65%, 70%, 80%, 85%, or even 90%.

[0328] In some embodiments, the preparation method of the D6+D2 dual-payload ADC comprises the following steps:

[0329] (c2) incubating the D6-type ADC and a second reducing agent in a buffer system to reduce the interchain disulfide bonds within the D6-type ADC;

[0330] (d2) introducing the incubation product from step (c2) and the second linker-payload to react with the reduced thiol group from step (c2).

[0331] In some embodiments, the uniformity of the D6+D2 dual-payload ADC is as high as 80%, 85%, or even 90%.

[0332] In some embodiments, the preparation method of the D6+D1 dual-payload ADC comprises the following steps:

[0333] (d3) introducing the incubation product from step (c2) and a second sulfide bridging reagent carrying the second linker-payload to react with the reduced thiol group from step (c2).

[0334] In some embodiments, the preparation method of the D6+D1 dual-payload ADC comprises the following steps:

[0335] (d3`) introducing the incubation product from step (c2) and the second sulfide bridging reagent carrying a reactive group to re-bridge the reduced thiol group from step (c2), and then incubating the second linker-payload in the buffer system to react with the reactive group of the second sulfide bridging group.

[0336] In some embodiments, the uniformity of the D6+D1 dual-payload ADC is as high as 80%, 85%, or even 90%.

[0337] In some embodiments, the preparation method of the D3-type ADC comprises the following steps:

[0338] (b4) introducing a metal chelator and a first sulfide bridging reagent carrying the first linker-payload to react with the reduced thiol group from step (a).

[0339] In some embodiments, the preparation method of the D3-type ADC comprises the following steps:

[0340] (b4') introducing the metal chelator and the first sulfide bridging reagent carrying the reactive group to re-bridge the reduced thiol group from step (a), and then incubating the first linker-payload in the first buffer system to react with the reactive group of the sulfide bridging group.

[0341] In some embodiments, the uniformity of the D3-type ADC is as high as 80%, 82%, or even 86%.

[0342] In some embodiments, the preparation method of the D3+D2 dual-payload ADC comprises the following steps:

[0343] (c5) incubating the D3-type ADC and a second reducing agent in a buffer system to reduce the interchain disulfide bonds within the D3-type ADC;

[0344] (d5) introducing the incubation product from step (c5) and the second linker-payload to react with the reduced thiol group from step (c5).

[0345] In some embodiments, the preparation method of the D3+D1 dual-payload ADC comprises the following steps:

[0346] (d6) introducing the incubation product from step (c5) and a second sulfide bridging reagent carrying the second linker-payload to react with the reduced thiol group from step (c5).

[0347] In some embodiments, the preparation method of the D3+D1 dual-payload ADC comprises the following steps:

[0348] (d6`) introducing the incubation product from step (c5) and the second sulfide bridging reagent carrying a reactive group to re-bridge the reduced thiol group from step (c5), and then incubating the second linker-payload in the buffer system to react with the reactive group of the second sulfide bridging group.

[0349] In some embodiments, the preparation method of the D0+D2 type ADC comprises the following steps:

[0350] (b7) introducing the metal chelator and the first sulfide bridging reagent to react with the reduced thiol group from step (a).

[0351] (c7) incubating the reaction product from (b7) and a second reducing agent in the buffer system to reduce the interchain disulfide bonds in the product from (b7);

[0352] (d7) introducing the incubation product from step (c7) and the second linker-payload to react with the reduced thiol group from step (c7).

[0353] In some embodiments, the uniformity of the D0+D2 ADC is as high as 60%, 65%, or even 68% or 70%.

[0354] In some embodiments, the preparation method of the D0+D1 type ADC comprises the following steps:

[0355] (d8) introducing the incubation product from step (c7) and a second sulfide bridging reagent carrying the second linker-payload to react with the reduced thiol group of step (c7).

[0356] In some embodiments, the preparation method of the D0+D1 type ADC comprises the following steps:

[0357] (d8`) introducing the incubation product from step (c7) and the second sulfide bridging reagent carrying a reactive group to re-bridge the reduced thiol group from step (c7), and then incubating the second linker-payload in the buffer system to react with the reactive group of the second sulfide bridging group.

[0358] In some embodiments, the uniformity of the D0+D1 ADC is as high as 85%, 87%, or even 90% or 92%.

[0359] In some embodiments, the first sulfide bridging reagent and the second sulfide bridging reagent each independently contain at least two substituents that allow for re-bridging of the thiol group.

[0360] In some embodiments, the first sulfide bridging reagent and the second sulfide bridging reagent are each independently selected from, but not limited to, the group consisting of:

[0361]

[0362] In some embodiments, the reactive groups each independently contain an azide and / or a dibenzocyclooctyne (DBCO).

[0363] In some embodiments, the sulfide bridging agent is connected to the reactive group through an alkyl group or polyethylene glycol (PEG). In some embodiments, the first sulfide bridging agent carrying a reactive group and the second sulfide bridging agent carrying a reactive group are selected from, but not limited to, the group consisting of:

[0364]

[0365]

[0366] wherein n is 0-20, 0-18, 0-15, 0-13, 0-10, 0-7, 0-5 or 0-3.

[0367] In some embodiments, the first sulfide bridging reagent carrying a reactive group can be different from the second sulfide bridging reagent carrying a reactive group. In some embodiments, the first sulfide bridging reagent carrying a reactive group can be the same as the second sulfide bridging reagent carrying a reactive group.

[0368] In some embodiments, the first sulfide-bridging reagent carrying a reactive group and the second sulfide-bridging reagent carrying a reactive group are dibromomaleimide-PEG4-N3 having the following structure:

[0369]

[0370] In some embodiments, the linkers in the first linker-payload and the second linker-payload are selected from any one of the linkers having the following characteristics: one end is capable of being linked to the reduced thiol group of the antibody or the reactive group of the sulfide bridging reagent, and the other end is capable of being linked to the payload.

[0371] The term "linker" herein refers to a reactive molecule containing at least two substituents, one of which can covalently bind to a drug molecule and the other can covalently couple to a reactive group of an antibody or a sulfide-bridging reagent.

[0372] In some embodiments, when the first linker-payload and the second linker-payload react with a reduced thiol group, the linkers of the first linker-payload and the second linker-payload independently comprise a cleavable linker or a non-cleavable linker. The cleavable linker can be a chemically unstable linker and an enzymatically cleavable linker. Due to its high plasma stability and good intracellular cleavage selectivity and effectiveness, enzymatically cleavable linkers are widely selected as cleavable linker candidates in ADCs. In some embodiments, the enzymatically cleavable linker comprises the following structures: -maleimidocaproyl-(-MC-), -maleimidocaproyl-peptide moiety-(-MC-peptide moiety-), -p-aminobenzyl alcohol-(-PAB-), or -peptide moiety- or -MC-peptide moiety-PAB-. In some embodiments, the peptide moiety is a dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide.

[0373] 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).

[0374] 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).

[0375] In some embodiments, the tetrapeptide can be, but is not limited to, glycine-glycine-phenylalanine-glycine (GGFG).

[0376] In some embodiments, 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 independently be, but are not limited to, 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. 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 second linker-payload can independently be, but is not limited to, MC-AAD-PAB, MC-GVC-PAB, MC-GGG-PAB, MC-FFK-PAB, MC-EVC-PAB, or MC-GFK-PAB. In some embodiments, when the first linker-payload and the second linker-payload react with a reduced thiol group, the linker of the first linker-payload and the second linker-payload can be, but is not limited to, MC-GGFG.

[0377] In some embodiments, the linker comprises a drug-carrying maleimide, a drug-carrying organic chloride, a drug-carrying organic bromide, a drug-carrying organic iodide, and / or a drug-carrying vinyl pyrimidine.

[0378] In some embodiments, when the first linker-payload and / or the second linker-payload reacts with the reactive group in the sulfide bridging reagent, the linker of the first linker-payload and / or the second linker-payload further comprises an azide group and / or dibenzocyclooctyne (DBCO). In some embodiments, when the linker of the first linker-payload and / or the second linker-payload contains an azide group, the reactive group of the sulfide bridging group contains DBCO. In some embodiments, when the linker of the first linker-payload and / or the second linker-payload contains DBCO, the reactive group of the sulfide bridging group contains an azide group.

[0379] In some embodiments, when the linker of the first linker-payload and / or the second linker-payload reacts with the reactive group in the sulfide bridging reagent, the linker of the first linker-payload and the second linker-payload is independently selected from, but not limited to, any one of the group consisting of:

[0380]

[0381] wherein n is 0-20, 0-18, 0-15, 0-13, 0-10, 0-7, 0-5 or 0-3, and m is 0-20, 0-18, 0-15, 0-13, 0-10, 0-7, 0-5 or 0-3; 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.

[0382] As used herein, the term "capping agent" refers to a compound that does not carry a drug but contains at least one substituent that can be covalently coupled to an antibody.

[0383] In some embodiments, the capping agent is a cleavable linker or a non-cleavable linker. In some embodiments, the capping agent is (2-aminoethyl)maleimide.

[0384] In some embodiments, the first sulfide bridging reagent carrying the first linker-payload and the second sulfide bridging reagent carrying the second linker-payload have the following formula:

[0385] QST

[0386] Wherein, Q is selected from the group consisting of:

[0387]

[0388] S is selected from, but not limited to, a cleavable linker or a non-cleavable linker, S is selected from the group consisting of:

[0389]

[0390]

[0391] wherein n is 0-20, m is 0-20, optionally, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

[0392] T is the payload.

[0393] In some embodiments, the payload is selected from any one of the payloads containing at least one substituent that allows attachment from the payload to the linker.

[0394] The term "payload" as used herein refers to any cytotoxic molecule or any molecule of medical interest that carries at least one substituent or moiety that allows 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 alleviating, relieving, or eliminating one or more symptoms of a disease or condition.

[0395] 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 can be, but is not limited to, an anticancer agent, an antiviral agent, or an antimicrobial agent.

[0396] In some embodiments, the cancer is a carcinoma, a lymphoma, a blastoma, a sarcoma, and a leukemia or lymphoid malignancy. More specific examples of the cancer include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or gastric tumor (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, 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 malignancies, anal cancer, penile cancer, and head and neck cancer.

[0397] Exemplary payloads are monomethyl auristatin E (MMAE), monomethyl auristatin D (MMAD), monomethyl auristatin EF (MMAF), calicheamicin (CLM), maytansine (DM1), maytansine alkaloid, duocarmycin, anthracycline, pyrrolobenzodiazepine dimer, amatoxin, quinoline alkaloid, Dxd, doxorubicin hydrochloride, methotrexate, erlotinib, bortezomib, fulvestrant, sunitinib mesylate imatinib, letrozole, finasunate, platinums (such as oxaliplatin, carboplatin and cisplatin), finasunate, fluorouracil, rapamycin, folinic acid, lapatinib, lonafarnib, sorafenib, gefitinib, captothecin, topotecan, moss statin, adezelesin, anthracycline, kazelesin, bisoprolol, dolastatin, auristatin, duocarmycin, eleutherobin, taxanes (such as paclitaxel or docetaxel), cyclophosphamide, doxorubicin, vincristine, prednisone or prednisolone, other alkylating agents (such as nitrogen mustard, chlorambucil and ifosfamide), antimetabolites (such as azathioprine or mercaptopurine), vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, etoposide phosphate, epipodophyllotoxin, actinomycin, daunorubicin, valrubicin, idarubicin, edrecolomab, epirubicin, bleomycin, plicamycin or mitomycin, and their salts.

[0398] In some embodiments, the payload is deludecanoic acid (DXd), Cy3 (cyanine 3), MMAE, MMAD or MMAF. In some embodiments, the payload is MMAE, DXd or Cy3.

[0399] The linker-payload is a chemical moiety that is synthesized by connecting a linker to a payload. Depending on the desired payload and the selected linker, those skilled in the art can select a suitable method to couple them together. For example, some conventional coupling methods (such as amine coupling methods) can be used to form the desired linker-payload, which still contains a reactive group for coupling to the antibody by a covalent bond. In the present invention, a drug-maleimide complex (i.e., a maleimide-linked drug) is used as an example of a payload carrying a reactive group. In ADC preparation, the most common reactive group that can be combined with a thiol group is maleimide. In addition, organic chlorides, bromides, and iodides are also frequently used.

[0400] The linker-payload can be any physically active compound, or any compound useful for diagnosing, preventing or treating disease.

[0401] 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 independently MC-GGFG-DXd, MC-VC-PAB-MMAE, MC-VC-PAB-MMAD, and MC-VC-PAB-MMAF. In some embodiments, the first sulfide bridging reagent carrying the first linker-payload and the second sulfide bridging reagent carrying the second linker-payload are independently selected from, but not limited to, the group consisting of:

[0402]

[0403]

[0404] In some embodiments, the payload of the first sulphur bridging reagent carrying the first linker-payload and the payload of the second sulphur bridging reagent carrying the second linker-payload are different. In some embodiments, the linker of the first sulphur bridging reagent carrying the first linker-payload and the linker of the second sulphur bridging reagent carrying the second linker-payload may be different. In some embodiments, the linker of the first sulphur bridging reagent carrying the first linker-payload and the linker of the second sulphur bridging reagent carrying the second linker-payload may be the same. In some embodiments, the sulphur bridging reagent of the first sulphur bridging reagent carrying the first linker-payload and the second sulphur bridging reagent carrying the second linker-payload may be different. In some embodiments, the first sulphur bridging reagent carrying the first linker-payload and the second sulphur bridging reagent carrying the second linker-payload may be the same.

[0405] In some embodiments, there is no specific limitation on the antibody. According to the antigen associated with the disease, those skilled in the art can select a suitable antibody that can be used in the bioconjugation process of the present invention.

[0406] In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a monospecific antibody, or a multispecific antibody.

[0407] As used herein, the term "antibody" refers to any immunoglobulin that binds to a specific antigen. A natural intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second, and third constant regions, while each light chain consists of a variable region and a constant region. Heavy chains from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM.

[0408] The term "Fc region" herein refers to a monomeric protein, a dimeric protein, or a heterodimeric protein having at least the CH2 and CH3 domains of an immunoglobulin. The CH2 and CH3 domains can form at least a portion of the dimer region of a protein / molecule (eg, an antibody).

[0409] In some embodiments, the antibody is a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding portion thereof.

[0410] The term "human antibody" as used herein refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or an amino acid sequence of an antibody derived from a non-human source (utilizing a human antibody repository), or other human antibody coding sequences. The definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0411] The term "humanized antibody" herein refers to a chimeric antibody comprising amino acid residues from a non-human heavy chain variable region (HVR) and amino acid residues from human FR. In certain embodiments, a humanized antibody will comprise substantially all or at least one (typically two) variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of non-human antibodies, and all or substantially all FRs correspond to the FRs of human antibodies. Alternatively, a humanized antibody may 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 a humanized antibody.

[0412] The term "chimeric antibody" as used herein refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0413] In some embodiments, the antibody refers to an immunoglobulin and is a molecule containing an antigen binding site that immunospecifically binds to an antigen. In some embodiments, the antibody is classified as IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments of the invention, the antibody is classified as IgG.

[0414] In some embodiments, the class of the antibody is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody is IgG1 or IgG4.

[0415] In some embodiments of the invention, 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.

[0416] In some embodiments, the one or more mutations are to stabilize the antibody and / or increase half-life. In some instances, the one or more mutations are to modulate Fc receptor interactions to reduce or eliminate Fc effector functions, such as FcγR, antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In other instances, the one or more mutations are to modulate glycosylation.

[0417] In some embodiments, the one or more mutations are located in the Fc region. In some instances, the Fc region comprises a mutation at residue position L234, L235, or a combination thereof. In some instances, the mutation comprises L234 and L235. In some instances, the mutation comprises L234A and L235A. In some cases, the residue positions are referenced to IgG1.

[0418] In some embodiments, the Fc region comprises a mutation at residue positions L234, L235, D265, N21, K46, L52, or P53, or a combination thereof. In some instances, the mutation comprises a combination of L234 and L235 with a mutation at residue positions K46, L52, or P53.

[0419] In some embodiments, the Fc region comprises mutations at L234, L235, and K46. In some cases, the Fc region comprises mutations at L234, L235, and L52. In some cases, the Fc region comprises mutations at L234, L235, and P53. In some cases, the Fc region comprises mutations at D265 and N21. In some cases, the residue positions are with reference to IgG1.

[0420] In some instances, the Fc region comprises L234A, L235A, D265A, N21G, K46G, L52R, or P53G, or a combination thereof. In some instances, the Fc region comprises a combination of L234A and L235A with K46G, L52R, or P53G. In some cases, the Fc region comprises L234A, L235A, and K46G. In some cases, the Fc region comprises L234A, L235A, and L52R. In some cases, the Fc region comprises L234A, L235A, and P53G. In some cases, the Fc region comprises D265A and N21G. In some cases, the residue positions are with reference to IgG1.

[0421] In some embodiments, the Fc region comprises a mutation at residue positions L233, L234, D264, N20, K45, L51 or P52. In some instances, the Fc region comprises a mutation at L233 and L234. In some instances, the Fc region comprises a combination of a mutation at L233 and L234 and a mutation at residue positions K45, L51 or P52. In some cases, the Fc region comprises a mutation at L233, L234 and K45. In some cases, the Fc region comprises a mutation at L233, L234 and L51. In some cases, the Fc region comprises a mutation at L233, L234 and K45. In some cases, the Fc region comprises a mutation at L233, L234 and K45. In some cases, the Fc region comprises a mutation at L233, L234 and P52. In some instances, the Fc region comprises a mutation at D264 and N20. In some cases, positions equivalent to residues L233, L234, D264, N20, K45, L51, or P52 in the IgG1, IgG2, IgG3, or IgG4 framework are contemplated.

[0422] In some embodiments, the Fc region comprises L233A, L234A, D264A, N20G, K45G, L51R, or P52G. In some instances, the Fc region comprises L233A and L234A. In some instances, the Fc region comprises a combination of L233A and L234A with K45G, L51R, or P52G. In some instances, the Fc region comprises L233A, L234A, and K45G. In some instances, the Fc region comprises L233A, L234A, and L51R. In some instances, the Fc region comprises L233A, L234A, and K45G. In some instances, the Fc region comprises L233A, L234A, and P52G. In some instances, the Fc region comprises D264A and N20G.

[0423] In some embodiments, the human IgG constant region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), for example, using amino acid modifications as described in 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, 114(10):1571-1572; Res, 67(18):8882-8890; Stavenhagen et al., 2008 Adv. Enzyme Regul., 48:152-164; Alegre et al., 1992 J Immunol, 148:3461-3468; Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11.

[0424] In some embodiments, the antibody of IgG1, IgG2, IgG3 or IgG4 is a human antibody or a humanized antibody. Information on IgG1, IgG2, IgG3 or IgG4 can be obtained at NCBI or UniProt (https: / / www.uniprot.org / ).

[0425] In some embodiments, the antibody is a bispecific antibody. In some embodiments of the invention, the antibody is an IgG1-like bispecific antibody.

[0426] In some embodiments, those skilled in the art can select a suitable method to prepare a bispecific antibody. In some embodiments of the present invention, the bispecific antibody can be prepared by knobs-in-holes technology (Ridgway JBB, Presta LG, Paul C. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization ("knobs-into-holes" engineering of heavy chain heterodimerization antibody CH3 domain) [J]. Protein Engineering (7): 617 (2023-08-11)., format chain exchange (FORCE) technology, universal 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 (a new method for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1) [J]. Journal of Biological Chemistry (Journal of Biological Chemistry) 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]. Antibodies (mAbs), 2022, 14(1), e2018960), CrossMAb technology (Klein C, Schaefer W, Regula JT. The use of CrossMAb technology for the generation of bi-and multispecific antibodies [J]. Antibodies (mAbs), 2016, 8(6), P1010-P1020.) or a combination thereof.

[0427] The term "knob-to-hole structure" herein is used in its broadest sense and encompasses various situations, such as a CH1 domain of one heavy chain having a knob mutation and a CH1 domain of another heavy chain having a hole mutation, a CH2 domain of one heavy chain having a knob mutation and a CH2 domain of another heavy chain having a hole mutation, and / or a CH3 domain of one heavy chain having a knob mutation and a CH3 domain of another heavy chain having a hole mutation. For example, in general, a "knob-in-hole structure" can refer to a modification within the interface of the CH3 domains of two antibody heavy chains: i) in the CH3 domain of one heavy chain (the first CH3 domain), one amino acid residue is substituted with another amino acid residue carrying a large side chain, thereby generating a protrusion ("knob") in the interface of the first CH3 domain; ii) in the CH3 domain of the other heavy chain (the second CH3 domain), one amino acid residue is substituted with another amino acid residue carrying a smaller side chain, thereby generating a cavity ("hole") in the interface of the second CH3 domain, into which the protrusion ("knob") in the first CH3 domain can be placed.

[0428] In some embodiments, the antibody is selected from any one of a cytotoxic antibody, a cell proliferation inhibitor, a cell activation and interaction regulator, a human immune system regulator, an antigen neutralizer, an antibody immunospecific for a viral antigen, or an antibody immunospecific for a microbial antigen.

[0429] In some embodiments of the present invention, the antibody may be a target-specific antibody. In some embodiments of the present invention, the antibody may be, but is not limited to, anti-HER2 antibody, anti-FAP antibody, anti-OX-40 antibody, anti-41BB antibody, anti-angiopoietin 2 antibody, anti-ant-IL-4Rα antibody, anti-BCMA antibody, anti-Blys antibody, anti-BTNO2 antibody, anti-C5 antibody, anti-CD122 antibody, anti-CD13 antibody, anti-CD133 antibody, anti-CD137 antibody, anti-CD138 antibody, anti-CD16a antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD 22 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-CD-8 antibody, anti-CD79 antibody, anti-CEA antibody, anti-CGPR / 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 Antibodies, anti-GP130 antibodies, anti-Her3 antibodies, anti-HSG antibodies, anti-ICOS antibodies, anti-IGF1 antibodies, anti-IGF1 / 2 antibodies, anti-IGF-1R antibodies, anti-IGF2 antibodies, anti-IGFR antibodies, anti-IL-1 antibodies, anti-IL-12 antibodies, anti-IL-12p40 antibodies, anti-IL-13 antibodies, anti-IL-17A antibodies, anti-IL-1β antibodies, anti-IL-23 antibodies, anti-IL-5 antibodies, anti-IL-6 antibodies, anti-IL-6R antibodies, anti-Lag-3 antibodies, anti-LAG 3 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 antibody, anti-SARS-CoV-2 antibody, anti-Te38 antibody, anti-TIM-3 antibody, anti-TNF antibody, anti-TNFα antibody, anti-TROP-2 antibody, anti-TWEAK antibody, anti-VEGF antibody, or anti-VEGFR antibody.

[0430] In some embodiments, the antibody has targeting specificity, targeting HER2 (human epidermal growth factor receptor 2), TROP2 (TACSTD2, tumor-associated calcium signal transducer 2), BCMA (TNFRSF17, TNF receptor superfamily member 17).

[0431] In some embodiments of the present invention, the antibody can be Transtuzumab, Sacituzumab, Belantamab, Risankizumab, Eptinezumab, Teprotumumab, Polatuzumab, Tafasitamab, Rovelizumab, Romosozumab, Dostarlimab, Enfortumab or Ublituximab.

[0432] In some embodiments, the antibody is trastuzumab, gosartan, or mabelantuzumab.

[0433] In some embodiments, the antibodies can be purchased commercially or produced by any method known to those skilled in the art.

[0434] In some embodiments, the preparation method of the D6-type ADC comprises the following steps:

[0435] (1) TCEP (4 to 15 equivalents) and ZnCl2 (Zn / TCEP molar ratio of 0.1:1 to 30:1) were added to a solution of trastuzumab (0.01 mM to 0.2 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 4 to 18 hours;

[0436] (2) EDTA (0.6 mM) and MC-VC-PAB-MMAE (0.08 mM) dissolved in DMA were introduced, and the reaction was incubated at room temperature for 1 h;

[0437] (3) Optionally, cysteine ​​is introduced to consume excess MC-VC-PAB-MMAE;

[0438] (4) Purify the reaction mixture using a desalting column.

[0439] In some embodiments, the preparation method of the D6+D2 dual-payload ADC comprises the following steps:

[0440] (1) ZnCl2 (Zn / TCEP molar ratio of 0.1:1 to 30:1) and the first reducing agent TCEP (4 equivalents to 15 equivalents) were added to a solution of the monoclonal antibody trastuzumab (0.01 mM to 0.2 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was placed at 4°C for 18 h;

[0441] (2) EDTA-2Na (0.6 mM) and MC-GGFG-DXd (0.1 mM) dissolved in DMA were introduced and the reaction was continued at room temperature for 1 h;

[0442] (3) The resulting product was incubated with the second reducing agent TCEP (0.02 mM) at room temperature for 2 h; the second linker-payload MC-VC-PAB-MMAE (0.05 mM) was added and the reaction continued at room temperature for 2 h.

[0443] (4) Purify the obtained product using a desalting column.

[0444] In some embodiments, the preparation method of the D6+D1 dual-payload ADC comprises the following steps:

[0445] (1) ZnCl2 (Zn / TCEP molar ratio of 0.1:1 to 30:1) and the first reducing agent TCEP (4 equivalents to 15 equivalents) were added to a solution of the monoclonal antibody trastuzumab (0.01 mM to 0.2 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was placed at 4°C for 18 h;

[0446] (2) EDTA-2Na (0.6 mM) and MC-GGFG-DXd (0.1 mM) dissolved in DMA were introduced and the reaction was continued at room temperature for 1 h;

[0447] (3) The resulting product and the second reducing agent TCEP (0.02 mM) were incubated at room temperature for 2 h; a sulfur bridge reagent carrying the reactive group dibromomaleimide-PEG4-N3 (0.012 mM) was added, and the reaction continued at room temperature for 2 h; then the second linker-payload DBCO-Cy3 (0.05 mM) was added, and the reaction continued at room temperature for 4 h.

[0448] (4) Purify the obtained product using a desalting column.

[0449] In some embodiments, the preparation method of the D3-type ADC comprises the following steps:

[0450] (1) ZnCl2 (Zn / TCEP molar ratio of 0.1:1 to 30:1) and the first reducing agent TCEP (4 equivalents to 15 equivalents) were added to a solution of the monoclonal antibody trastuzumab (0.01 mM to 0.2 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was placed at 4°C for 18 h;

[0451] (2) introducing EDTA (0.6 mM) and bismaleimide-DBCO (0.045 mM) to react with the reduced thiol group from step (1) at room temperature for 3 h, and then recovering the product using a desalting column to obtain trastuzumab-[bismaleimide-DBCO]3;

[0452] (3) Trastuzumab-[maleimide-DBCO]3 and N3-Cy3 (0.06 mM) were incubated in BES buffer (20 mM, pH 6.7) at 25 °C for 6 h, and then trastuzumab-[maleimide-DBCO-N3-Cy3]3 was recovered using a desalting column.

[0453] In some embodiments, the preparation method of the D0+D2 type ADC comprises the following steps:

[0454] (1) ZnCl2 (Zn / TCEP molar ratio of 0.1:1 to 30:1) and the first reducing agent TCEP (4 equivalents to 15 equivalents) were added to a solution of the monoclonal antibody trastuzumab (0.01 mM to 0.2 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was placed at 4°C for 18 h;

[0455] (2) introducing EDTA (0.6 mM) and (2-aminoethyl)maleimide (0.04 mM) to react with the reduced thiol group from step (1) at room temperature for 3 h, and then recovering the product using a desalting column to obtain trastuzumab-maleimide;

[0456] (3) Trastuzumab-maleimide and reducing agent TCEP (0.02 mM) were incubated at room temperature for 2 h; the second linker-payload MC-VC-PAB-MMAE (0.05 mM) was added and the reaction continued at room temperature for 2 h.

[0457] (4) Purify the obtained product using a desalting column.

[0458] In some embodiments, the preparation method of the D0+D1 type ADC comprises the following steps:

[0459] (1) ZnCl2 (Zn / TCEP molar ratio of 0.1:1 to 30:1) and the first reducing agent TCEP (4 equivalents to 15 equivalents) were added to a solution of the monoclonal antibody trastuzumab (0.01 mM to 0.2 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was placed at 4°C for 18 h;

[0460] (2) introducing EDTA (0.6 mM) and (2-aminoethyl)maleimide (0.04 mM) to react with the reduced thiol group from step (1) at room temperature for 3 h, and then recovering the product using a desalting column to obtain trastuzumab-maleimide;

[0461] (3) Trastuzumab-maleimide and the second reducing agent TCEP (0.02 mM) were incubated at room temperature for 2 h. The second sulfide bridge reagent with the reactive group dibromomaleimide-PEG4-N3 (0.013 mM) reacted with the reduced thiol group at 24 °C for 3 h.

[0462] (4) The resulting product was incubated with DBCO-Cy3 (0.02 mM) in MES (20 mM, pH 6.7) at 25 °C for 8 h, and then trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1 was recovered using a desalting column.

[0463] Various analytical methods can be used to determine the yield and isomer mixture of the ADC. In some embodiments of the present invention, the analytical method is HIC-HPLC. HIC-HPLC is capable of separating ADCs with different numbers of antibody-loaded drugs. The drug loading level can be determined based on the ratio of absorbance (e.g., at 250nm and 280nm). For example, if the drug can absorb at 250nm, while the antibody absorbs at 280nm. Therefore, the ratio of 250 / 280 will increase with the increase of drug loading. Using the bioconjugation process described herein, it is generally observed that antibodies with an even number of drugs are coupled to the antibody because the reduction of disulfides produces an even number of free cysteine ​​thiols. Compared with the ADC produced by the conventional coupling process, the uniformity of the ADC of the present invention is improved.

[0464] Antibodies with site-specific modifications

[0465] In a sixth aspect, the present invention provides an antibody with site-specific modification prepared by the method of the present invention.

[0466] In some embodiments, the sequence of the antibody with the site-specific modification is wild-type.

[0467] The term "wild-type antibody" herein refers to a naturally occurring antibody that has no mutations.

[0468] In some embodiments, the glycans of the antibody with site-specific modifications are unmodified.

[0469] In some embodiments, antibodies with site-specific modifications do not involve antibody engineering, enzyme technology, and glycan modification.

[0470] In some embodiments, an antibody having a site-specific modification is conjugated with Modifying Agent 1 to form a D6-type ADC, a D1-type ADC, a D3-type ADC, a D2-type ADC, a D4-type ADC, or a D6-type ADC. In some embodiments, an antibody having a site-specific modification is conjugated with Modifying Agent 1 and Modifying Agent 2 to form a D6+D2-type dual payload ADC, a D6+D1-type dual payload ADC, a D3+D1-type dual payload ADC, a D3+D2-type dual payload ADC, a D0+D2-type ADC, a D0+D1-type ADC, a D2+D6-type dual payload ADC, a D2+D3-type dual payload ADC, a D1+D6-type dual payload ADC, a D1+D3-type dual payload ADC, a D0+D6-type ADC, a D0+D3-type ADC, a D2+D4-type dual payload ADC, a D2+D2-type dual payload ADC, a D1+D4-type dual payload ADC, or a D1+D2-type dual payload ADC.

[0471] In some embodiments, the antibody with site-specific modification is a D2-type ADC, a D4-type ADC, a D1-type ADC, a D6-type ADC, a D3-type ADC, a D1+D6-type ADC, a D6+D2-type ADC, a D6+D1-type ADC, a D3+D1-type ADC, a D3+D2-type ADC, a D0+D6-type ADC, a D0+D2-type ADC, a D1+D2-type ADC, a D1+D4-type ADC, or a D2+D4-type ADC.

[0472] In some embodiments, the antibodies with site-specific modifications are trastuzumab-[MC-VC-PAB-MMAE]6, gosartuzumab-[MC-VC-PAB-MMAE]6, mabelantuzumab-[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.

[0473] Pharmaceutical compositions comprising antibodies with site-specific modifications

[0474] In a seventh aspect, the present invention provides a pharmaceutical composition comprising an antibody with site-specific modification according to the present invention and one or more pharmaceutically acceptable carriers.

[0475] The pharmaceutical compositions provided herein can be formulated in any manner known in the art, such as for example, the pharmaceutical compositions provided herein can be formulated in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniformity of dosage) for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration.

[0476] A pharmaceutical composition is formulated to be compatible with its intended route of administration (eg, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal administration).

[0477] Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0478] Suitable components can include, for example, antioxidants, fillers, adhesives, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers (such as sugar and cyclodextrins). Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated benzyl alcohol and / or propyl gallate. As disclosed herein, in the composition comprising antibody or its Fab provided herein and conjugate, including one or more antioxidants (such as methionine) reduces the oxidation of antibody or its Fab. This reduction of oxidation prevents or reduces the loss of binding affinity, thereby improving antibody stability and extending the shelf life to the greatest extent. Therefore, in certain embodiments, there is provided a pharmaceutical composition comprising one or more antibodies or its Fab disclosed herein and one or more antioxidants (such as methionine).

[0479] In some embodiments, the pharmaceutical composition can be a liquid solution, suspension or emulsion. In some embodiments, the pharmaceutical composition is formulated as an injectable composition. The injectable pharmaceutical composition can be prepared in any conventional form, such as, for example, a liquid solution, suspension, emulsion or a solid form suitable for generating a liquid solution, suspension or emulsion. Injectable preparations can include sterile and / or non-pyrogenic solutions for injection, sterile soluble dry products (such as lyophilized powders) that can be mixed with a solvent before use, including subcutaneous injection tablets, sterile suspensions for injection, sterile insoluble dry products that can be combined with a solvent before use, and sterile and / or non-pyrogenic emulsions. The solution can be aqueous or non-aqueous.

[0480] In some embodiments, the pharmaceutical composition is combined with other therapeutic agents. There are no specific limitations on the other therapeutic agents, as long as the other therapeutic agents can reduce the side effects of the pharmaceutical composition or increase the effectiveness of the pharmaceutical composition. The other therapeutic agents include anticancer agents, anti-autoimmune disease agents, antiemetics, antiallergic agents, and the like.

[0481] In some embodiments, anticancer agents may include, but are not limited to, erlotinib, bortezomib, fulvestrant, sunitinib mesylate, imatinib, letrozole, finasulate, platinums (such as oxaliplatin, carboplatin, and cisplatin), finasulate, fluorouracil, rapamycin, folinic acid, lapatinib, lonafarnib, sorafenib, gefitinib, capmtothecin, topotecan, bryostatin, adezelesin, anthracyclines, kazelesin, bisoprolol, dolastatin, auristatin, bisoprolol ... Carbomycin, eleutherobin, taxanes (such as paclitaxel or docetaxel), cyclophosphamide, doxorubicin, vincristine, prednisone or prednisolone, other alkylating agents (such as nitrogen mustard, chlorambucil and ifosfamide), antimetabolites (such as azathioprine or mercaptopurine), other microtubule inhibitors (vinca alkaloids, like vincristine, vinblastine, vinorelbine and vindesine, and taxanes), podophyllotoxins (etoposide, teniposide, etoposide phosphate and epipodophyllotoxin), topoisomerase inhibitors, other cytotoxins (such as actinomycin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin, mitomycin, etc.).

[0482] In some embodiments, anti-autoimmune disease agents may include, but are not limited to, ibuprofen, loxoprofen, naproxen, diclofenac, indomethacin, meloxicam, lornoxicam, nabumetone, celecoxib, acetaminophen, glucocorticoids, azathioprine, cyclophosphamide, and the like.

[0483] In some embodiments, during and after administration of the ADC of the present invention, the patient may experience nausea. Therefore, an antiemetic may be administered to prevent nausea (upper abdomen) and vomiting. The antiemetic may include, but is not limited to, aprepitant, ondansetron, granisetron hydrochloride, lorazepam, dexamethasone, prochlorperazine, casopitant, and the like.

[0484] In some embodiments, during and after administration of the ADC of the present invention, the patient may experience an allergic reaction. Therefore, an antiallergic agent may be used to minimize the risk of an allergic reaction. Antiallergic agents include dexamethasone, beclomethasone, hydrocortisone, prednisolone, prednisone, methylprednisolone, hydroxyzine, cyproheptadine, bronchodilators, terbutaline, and the like.

[0485] Uses of TCEP or its salts

[0486] In an eighth aspect, the present invention provides use of TCEP or a salt thereof in preparing an antibody with site-specific modification.

[0487] In some embodiments, antibodies with site-specific modifications are modified by selectively reducing interchain SS bonds. In some embodiments, antibodies with site-specific modifications are modified by selectively reducing three interchain SS bonds.

[0488] In some embodiments, TCEP or a salt thereof is used with a transition metal ion.

[0489] In some embodiments, TCEP and transition metal ions together selectively reduce three of the four interchain disulfide bonds of the antibody at a specific molar ratio of TCEP to antibody and a specific incubation time of TCEP, and in step (a), the metal chelator is introduced after the incubation reaction.

[0490] Uses of antibodies with site-specific modifications

[0491] In a ninth aspect, the present invention provides use of an antibody having site-specific modification according to the present invention in the production of a therapeutic agent for diagnosing, preventing or treating a disease.

[0492] In a tenth aspect, the present invention 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 invention.

[0493] The term "treat" as used herein for any disease refers to alleviating or ameliorating the disease (i.e., slowing or arresting the progression of the disease or at least one of its clinical symptoms); or alleviating or improving at least one physical parameter or biomarker associated with the disease, including physical parameters or biomarkers that may not be discernible by the patient. For cancer, "treating" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination of the above effects. For tumors, "treating" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, delaying tumor progression, or a combination of these means.

[0494] As used herein, the term "prevention" as applied to any disease refers to preventative treatment of the disease; or delaying the onset or progression of the disease.

[0495] The term "subject" herein refers to mammals, primates (e.g., human male or female), dogs, rabbits, guinea pigs, pigs, rats, and mice. In certain embodiments, the subject is a primate. In other embodiments, the subject is a human.

[0496] The term "therapeutically effective amount" as used herein refers to the amount of the antibody of the present invention having a site-specific modification that will elicit a biological or medical response in a subject, such as improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease. The therapeutically effective amount will vary depending on the type and severity of the condition to be alleviated. It should also be understood that for any particular subject, the specific dosing regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the composition. In some embodiments of the present invention, the therapeutically effective amount is based on various factors, such as the type of disease, the patient's age, weight, sex, medical condition, severity, route of administration, and the specific antibody employed. In some embodiments of the present invention, the therapeutically effective amount can vary widely, but can be routinely determined using standard methods. In some embodiments of the present invention, the therapeutically effective amount can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which can include clinical effects and / or laboratory values ​​such as toxic effects.

[0497] In some embodiments, the disease is a tumor or cancer. In some embodiments, the disease is an autoimmune disease, etc.

[0498] In some embodiments, cancer can include, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or gastric tumors (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, 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 malignancies, anal cancer, penile cancer, and head and neck cancer.

[0499] It will be apparent to those skilled in the art that other suitable modifications and alterations to the invention described herein will be readily apparent and can be made using suitable equivalents without departing from the scope of the invention or the embodiments disclosed herein. The invention has now been described in detail and will be more clearly understood by reference to the following examples, which are intended to be illustrative only and are not intended to be limiting. Further, unless otherwise specifically stated, the reagents and solvents described in this specification can be readily obtained from commercial suppliers.

[0500] Example

[0501] The present invention is further described in the following examples, which are not intended to limit the scope of the invention described in the claims.

[0502] Reagents and manufacturers

[0503] Trastuzumab is commercially available from Roche.

[0504] Gosartuzumab and mabelantuzumab are commercially available from MedChemExpress.

[0505] MC-VC-PAB-MMAE can be purchased from Levena biopharma.

[0506] DMA (dimethylacetamide) can be purchased from Aldrich Sigma.

[0507] TCEP is commercially available from Bidepharm.

[0508] EDTA is commercially available from Aladdin.

[0509] DHAA is commercially available from Aladdin.

[0510] (2-Aminoethyl)maleimide is commercially available from Bidepharma.

[0511] Buffers are available commercially from Macklin.

[0512] Homogeneity determination:

[0513] The distribution of ADC was analyzed using HIC-HPLC (Agilent 1200) with a TSK gel butyl-NPR column (2.5 μm, 4.6 mm*35 mm) (available 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 K₂HPO₄, 28.6 mM K₂HPO₄, and 25% v / v isopropanol. The sampler temperature was 4°C. The flushing procedure was as follows:

[0514] Time [min] Solvent A[%] Solvent B[%] 0.00 100.0 0.0 2.00 100.0 0.0 15.00 0.0 100.0 17.00 0.0 100.0 18.00 100.0 0.0 20.00 100.0 0.0

[0515] Example 1: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugate Using the Process of the Invention

[0516] (1) TCEP (0.048 mM) and ZnCl2 (0.024 mM) were added to a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 18 h;

[0517] (2) EDTA (0.6 mM) and MC-VC-PAB-MMAE (0.08 mM) dissolved in DMA were introduced, and the reaction was incubated at room temperature for 1 h;

[0518] (3) The reaction mixture was purified using a desalting column (Thermo, type: 40K, 0.5 mL, reference number: 87766, batch number SJ251704).

[0519] Example 2: Preparation of gosartuzumab-[MC-VC-PAB-MMAE]6 conjugate using the process of the present invention The method of Example 2 is similar to that of Example 1, except that the antibody is gosartuzumab, the buffer system is MES buffer, and Zn 2+ The molar ratio of TCEP is 0.87:1.

[0520] Example 3: Preparation of Mabelantuzumab-[MC-VC-PAB-MMAE]6 conjugate using the process of the present invention The method of Example 3 is similar to that of Example 1, except that the antibody is Mabelantuzumab, the buffer system is MES buffer, and Zn 2+ The molar ratio of TCEP is 0.87:1.

[0521] The results of the homogeneity test are as follows:

[0522] Table 1: Homogeneity test results of Examples 1 to 3

[0523]

[0524] As shown in Table 1 and Figures 1 to 3 As shown, the results confirmed that MC-VC-PAB-MMAE was successfully linked to trastuzumab, gosartan, and mabelantuzumab, and the D6 content in Examples 1 to 3 was generally as high as 80%, such as 90%, 85%, and 82%. These results clearly show that the D6-type ADC prepared by the method of the present invention has significantly improved uniformity.

[0525] Examples 4 to 13: Preparation of Antibody-[MC-VC-PAB-MMAE]6 Conjugates (Molar Ratio of TCEP to Antibody and / or Reduction Time in Step (1) Different)

[0526] 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 mabelantuzumab. The concentrations of ZnCl2 and TCEP and the reduction time in step (1) are as follows:

[0527]

[0528] "E" is short for "Example".

[0529] Comparative Example 11: Preparation of Antibody-[MC-VC-PAB-MMAE]2 Conjugate (D2-type ADC)

[0530] (1) ZnCl2 (0.24 mM) and TCEP (0.02 mM) were added to a trastuzumab solution (0.012 mM, dissolved in MES buffer, pH 6.7, 20 mM), and the reaction mixture was placed at 4 °C for 4 h;

[0531] (2) Add EDTA (0.6 mM) to capture Zn 2 +;

[0532] (3) MC-VC-PAB-MMAE (0.06 mM) dissolved in DMA was introduced and the reaction was continued at 24°C for 30 min; (4) Cysteine ​​(available from Aladdin, 0.08 mM) was added to consume excess MC-VC-PAB-MMAE; (5) The reaction mixture was purified using a desalting column.

[0533] The results of the uniformity measurements of Examples E4 to E13 and Comparative Example 11 are as follows:

[0534] Table 2: Uniformity measurement results of Examples 4 to 13 and Comparative Example 11

[0535]

[0536] "C" is an abbreviation for comparative example.

[0537] As shown in Table 2, compared with Comparative Example 11, when the molar ratio of TCEP to antibody was 3:1 to 13:1, the content of D6 was as high as 55%, 80%, and even 85% and 87%. When the molar ratio of TCEP to antibody was 8:1 to 13:1, the reduction time in step (1) was shortened to 6 hours. When the molar ratio of TCEP to antibody was 8:1 to 12:1 and the reduction time in step (1) was 6 hours, the content of D6 was as high as 87% or 89%. The results show that the molar ratio of TCEP / antibody plays an important role in determining the D6 content and selective reduction.

[0538] Examples 14 to 28: Trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (Zn 2+ Preparation of different molar ratios with TCEP and / or different reduction times in step (1)

[0539] The methods of Examples 14 to 28 are similar to those of Example 1, except for the concentration of ZnCl2 and / or TCEP and / or the reduction time in step (1), as shown in Table 3.

[0540] Table 3: Zn of Examples 14 to 28 2+ Concentration

[0541]

[0542] Comparative Examples 1 to 4: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugates (Zn 2+ Different molar ratios with TCEP)

[0543] 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 ZnCl2 in step (1) is 0.

[0544] The results of the homogeneity test are as follows:

[0545] Table 4: Uniformity measurement results of Examples 14 to 28 and Comparative Examples 1 to 4

[0546]

[0547]

[0548] The comparative example is abbreviated as "C".

[0549] As shown in Table 4, the results show that as Zn 2+ When the molar ratio of Zn to TCEP increases from 0.11:1 to 27.27:1, the content of D6 is as high as 75%, and even reaches 80%, 85% and 90%. 2+ When the concentration was 0, the D6 content was as low as 10%, which indicated that metal transition ions were very important for improving the selective reduction and uniformity of D6-type conjugates.

[0550] Examples 29 to 32: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugates (Different Reduction Times in Step (1))

[0551] The methods of Examples 29 to 32 are similar to those of Example 1, except for the reduction time in step (1), as shown in Table 5. 2+ The molar ratio of TCEP to TCEP was 0.22:1, and the molar ratio of TCEP to trastuzumab was 9:1.

[0552] The results of the homogeneity test are as follows:

[0553] Table 5: Homogeneity test results for Examples 29 to 32

[0554]

[0555] As shown in Table 5, the results show that when the molar ratio of TCEP to antibody is 9:1 and the reducing agent time in step (1) is 4 hours to 10 hours, the content of D6 is as high as 86%, and even reaches 90%, which indicates that by increasing the molar ratio of TCEP to antibody, the method shown here reduces the reduction time cost.

[0556] Examples 33 to 36: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugates (Different Reduction Temperatures in Step (1))

[0557] The methods of Examples 33 to 36 are similar to those of Example 1, except that the reduction temperature, Zn 2+ The molar ratio of TCEP to TCEP and / or the molar ratio of TCEP to antibody in step (1) are as follows.

[0558]

[0559] The results of the homogeneity test are as follows:

[0560] Table 6: Homogeneity test results of Examples 33 to 36

[0561]

[0562] As shown in Table 6, the results show that when the reduction temperature in step (1) is between 0°C and 25°C, the content of D6 is as high as 80%, 85%, and even 88%.

[0563] Comparative Examples 37 to 47: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugates Using Different Buffer Systems

[0564] The methods of Examples 37 to 47 are similar to those of Example 1, except that the BES buffer of Example 1 is replaced with different buffers of Examples 37 to 47.

[0565] Table 7: Buffer systems and pH values ​​of Examples 37 to 47

[0566] Example No. Buffer system pH value of the buffer system E37 Bis-Tris buffer 6.7 E38 PIPES buffer 6.7 E39 MOPS buffer 6.7 E40 BES buffer 6.7 E41 HEPES buffer 6.7 E42 DIPSO buffer 6.7 E43 MOBS buffer 7.4 E44 MOPSO buffer 7.4 E45 TES buffer 7.4 E46 ACES Buffer 7.4 E47 TAPSO buffer 7.4

[0567] Comparative Examples 5 to 6: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugates Using Different Buffer Systems

[0568] The method of Comparative Examples 5 to 6 is similar to that of Example 1, except that the BES buffer of Example 1 is replaced with different buffers of Comparative Examples 5 to 6.

[0569] Table 8: Buffer systems and pH values ​​of buffer systems in Comparative Examples 5 to 6

[0570] Comparison Example No. Buffer system pH value of the buffer system C5 PB 6.7 C6 ADA buffer 6.7

[0571] The results of the homogeneity test are as follows:

[0572] Table 9: Uniformity measurement results of Examples 37 to 47 and Comparative Examples 5 to 6

[0573]

[0574] As shown in Table 9 and Figures 18 to 30 As shown, the results indicate that the type of buffer system will affect the content of D6 by affecting the reduction kinetics and selectivity. The buffer systems of Examples 37 to 47 help to increase the content of D6.

[0575] Comparative Examples 48 to 51: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugates at Different pH Values ​​The methods of Examples 48 to 51 are similar to those of Example 1, except for the pH value of the buffer system.

[0576] Table 10: pH values ​​of the buffer systems of Examples 48 to 51

[0577] Example No. Buffer system pH value of the buffer system E48 BES buffer 6.4 E49 BES buffer 6.7 E50 BES buffer 7.0 E51 BES buffer 7.4

[0578] The results of the homogeneity test are as follows:

[0579] Table 11: Homogeneity test results of Examples 48 to 51

[0580] Example No. Attached photos Buffer system pH D0(％) D2(％) D4(％) D6(％) D8(％) E48 31 BES buffer 6.4 0 4 10 79 7 E49 32 BES buffer 6.7 0 0 7 89 4 E50 33 BES buffer 7.0 0 0 6 90 4 E51 34 BES buffer 7.4 0 0 5 90 5

[0581] As shown in Table 11 and Figures 31 to 34 As shown, the results indicate that the pH value of the buffer system of Examples 48 to 51 is helpful to increase the content of D6.

[0582] Comparative Examples 52 to 55: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugates with Different Buffer System Concentrations

[0583] The methods of Examples 52 to 55 are similar to those of Example 1, except for the concentration of the buffer system.

[0584] Table 12: Concentrations of buffer systems for Examples 52 to 55

[0585] Example No. Buffer system Buffer concentration E52 BES buffer 20mM E53 BES buffer 40mM E54 BES buffer 60mM E55 BES buffer 80mM

[0586] The results of the homogeneity test are as follows:

[0587] Table 13: Homogeneity test results of Examples 52 to 55

[0588]

[0589] As shown in Table 13 and Figures 35 to 38 As shown, the results indicate that the concentration of the buffer systems of Examples 51 to 54 helps to increase the content of D6.

[0590] Example 56: Preparation of Trastuzumab-[Bismaleimide-DBCO]3

[0591] 1. Synthesis of Bismaleimide-DBCO (Sulfur Bridge Reagent)

[0592]

[0593] Intermediate 2: To a mixture of 1 (6.5 g, 50.0 mmol, 1.0 equiv) in DMF (100 mL) was added DBU (30.7 g, 0.2 mol, 4.0 equiv). The mixture was stirred at 80°C for 1 h, and 2-benzyl bromoacetate (25.4 g, 0.11 mol, 2.2 equiv) was added. The 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 organic layers were combined, washed with brine (200 mL), dried over Na2SO4, and filtered. The filtrate was concentrated and purified by flash column (EtOAc / petroleum ether = 0-40%) to give product 2 (4.5 g, 21.0%) as a white solid.

[0594] Intermediate 3: To a mixture of Intermediate 2 (4.5 g, 10.58 mmol, 1.0 equiv) in DMF (50 mL) was added DBU (3.22 g, 21.16 mol, 2.0 equiv). 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 equiv) was added. The 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 organic layers were combined, washed with brine (100 mL), dried over Na2SO4, and filtered. The filtrate was concentrated and purified by flash column (EtOAc / petroleum ether = 0-40%) to give product 3 (4.8 g, 79.8%) as a white solid.

[0595] Intermediate 4: Pd / C (500 mg, 10% Pd / C, moistened with about 55% water) was added to a mixture of intermediate 3 (4.8 g, 8.44 mmol, 1.0 equivalent) in THF (50 mL). The mixture was degassed 3 times and purged with H2. Under an H2 atmosphere, the resulting mixture was stirred at room temperature for 2 h. 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.

[0596] Intermediate 5: To a mixture of intermediate 4 (50 mg, 1.00 mmol, 1.0 equiv) in DCM (5 mL) was added HATU (0.92 g, 2.4 mmol, 2.4 equiv) and DIPEA (0.65 g, 5.0 mmol, 5.0 equiv). The mixture was stirred at room temperature for 0.5 h, and 4a (0.51 g, 2.0 mmol, 2.0 equiv) was added. The resulting mixture was stirred at room temperature for 2 h. LCMS showed that intermediate 4 was completely consumed. The mixture was quenched by the addition of HCl (0.5 M, 2 mL) and purified by RP column (water / MeCN = 10% to 70%), and the eluate was lyophilized to give the product 5 (0.51 g, 80.3%) as a white solid.

[0597] Intermediate 6: To a mixture of intermediate 5 (100 mg, 0.16 mmol, 1.0 equiv) in DCM (2 mL) was added TFA (100 uL). The mixture was stirred at room temperature for 1 hour. LCMS showed that intermediate 5 was completely consumed. The mixture was concentrated, and the residue was dissolved in water (10 mL) and then lyophilized to give product 6 (100 mg, 97.8%) as a white solid.

[0598] Compound 7 (bismaleimide-DBCO): To a mixture of intermediate 6 (50 mg, 93.9 umol, 1.0 equiv) in DCM (2 mL) was added 6a (37.8 mg, 93.9 umol, 1.0 equiv) followed by DIPEA (17 μL, 93.9 umol, 1.0 equiv). 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%), and 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 accurate mass is 819.26. 1 H NMR (400MHz, DMSO-d6): δ8.17(t,J=6.0Hz,2H),7.85(d,J=6.4Hz,1H),7.64(dd,J=18.4,7.3Hz,2H),7.47(d,J=10.3Hz,3H),7.38–7.24(m,3H),6.96 (s,4H),5.02(d,J=14.0Hz,1H),4.22(s,4H),3.68(t,J=6.6Hz,2H),3.65- 3.58(m,4H),3.43(t,J=6.2Hz,3H),3.24-3.20(m,3H),3.16-3.10(m,5H).

[0599] 2. Preparation of Trastuzumab-[Bismaleimide-DBCO]3

[0600] (1) TCEP (0.048 mM) and ZnCl2 (0.024 mM) were added to a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 18 h;

[0601] (2) introducing EDTA (0.6 mM) and bismaleimide-DBCO (0.045 mM) to react with the reduced thiol group from step (1) at room temperature for 1 h, and then recovering the product using a desalting column to obtain trastuzumab-[bismaleimide-DBCO]3;

[0602] The results of the homogeneity test are as follows:

[0603] serial number Attached photos ADC type D3(％) D4(％) E56 39 D3 86.55 13.45

[0604] As shown in the table, the results confirmed that the content of trastuzumab-[bismaleimide-DBCO]3 was generally as high as 86%, indicating that the process of this method is conducive to site-specific modification of D3 type antibodies and can improve uniformity. Example 57: Preparation of trastuzumab-[MC-GGFG-DXd]6[MC-VC-PAB-MMAE]2 (D6+D2 type ADC)

[0605] (1) ZnCl2 (0.048 mM) and the reducing agent TCEP (0.048 mM) were added to the monoclonal antibody trastuzumab (0.012 mM) in BES (pH 7.0, 20 mM) buffer, and the reaction mixture was placed at 4°C for 18 h;

[0606] (2) EDTA-2Na (0.6 mM) and MC-GGFG-DXd (0.1 mM) dissolved in DMA were introduced and the reaction was continued at room temperature for 1 h, and then the obtained product was purified using a desalting column;

[0607] (3) The resulting product was incubated with the second reducing agent TCEP (0.02 mM) at room temperature for 3 h; the second linker-payload MC-VC-PAB-MMAE (0.05 mM) was added and the reaction continued at room temperature for 2 h;

[0608] (4) Purify the obtained product using a desalting column.

[0609] Among them, an average of about six drug molecules MC-GGFG-DXd were coupled to trastuzumab, and an average of about two drug molecules MC-VC-PAB-MMAE were coupled to trastuzumab.

[0610] The results of the homogeneity test are as follows:

[0611]

[0612]

[0613] As shown in the table above, the results confirmed that the D6+D2 ADC content was generally as high as 87.10%, indicating that the process of this method is conducive to the D6+D2 site-specific modification of antibodies and can improve uniformity.

[0614] Example 58: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6[Maleimide-PEG4-N3-DBCO-Cy3]1 (D6+D1 type ADC)

[0615] 1. Synthesis of dibromomaleimide-PEG4-N3

[0616]

[0617] 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 minutes. DCM (10 mL) was then added, the organic phase was washed with H2O, dried over MgSO4, and the solvent was removed in vacuo to give the title compound 1 (139 mg, 0.4 mmol, 80%).

[0618] A solution of azido-PEG4-amine (105 mg, 0.4 mmol, 1 eq., Xi'an Kangfuno Biotechnology 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).

[0619] After 30 minutes, 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 4. After vacuum concentration, column chromatography purification (100% EtOAc as mobile phase) was performed to give dibromomaleimide-PEG 4 -N 3 (title product 2) as a light yellow oil (150 mg, 0.3 mmol, 75%).

[0620] 2. Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6[Maleimide-PEG4-N3-DBCO-Cy3]1

[0621] (1) ZnCl2 (0.0408 mM) and TCEP (0.048 mM) were added to a solution of monoclonal antibody trastuzumab (0.012 mM) in BES buffer (pH 7.0, 20 mM), and the reaction mixture was placed at 4°C for 16 h;

[0622] (2) EDTA-2Na (0.6 mM) and MC-VC-PAB-MMAE (0.1 mM) dissolved in DMA were introduced and the reaction was continued at room temperature for 1 h, and then the product was recovered using a desalting column;

[0623] (3) The resulting product and the reducing agent TCEP (0.09 mM) were incubated at room temperature for 20 h; a second sulfur bridge reagent having a reactive group, dibromomaleimide-PEG4-N3 (0.012 mM), was added, and the reaction continued at room temperature for 2 h; then the second linker-payload DBCO-Cy3 (0.05 mM) was added, and the reaction continued at room temperature for 4 h.

[0624] (4) Purify the obtained product using a desalting column.

[0625] Among them, an average of about six drug molecules MC-VC-PAB-MMAE were coupled to trastuzumab, and an average of about one drug molecule maleimide-PEG4-N3-DBCO-Cy3 was coupled to trastuzumab.

[0626] The results of the homogeneity test are as follows:

[0627] serial number Attached photos D2(％) D4(％) D6(％) D8(％) E58-Step (2) 41A 4.48 4.97 86.49 4.06 serial number Attached photos D6+D1(%) D8(％) / / E58-Step (4) 41B 89.89 10.11 / /

[0628] As shown in the table above, the results confirmed that the content of D6+D1 type ADC is usually as high as 80%, 85%, and even 90%, which indicates that the process of this method is conducive to the D6+D1 type site-specific modification of antibodies and can improve the uniformity. Example 59: Preparation of Trastuzumab-[Maleimide]6[MC-VC-PAB-MMAE]2 (D0+D2 type ADC)

[0629] (1) TCEP (0.048 mM) and ZnCl2 (0.024 mM) were added to a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 18 h;

[0630] (2) EDTA (0.6 mM) and (2-aminoethyl)maleimide (0.1 mM) were introduced to react with the reduced thiol group from step (1) at room temperature for 1 h, and then the product was recovered using a desalting column to obtain trastuzumab-[maleimide] 6;

[0631] (3) Trastuzumab-[maleimide]6 and the reducing agent TCEP (0.02 mM) were incubated at room temperature for 18 h; the second linker-payload MC-VC-PAB-MMAE (0.05 mM) was added and the reaction continued at room temperature for 2 h.

[0632] (4) Purify the obtained product using a desalting column.

[0633] The results of the homogeneity test are as follows:

[0634] serial number Attached photos D0(％) / / / E59-Step (2) 42A 100 / / / serial number Attached photos D0(％) D0+D1(%) D0+D2(%) D0+D3(%) E59-Step (4) 42B 11.66 6.31 70.92 11.11

[0635] As shown in the table above, the results confirmed that the D0+D2 ADC content was generally as high as 70.92%, indicating that the process of this method is conducive to D0+D2 site-specific modification of antibodies and can improve uniformity.

[0636] Example 60: Preparation of Trastuzumab-[Maleimide]6[Maleimide-PEG4-N3-DBCO-Cy3]1 (D0+D1 Type ADC)

[0637] (1) TCEP (0.048 mM) and ZnCl2 (0.024 mM) were added to a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 18 h;

[0638] (2) EDTA (0.6 mM) and (2-aminoethyl)maleimide (0.1 mM) were introduced to react with the reduced thiol group from step (1) at room temperature for 1 h, and then the product was recovered using a desalting column to obtain trastuzumab-[maleimide] 6;

[0639] (3) Trastuzumab-[maleimide]2 and the second reducing agent TCEP (0.02 mM) were incubated at room temperature for 18 h. The second sulfide bridge reagent with the reactive group dibromomaleimide-PEG4-N3 (0.013 mM) reacted with the reduced thiol group at 24 °C for 3 h.

[0640] (4) The resulting product was incubated with DBCO-Cy3 (0.02 mM) in MES (20 mM, pH 6.7) at 25 °C for 8 h, and then trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3] was recovered using a desalting column.

[0641] The results of the homogeneity test are as follows:

[0642] serial number Attached photos D0(％) D0+D1(%) E60 43 7.04 92.96

[0643] As shown in the table above, the results confirmed that the D0+D1 type ADC content was generally as high as 92.96%, indicating that the process of this method is conducive to the D0+D1 type site-specific modification of antibodies and can improve uniformity.

[0644] Example 61: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugate Using the Process of the Invention

[0645] (1) TCEP (0.048 mM) and ZnCl2 (0.012 mM) were added to a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 18 h;

[0646] (2) Add DHAA (0.096 mM) to selectively reoxidize the reduced thiol groups in the Fab region in step (1) at 25 °C in the dark for 1 h;

[0647] (3) EDTA (0.6 mM) and MC-VC-PAB-MMAE (0.048 mM) dissolved in DMA were introduced to react with the remaining thiol groups from step (2), and the reaction was incubated at room temperature for 1 h;

[0648] (4) The reaction mixture was purified using a desalting column (Thermo, type: 40K, 0.5 mL, reference number: 87766, batch number SJ251704).

[0649] The results of the homogeneity test are as follows:

[0650] Table 14: Homogeneity test results of Example 61

[0651] Example No. Attached photos Antibody D0(％) D2(％) D4(％) D8(％) 61 44 Trastuzumab 5 92 3 0

[0652] As shown in Table 12 and Figure 44 As shown, the results confirmed that MC-VC-PAB-MMAE was successfully linked to trastuzumab, and the content of D2 in Example 61 was 92%. These results clearly show that the D2-type ADC prepared by the method of the present invention has significantly improved uniformity.

[0653] Examples 62 to 90: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugates Using the Process of the Invention The methods of Examples 62 to 90 were similar to those of Example 61, except for the parameters in steps (1) and (2). The different parameters are shown in Table 15. Also, in Examples 76 to 90, the oxidation time in step (2) was 2 h, and in Example 90, the molar ratio of ZnCl2 to antibody was 2:1.

[0654] Table 15: Parameters in steps (1) and (2) of Examples 62 to 75

[0655]

[0656] Room temperature is abbreviated as "RT"

[0657] Table 16: Parameters in steps (1) and (2) of Examples 76 to 90

[0658]

[0659] Comparative Examples 7 to 9: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugates (Zn 2+ Different molar ratios with TCEP)

[0660] 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.

[0661] Table 17: Homogeneity test results for Examples 62 to 75

[0662]

[0663] Table 18: Uniformity measurement results of Examples 76 to 90 and Comparative Examples 7 to 9

[0664]

[0665]

[0666] As shown in Examples 76 to 90, when the molar ratio of TCEP to antibody is 4:1 to 10:1, the content of D6 is as high as 80%, and even reaches 85% and 90%. At the same time, the reduction time in step (1) is shortened to 1 hour, and the reduction time cost is lower.

[0667] As shown in Tables 17 and 18, the results indicate that the molar ratio of DHAA to antibody plays an important role in determining D2 content and selective oxidation. When the molar ratio of DHAA to antibody ranged from 4:1 to 24:1, D2 content reached as high as 60%, 70%, 80%, 85%, and even 90% or 95%.

[0668] Zn 2+ The molar ratio of Zn to antibody also affects the content and selective oxidation of D2. 2+ When the concentration is 0, the content of D2 is as low as 7.87%. 2+ When the molar ratio of D2 to antibody is 1:1 or 2:1, it helps to improve the uniformity of D2-type ADC.

[0669] As shown in Examples 65 and 67 or Examples 68 and 70, the content of D2-type ADC increased from 89% to 95% or from 88% to 94% by purifying the oxidation product. These results confirm that it is important to purify the oxidation product before step (3) in order to increase the content of D2-type ADC.

[0670] Examples 91 to 94: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugates (Oxidation Temperature and / or Oxidation Time in Step (2) Different)

[0671] The methods of Examples 91 to 94 are similar to Example 61, except for the oxidation temperature and / or oxidation time in step (2), as shown in Table 19.

[0672] The results of the homogeneity test are as follows:

[0673] Table 19: Homogeneity test results of Examples 91 to 94

[0674]

[0675] As shown in Table 19, the results show that when the oxidation temperature in step (2) is 4°C to 37°C, the content of D6 is as high as 70%, even reaching 90%, and the oxidation time in step (2) is 1h to 48h.

[0676] Examples 95 to 110 and Comparative Example 10: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugates Using Different Buffer Systems

[0677] The methods of Examples 95 to 110 and Comparative Example 10 were similar to those of Example 61, except that the BES buffer of Example 61 was replaced with different buffers in Examples 95 to 110 and Comparative Example 10. Meanwhile, in Examples 95 to 110 and Comparative Example 10, the oxidation time in step (2) was 2 h.

[0678] The results of the homogeneity test are as follows:

[0679] Table 20: Uniformity measurement results of Examples 95 to 110 and Comparative Example 10

[0680] serial number Attached photos Buffer system pH D0(％) D2(％) D4(％) D6(％) D8(％) E95 63A Bis-Tris 6.7 4.68 83.54 11.78 0 0 E96 63B PIPES 6.7 20.77 75.71 3.51 0 0 E97 63C PB 6.7 26.00 74.00 0 0 0 E98 63D HEPES 7.0 9.80 90.20 0 0 0 E99 63E MOPS 7.0 12.48 81.43 6.09 0 0 E100 63F DIPSO 7.4 5.23 91.09 3.68 0 0 E101 63G MOBS 7.4 7.89 92.11 0 0 0 E102 63H MOPSO 7.4 7.45 88.78 3.77 0 0 E103 64A TES 7.4 9.20 86.09 4.71 0 0 E104 64B ACES 7.4 6.64 93.36 0 0 0 E105 64C TAPSO 7.4 8.16 88.92 2.93 0 0 E106 64D MES 5.8 8.43 91.57 0 0 0 E107 64E MES 6.1 6.47 93.53 0 0 0 E108 64F BES 6.4 8.10 91.90 0 0 0 E109 64G BES 7.4 4.08 95.92 0 0 0 E110 64H BES 7.4 7.78 92.22 0 0 0 C10 65 ADA 6.7 67.96 25.96 6.08 0 0

[0681] As shown in Table 20, the results show that the type and pH value of the buffer system will affect the content of D2 by affecting the reduction kinetics and selectivity. The buffer systems of Examples 95 to 110 can be used to increase the content of D2, and the pH value of the buffer system is 5.8 to 7.4.

[0682] Example 111: Preparation of Trastuzumab-[Maleimide-PEG4-N3-DBCO-MMAE]1 (D1-type ADC)

[0683] (1) TCEP (0.048 mM) and ZnCl2 (0.012 mM) were added to a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 18 h;

[0684] (2) Add DHAA (0.066 mM) to selectively reoxidize the reduced thiol groups in the Fab region in step (1) at 25 °C for 2 h;

[0685] (3) introducing EDTA (0.6 mM) and the first sulfur bridge reagent dibromomaleimide-PEG4-N3 (0.013 mM) to react with the reduced thiol group from step (3), the reaction temperature is 25 ° C and the reaction time is 1 h, and then the product is recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1;

[0686] (4) Trastuzumab-[maleimide-PEG4-N3]1 and DBCO-MMAE (0.02 mM) were incubated in BES buffer (20 mM, pH 7.0) at a reaction temperature of 25°C and a reaction time of 8 h.

[0687] (5) The reaction mixture was purified by HIC chromatography using a desalting column and AKTA.

[0688] AKTA with HIC chromatography: A polar MC30-HIC butyl column (4.2 mL, ADC purification was performed using an AKTA explorer (available from Sepax Technologies, Inc., 30 μm) (available 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 volume ratio, filtered, and then loaded at a flow rate of 2 mL / min. The target component was washed with solvent A / solvent B between v / v 65% / 35% and v / v 0% / 100% at a flow rate of 3 mL / min at 25°C, and the collected product solution was concentrated and exchanged into His buffer (20 mM, pH 5.5) by centrifugation.

[0689] The results of the homogeneity test are as follows:

[0690] serial number Attached photos D0(％) D1(％) E111 66 2.42 97.58

[0691] As shown in the table above, the results confirmed that the D1-type ADC content was generally as high as 97.58%, indicating that the process of this method is conducive to the D1-type site-specific modification of antibodies and can improve uniformity.

[0692] Example 112: Preparation of Trastuzumab-[Maleimide-PEG4-N3-DBCO-MMAE]1[MC-GGFG-DXd]6 (D1+D6 type ADC)

[0693] (1) In BES buffer (20 mM, pH 7.0), trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1 prepared in Example 111 (0.008 mM) and TCEP (0.08 mM) were introduced at a reaction temperature of 37° C. and a reaction time of 16 h;

[0694] (2) MC-GGFG-DXd (0.14 mM) was introduced into the solution from step (1), and the reaction mixture was placed at 24°C for 1 h, and then trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1[MC-GGFG-DXd]6 was recovered using a desalting column.

[0695] The results of the homogeneity test are as follows:

[0696] serial number Attached photos D1+D2(%) D1+D4(%) D1+D6(%) D8(％) E112 67 5.66 5.98 82.42 5.94

[0697] As shown in the table above, the results confirmed that the D1+D6 ADC content was generally as high as 82.42%, indicating that the process of this method is conducive to site-specific modification of D1+D6 antibodies and can improve uniformity.

[0698] Example 113: Preparation of Trastuzumab-[Maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2 (D1+D2 Type ADC)

[0699] (1) TCEP (0.048 mM) and ZnCl2 (0.012 mM) were added to a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 18 h;

[0700] (2) Add DHAA (0.096 mM) to selectively reoxidize the reduced thiol groups in the Fab region in step (1) at 25 °C for 2 h;

[0701] (3) introducing EDTA (0.6 mM) and the first sulfur bridge reagent dibromomaleimide-PEG4-N3 (0.013 mM) to react with the reduced thiol group from step (3), the reaction temperature is 25 ° C and the reaction time is 1 h, and then the product is recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1;

[0702] (4) incubating with trastuzumab-[maleimide-PEG4-N3]1 (0.013 mM) at 25°C for 1 h, and then incubating with DBCO-Cy3 (0.02 mM) in BES buffer (20 mM, pH 7.0) at 25°C for 8 h;

[0703] (5) purifying the reaction mixture using a desalting column;

[0704] (6) Incubate ZnCl2 (0.8 mM), the second reducing agent TCEP (0.011 mM), and the product from step (5) (0.008 mM) in BES buffer (pH 7.0, 20 mM), and place the reaction mixture at 4 °C for 4 h;

[0705] (7) Introducing EDTA (3 mM) to capture Zn 2+ , and MC-VC-PAB-MMAE (0.048 mM) was introduced to react with the reduced thiol groups from step (6), the reaction temperature was 25 ° C and the reaction time was 2 h;

[0706] (8) Purify the reaction mixture using a desalting column.

[0707] The results of the homogeneity test are as follows:

[0708] serial number Attached photos D1(％) D2(％) D1+D2(%) D4(％) D1+D4(%) E113 68 2.84 2.39 71.97 2.49 20.31

[0709] In step (6), one of the interchain disulfide bonds in the D1-type ADC is reduced. As shown in the table above, the results confirm that the D1+D2-type ADC content is generally as high as 70%, indicating that this process is conducive to D1+D2-type site-specific modification of antibodies and can improve homogeneity.

[0710] Examples 114 to 115: Preparation of Trastuzumab-[Maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 (D1+D4 type ADC)

[0711] (1) TCEP (0.048 mM) and ZnCl2 (0.024 mM) were added to a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 18 h;

[0712] (2) Add DHAA (0.12 mM) to selectively reoxidize the reduced thiol groups in the Fab region in step (1) at 25 °C for 2 h;

[0713] (3) introducing EDTA (0.6 mM) and a mixture of the first sulfur bridge reagent dibromomaleimide-PEG4-N3 (0.028 mM) and DBCO-Cy3 (0.033 mM) to react with the reduced thiol group from step (2) at a reaction temperature of 25 ° C. and a reaction time of 2 h, and then recovering the product using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1;

[0714] (4) Incubate ZnCl2 (Example 113: 0.024 mM; Example 114: 0.36 mM), a second reducing agent TCEP (0.024 mM), and the product from step (3) (0.008 mM) in BES buffer (pH 7.0, 20 mM), and place the reaction mixture at 4°C for 16 h;

[0715] (5) Introducing EDTA (3 mM) to capture Zn 2+ , and introduced MC-VC-PAB-MMAE (0.048 mM) to react with the reduced thiol groups from step (4), the reaction temperature was 25 ° C and the reaction time was 2 h;

[0716] (6) Purify the reaction mixture using a desalting column.

[0717] The results of the homogeneity test are as follows:

[0718] serial number Attached photos D0(％) D1(％) D2(％) / / E114-Step (3) 69A 5.54 87.75 6.71 / / serial number Attached photos D1+D2(%) D2+D2(%) D1+D4(%) D2+D4(%) D1+D6(%) E114-Step (6) 69B 8.81 7.74 79.18 4.26 / E115-Step (6) 69C 5.40 6.94 81.17 3.77 2.72

[0719] In step (4), two of the interchain disulfide bonds in the D1-type ADC are reduced. As shown in the table above, the results confirm that the D1+D4-type ADC content is generally as high as 80%, indicating that this process is conducive to D1+D4-type site-specific modification of antibodies and can improve homogeneity.

[0720] Example 116: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]4 Conjugate (D2+D4 Type ADC)

[0721] (1) TCEP (0.0672 mM) and ZnCl2 (0.024 mM) were added to a solution of trastuzumab (0.012 mM) in BES buffer (20 mM, pH 7.0), and the reaction mixture was vortexed and then incubated at 4°C for 16 h.

[0722] (2) Add DHAA (0.12 mM) to selectively reoxidize the reduced thiol groups in the Fab region in step (1) at 25 °C for 2 h;

[0723] (3) EDTA (0.6 mM) and MC-VC-PAB-MMAE (0.048 mM) dissolved in DMA were introduced to react with the remaining thiol groups from step (2), and the reaction was incubated at room temperature for 1 h;

[0724] (4) purifying the reaction mixture using a desalting column;

[0725] (5) Incubate ZnCl2 (0.552 mM), the second reducing agent TCEP (1.2 mM) and the product from step (4) in BES buffer (pH 7.0, 20 mM), and place the reaction mixture at 4 ° C for 2.5 h;

[0726] (6) Introducing EDTA (3 mM) to capture Zn 2+ , and MC-GGFG-DXd (0.048 mM) was introduced to react with the reduced thiol groups from step (5), the reaction temperature was 25 ° C and the reaction time was 2 h;

[0727] (7) Purify the reaction mixture using a desalting column.

[0728] The results of the homogeneity test are as follows:

[0729]

[0730]

[0731] As shown in the table above, the results indicate that the D2(DXd)+D4(MMAE) type ADC content is typically as high as 90%, indicating that the process of this method is conducive to site-specific modification of D2+D4 type antibodies and can improve uniformity.

[0732] In summary, the methods of the present invention provide diverse classes of highly homogeneous ADCs without the use of antibody and enzyme engineering. For example, D6-type ADCs exhibit homogeneity greater than 55%, 65%, 70%, 80%, 85%, and even 90%, while D2-type ADCs exhibit homogeneity as high as 60%, 70%, 75%, and even 80%, 85%, 90%, or 95%. Furthermore, the methods of the present invention are compatible with current thiol-reactive linker drug technology, exhibiting minimal conformational changes and intact Fc functionality. Furthermore, the methods are simple to operate and offer lower costs.

[0733] While particular embodiments have been described, presently unforeseen or potentially unforeseen alternatives, modifications, variations, improvements, and substantial equivalents may occur to the applicant or others skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to encompass all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. A method for preparing an antibody with site-specific modification, characterized in that: The site-specific modification is that three interchain disulfide bonds in the antibody are selectively reduced, and the method comprises using tris(2-carboxyethyl)phosphine (TCEP) or a salt thereof and a transition metal ion together.

2. A method for preparing an antibody with site-specific modification, characterized in that: The site-specific modification is that three interchain disulfide bonds in the antibody are selectively reduced, two of the three interchain disulfide bonds are located in the Fab region and one is located in the hinge region of the antibody, and the method comprises using TCEP or a salt thereof and a transition metal ion together.

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

1.

4. The method according to claim 3, characterized in that The method comprises introducing a metal chelating agent after step (a).

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

1.

6. The method according to claim 3, characterized in that 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. More optionally, in step (a), the incubation temperature is 0°C to 15°C.

7. The method according to claim 3, characterized in that In step (a), the incubation time is 3 h to 24 h, optionally, in step (a), the incubation time is 12 h to 24 h, more optionally, in step (a), the incubation time is 16 h to 20 h, most optionally, in step (a), the incubation time is 16 h to 18 h.

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

9. The method according to claim 3, characterized in that: In step (a), the molar ratio of the transition metal ion to the TCEP is 0.1:1 to 30:1, optionally, the molar ratio of the transition metal ion to the TCEP is 0.1:1 to 20:1, more optionally, the molar ratio of the transition metal ion to the TCEP is 0.5:1 to 8:

1.

10. The method according to claim 3, characterized in that: 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. The method according to claim 3, characterized in that The pH value of the buffer system is 5.5 to 8, preferably, the pH value of the buffer system is 5.8 to 7.4, and more preferably, the pH value of the buffer system is 6.7 to 7.

4.

12. The method according to claim 3, characterized in that The concentration of the buffer system is 10 mM to 100 mM, 20 mM to 80 mM or 20 mM to 40 mM.

13. The method according to claim 3, characterized in that The transition metal ion is selected from the group consisting of: Zn 2+ 、Cd 2+ , Hg 2+ 、Ni 2+ 、Co 2+ or a combination thereof, optionally, the transition metal ion is Zn 2+ .

14. A method for preparing an antibody with site-specific modification, characterized in that: The method comprises the method of claims 3 to 13, and further comprises the following steps: (B1) introducing an oxidant to selectively reoxidize the reduced thiol groups from step (a), optionally, reoxidizing the reduced thiol groups in the Fab region of the antibody, preferably, removing excess of the oxidant to purify the oxidation product; (C1) introducing the metal chelator and the modification reagent 1 to react with the reduced thiol group from step (B1), wherein the modification reagent 1 is a capping reagent, a first linker-payload or a first sulfide bridge reagent, and optionally, the first sulfide bridge reagent carries the first linker-payload or a reactive group.

15. The method according to claim 14, characterized in that The method comprises the following steps, (C2) introducing the metal chelator and a second reducing agent to selectively reduce the antibody from step (B1), optionally reducing the interchain disulfide bonds in the hinge region of the antibody; or (C2') introducing the second reducing agent to reduce the interchain disulfide bonds in the product from step (C1), optionally introducing the transition metal ion; (D2) introducing a modification reagent 2 to react with the reduced thiol group from step (C2) or step (C2'), optionally introducing the metal chelator, wherein the modification reagent 2 is a second linker-payload or a second sulfide bridge reagent, optionally, the second sulfide bridge reagent carries the second linker-payload or reactive group.

16. The method according to claim 14 or 15, characterized in that The first sulphur bridging reagent and the second sulphur bridging reagent independently contain at least two substituents that allow re-bridging of the thiol group.

17. The method according to claim 16, characterized in that The first sulphur bridging agent and the second sulphur bridging agent are each independently selected from the group consisting of:

18. The method according to claim 14 or 15, characterized in that The reactive groups each independently contain an azide group and / or dibenzocyclooctyne DBCO.

19. The method according to claim 14, characterized in that In step (a), the molar ratio of TCEP to the antibody is 4:1 to 15:1, and the incubation time is 1 h to 16 h. Optionally, in step (a), the molar ratio of TCEP to the antibody is 4:1 to 10:1, and the incubation time is 4 h to 8 h.

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

1.

21. The method according to claim 14, characterized in that In step (B1), the molar ratio of the oxidant to the antibody is 2:1 to 25:1, optionally, the molar ratio of the oxidant to the antibody is 2:1 to 20:1, more optionally, the molar ratio of the oxidant to the antibody is 8:1 to 15:

1.

22. The method according to claim 14, characterized in that In step (B1), the oxidation temperature is 0°C to 37°C, and the oxidation time is 1 h to 48 h. Optionally, the oxidation temperature is 0°C to 30°C, and the oxidation time is 1 h to 8 h.

23. The method according to claim 14, characterized in that In step (b1), the oxidation reaction is carried out in the dark.

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

1.

26. The method according to claim 15, characterized in that In step (C2), the reduction temperature is 0°C to 30°C, and the reduction time is 1 h to 8 h.

27. The method according to claim 15, characterized in that 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 h to 24 h.

28. The method according to claim 15, characterized in that 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 0.5h to 24h.

29. A method for preparing an antibody with site-specific modification, characterized in that: The method comprises the method of claims 3 to 13, and further comprises the following steps: (b) introducing the metal chelator and the modifying reagent 1 to react with the reduced thiol group from step (a).

30. The method according to claim 29, characterized in that The method also The following steps are included: (c) incubating the reaction product from (b) and the second reducing agent in the buffer system to reduce the interchain disulfide bonds in the reaction product from (b); (d) introducing the incubation product from step (c) and the modification reagent 2 to react with the reduced thiol group from step (c).

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

32. The method according to claim 14 or 29, characterized in that When the first sulphur bridging agent carries the reactive group, the step (C1) and / or the step (b) comprises the following steps: The metal chelator and the first sulfide bridging reagent carrying the reactive group are introduced to re-bridge the reduced thiol group from step (B1) or step (a), and then the first linker-payload is incubated in the buffer system to react with the reactive group of the first sulfide bridging group.

33. The method according to claim 15 or 30, characterized in that When the second sulphur bridging reagent carries the reactive group, the step (D2) and / or the step (d) comprises the following steps: The product from step (C2) or step (C2') or step (c) is introduced with the second sulfide bridging reagent carrying the reactive group to re-bridge the reduced thiol group from step (C2) or step (C2') or step (c), and then, in the buffer system, the second linker-payload is incubated to react with the reactive group of the second sulfide bridging group.

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

35. The method according to claim 34, characterized in that The antibody is a human antibody, a humanized antibody, a chimeric antibody or an antigen-binding portion thereof. Optionally, the antibody is IgG1 or IgG4.

36. The method according to any one of claims 14 to 15 and claims 29 to 30, characterized in that The linkers in the first linker-payload and the second linker-payload are selected from any one of the linkers having the following characteristics: one end is capable of being linked to the reduced thiol group of the antibody or the reactive group of the sulfide bridging reagent, and the other end is capable of being linked to the payload.

37. The method according to any one of claims 14 to 15 and claims 29 to 30, characterized in that The payload is selected from any one of the payloads having at least one substituent that allows attachment from the payload to the linker.

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

39. The site-specifically modified antibody according to claim 38, characterized in that The antibody with site-specific modification is D2-type ADC, D4-type ADC, D1-type ADC, D6-type ADC, D3-type ADC, D1+D6-type ADC, D6+D2-type ADC, D6+D1-type ADC, D3+D1-type ADC, D3+D2-type ADC, D0+D6-type ADC, D0+D2-type ADC, D1+D2-type ADC, D1+D4-type ADC or D2+D4-type ADC.

40. A pharmaceutical composition comprising the antibody with 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 with site-specific modification according to any one of claims 38 to 39.

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

43. Use of an antibody with site-specific modification according to any one of claims 38 to 39 in the production of a therapeutic agent for 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 according to any one of claims 38 to 39.

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