Methods for programmed management of disulfide key site-specific modification of antibodies

Abstract

The invention relates to a method for programmed management of disulfide key site specific modification of an antibody, a modified antibody prepared by the method and application of antibody modification.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to PCT Application No. PCT / CN2022 / 113992 filed on August 22, 2022, PCT Application No. PCT / CN2022 / 119999 filed on September 20, 2022, PCT Application No. PCT / CN2022 / 119955 filed on September 20, 2022, PCT Application No. PCT / CN2022 / 131519 filed on November 11, 2022, PCT Application No. PCT / CN2022 / 131521 filed on November 11, 2022, and PCT Application No. PCT / CN2023 / 073070 filed on January 19, 2023. The contents of the previous PCT applications are considered part of the present invention and are incorporated herein in their entirety. Technical Field

[0003] The present invention relates to a method for programmed management of site-specific modification of antibody disulfide bonds, a modified antibody prepared by the method and uses thereof. Background Art

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

[0005] Site-specific modification methods have been widely used in the development of protein-based technologies. Modification of proteins has become a valuable approach to interrogate and intervene in biological systems. Protein modification methods are essential for the development of protein-drug conjugates, including antibody-drug conjugates (ADCs), without altering protein structure or activity.

[0006] In general, the conjugation of antibodies to cytotoxic agents typically involves conjugation to exposed residues (including lysine), or reduction of disulfide bonds to expose free interchain cysteines on therapeutic IgG (immunoglobulin G) antibodies. There are other newer methods to introduce conjugation sites into mAbs, such as site-specific glycosyl conjugation, cysteine ​​engineering, incorporation of non-natural amino acids, and coupling of short peptide tags to drug linkers. There are usually about 80 lysine residues on antibodies; however, less than 10 residues can be chemically conjugated. Cysteine ​​conjugation ultimately achieves the reduction of four interchain disulfide bonds. These bonds are reduced under specific conditions, subsequently generating two, four, six or eight exposed sulfhydryl groups. Both Cys and Lys conjugation methods produce heterogeneous mixtures. ("Advances and Limitations of Antibody Drug Conjugates for Cancer" Biomedicines. 2021 August; 9(8): 872.).

[0007] The drug-to-antibody ratio (DAR), or the number of drug molecules conjugated to a single ADC, is important for the efficacy of the ADC. DARs vary widely and depend on other ADC variables. DAR values ​​also depend on the conjugation site and the conjugated light or heavy chain used. DAR values ​​can affect drug effectiveness, as low drug loadings can lead to reduced potency, while high drug loadings can affect toxicity and pharmacokinetics (“Introduction to Antibody-Drug Conjugates.” Antibodies (Basel) Dec 2021;10(4):42.).

[0008] Many approaches have been developed to improve the homogeneity of ADCs. For example, Synaffix has developed GlycoConnect TM The technology (US2015 / 0320882, synaffix.com / platform / technology / ) modifies native antibody glycans through a three-step process (enzyme digestion, enzyme-mediated ligation, and non-metal click chemistry) to convert antibodies into DAR2, DAR4, or DAR1 and DAR6 type stable conjugated ADCs.

[0009] US20210040145 discloses a 14-amino acid peptide Tub-tag for the C-terminus of any target protein (POI) and catalyzes the addition of various tyrosine derivatives. Tub-tag technology uses this enzyme to reuse tubulin-tyrosine ligase to connect the functional part of the C-terminus of the antibody, thereby homogeneously generating DAR2-type antibody conjugates.

[0010] However, these techniques suffer from several disadvantages, such as immunogenicity risk, complex purification, and / or high cost.

[0011] Therefore, the demand for antibody site-specific modification technology still exists. Summary of the invention

[0012] For the above purpose, this article provides a method for programmatic management of antibody disulfide bond site-specific modification, comprising the following steps:

[0013] (R1) contacting the antibody with a first reducing agent or a salt thereof in the presence of a transition metal ion to reduce at least one of the interchain disulfide bonds of the antibody.

[0014] In some embodiments, in step (R1), one of the interchain disulfide bonds of the antibody is reduced, optionally, one of the interchain disulfide bonds in the hinge region of the antibody is reduced.

[0015] In some embodiments, in step (R1), three of the interchain disulfide bonds of the antibody are reduced, optionally, two of the interchain disulfide bonds in the Fab region and one of the interchain disulfide bonds in the hinge region of the antibody are reduced.

[0016] In some embodiments, the method further comprises step (O1),

[0017] (O1) introducing an oxidizing agent to selectively reoxidize the reduced thiol groups produced in step (R1).

[0018] In some embodiments, the method further comprises step (R2),

[0019] (R2) Incubating a second reducing agent or a salt thereof in a buffer system to reduce one of the interchain disulfide bonds of the antibody produced in step (O1).

[0020] In some embodiments, the method further comprises the following steps:

[0021] (C1) introducing a certain amount of a metal chelator and at least an equimolecular ratio of a first coupling group to react with the reduced thiol group produced in step (R1), (O1) or (R2), calculated on a molar basis of the antibody, wherein the first coupling group is a first capping reagent, a first linker-payload or a first sulfide bridging reagent, and optionally, the first sulfide bridging reagent carries a first linker-payload or a reactive group.

[0022] In some embodiments, the method further comprises the following steps:

[0023] (C2) incubating at least an equimolecular ratio of the first coupling group to react with the reduced thiol group produced in step (R1), calculated on a molar basis of the antibody; then,

[0024] Optionally, a metal chelator and an oxidant are introduced, or

[0025] Optionally, a metal chelator and a first coupling group are introduced.

[0026] In some embodiments, the method further comprises the following steps:

[0027] (R3) incubating a second reducing agent or a salt thereof in a buffer system to reduce the interchain disulfide bonds of the antibody of step (C1) or (C2), and optionally, introducing transition metal ions;

[0028] (C3) introducing at least an equimolecular proportion of a second coupling group to react with the reduced thiol group produced in step (R3), calculated on a molar basis of the antibody, wherein the second coupling group is a second capping reagent, a second linker-payload or a second sulfide bridging reagent, and optionally, the second sulfide bridging reagent carries a second linker-payload or a reactive group.

[0029] The method provided herein is compatible with current thiol-reactive linker drug technology and has minimal conformational changes and intact Fc function. At the same time, the method is simple to operate and has lower costs, can be easily run without enzyme engineering, and is fully compatible with current thiol-reactive linker drug technology.

[0030] In one aspect, the present invention provides a modified antibody prepared by the above method. The modified antibody is coupled to one, two or three coupling groups.

[0031] In one aspect, provided herein is a pharmaceutical composition comprising a modified antibody prepared by the above method and at least one pharmaceutically acceptable ingredient.

[0032] In one aspect, provided herein is the use of a modified antibody prepared by the above method or a pharmaceutical composition provided herein in the preparation of a therapeutic agent for preventing, diagnosing or treating a disease.

[0033] In one aspect, provided herein is a method for preventing or treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a modified antibody prepared by the above method. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings are briefly described below and are intended to illustrate exemplary embodiments disclosed herein and not to limit the invention.

[0035] Figure 1-2 HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared by Example I.1 and Example I.4 are shown.

[0036] Figure 3 The HIC-HPLC chromatogram of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate of Example 1.125 is shown.

[0037] Figure 4 The HIC-HPLC chromatogram of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate prepared in Example 1.126 is shown.

[0038] Figure 5 The HIC-HPLC chromatogram of the trastuzumab-[maleimide] 1 conjugate of Example 1.127 is shown.

[0039] Figure 6 The HIC-HPLC chromatogram of the trastuzumab-[maleimide]1[MC-GGFG-DXd]6 conjugate of Example 1.127 is shown.

[0040] Figure 7 A shows the HIC-HPLC chromatogram of the trastuzumab-[maleimide] 1 conjugate of Example 1.128; B shows the HIC-HPLC of the trastuzumab-[maleimide] 1[MC-VC-PAB-MMAE] 6 conjugate of Example 1.128.

[0041] Figure 8 A shows the HIC-HPLC chromatogram of trastuzumab-[MC-VC-PAB-MMAE]2 of Example 1.129; B shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]6 conjugate of Example 1.129.

[0042] Fig. 9 The HIC-HPLC chromatogram of Trastuzumab-[MC-MMAF]2 of Example 1.130 is shown.

[0043] Fig.10 The HIC-HPLC chromatogram of the trastuzumab-[MC-MMAF]2[MC-GGFG-DXd]6 conjugate of Example 1.130 is shown.

[0044] Fig.11 A shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example I.131; BC shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]2 conjugate prepared in Examples I.131-I.132.

[0045] Fig.12 The HIC-HPLC chromatogram of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2 conjugate prepared in Example 1.133 is shown.

[0046] Fig.13 A shows the HIC-HPLC chromatogram of the trastuzumab-[maleimide] 1 conjugate prepared in Example I.134; BC shows the HIC-HPLC chromatogram of the trastuzumab-[maleimide] 1[MC-VC-PAB-MMAE] 4 conjugate prepared in Examples I.134-I.135.

[0047] Fig.14 A shows the HIC-HPLC chromatogram of the trastuzumab-[MC-GGFG-DXd]2 conjugate prepared in Example I.136; BC shows the HIC-HPLC chromatogram of the trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4 conjugate prepared in Examples I.136-I.137.

[0048] Fig.15 The HIC-HPLC chromatogram of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 conjugate prepared in Example 1.138 is shown.

[0049] Figure 16-17 The HIC-HPLC chromatogram of Trastuzumab-[MC-VC-PAB-MMAE]6 prepared by Example II.1 and Example II.4 is shown.

[0050] Figure 18-19HIC-HPLC of trastuzumab-[bismaleimide-DBCO]3 conjugates prepared by Example II.144 and Example II.145 is shown.

[0051] Fig. 20 A shows the HIC-HPLC of the trastuzumab-[MC-GGFG-DXd]6 conjugate of Example II.146; B shows the HIC-HPLC of the trastuzumab-[MC-GGFG-DXd]6[MC-VC-PAB-MMAE]2 conjugate of Example II.146.

[0052] Fig.21 A shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example II.147; B shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]6[MC-GGFG-DXd]2 conjugate of Example II.147.

[0053] Fig. 22 A shows the HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]6 of Example II.148; B shows the HIC-HPLC of trastuzumab-[MC-VC-PAB-MMAE]6[maleimide-PEG4-N3-DBCO-Cy3]1 of Example II.148.

[0054] Fig.23 A shows the HIC-HPLC of the trastuzumab-[MC-GGFG-DXd]6 conjugate of Example II.149; B shows the HIC-HPLC of the trastuzumab-[MC-GGFG-DXd]6[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate of Example II.149.

[0055] Fig.24 A shows the HIC-HPLC of the trastuzumab-[maleimide]6 conjugate of Example II.150; B shows the HIC-HPLC of the trastuzumab-[maleimide]6[MC-VC-PAB-MMAE]2 conjugate of Example II.150.

[0056] Fig.25 A shows the HIC-HPLC of the trastuzumab-[maleimide]6 conjugate of Example II.151; B shows the HIC-HPLC of the trastuzumab-[maleimide]6[MC-VC-PAB-MMAE]2 conjugate of Example II.151.

[0057] Figure 26-27HIC-HPLC of the trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1 conjugates of Examples II.152-II.153 is shown.

[0058] Fig.28 HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1 conjugate of Example III.93 is shown.

[0059] Fig.29 HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate of Example III.94 is shown.

[0060] Fig.30 HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1[MC-GGFG-DXd]6 conjugate of Example III.95 is shown.

[0061] Fig.31 HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]6 conjugate of Example III.96 is shown.

[0062] Figure 32-33 HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2 conjugate of Examples III.97-III.98 is shown.

[0063] Fig.34 A shows the HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate of Example III.99; BC shows the HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 conjugate of Examples III.99-III.100.

[0064] Fig.35 A shows the HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate of Example III.101; BC shows the HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 conjugate of Examples III.101-III.102.

[0065] Fig.36 A shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example III.103; B shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]4 conjugate of Example III.103.

[0066] Fig.37 A shows the HIC-HPLC of the trastuzumab-[MC-GGFG-DXd]2 conjugate of Example III.104; B shows the HIC-HPLC of the trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4 conjugate of Example III.104.

[0067] Fig.38 A shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example III.105; BC shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]4 conjugate of Examples III.105-III.106.

[0068] Fig.39 HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]4 conjugate of Example V.1 is shown.

[0069] Fig.40 A shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]4 conjugate of Example V.46; B shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]4[MC-GGFG-DXd]2 conjugate of Example V.46.

[0070] Fig.41 A shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]4 conjugate of Example V.47; B shows the HIC-HPLC of the trastuzumab-[MC-VC-PAB-MMAE]4[MC-GGFG-DXd]2 conjugate of Example V.47.

[0071] Fig.42A shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example I.139; B shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]2 conjugate of Example I.139 before AKTA; C shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]2 conjugate of Example I.139 before AKTA; D shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]2[MC-MMAF]2 conjugate of Example I.139. DETAILED DESCRIPTION

[0072] 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 added to the present invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated 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 invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to exhaust all permutations, combinations, and variations thereof.

[0073] Unless otherwise defined, all scientific and technological terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to the methods and materials 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.

[0074] Unless otherwise defined herein, scientific terms and technical terms related to 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 "one", "an" and "the" in this specification and the appended claims include plural referents unless the context clearly provides 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 stated, the "or" used means "and / or".

[0075] Throughout this document, unless the context requires otherwise, the words "comprises", "comprising", "including" and "containing" should be understood to imply the inclusion of 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.

[0076] Methods of Antibody Modification

[0077] Provided herein is a method for programmatic management (control) of antibody disulfide bond modification, which can modify proteins such as antibodies to covalently link target groups. The product can be an antibody drug conjugate (ADC), a target group (e.g., a cytotoxic drug) connected to a monoclonal antibody via a linker or a sulfide bridge reagent. In particular, provided herein is a method for programmatic management of antibody disulfide bond site-specific modification, and site-specific modified antibodies with improved homogeneity are beneficial for improving safety and efficacy.

[0078] The present invention provides an example of a method for programmed management of site-specific modification of antibody disulfide bonds, the method comprising the following steps:

[0079] (R1) contacting a first reducing agent or a salt thereof with an antibody in a buffer system under a transition metal ion environment to reduce at least one of the interchain disulfide bonds of the antibody.

[0080] The reducing agent (optionally, together with a transition metal ion) has reducing properties and reduces the disulfide bonds of the antibody to modify the protein or antibody. Optionally, the reducing agent selectively reduces the interchain disulfide bonds, thereby selectively modifying the antibody.

[0081] In some embodiments, provided herein is a method I or method II for programming the site-specific modification of antibody disulfide bonds. In some embodiments, in step (R1) of method I, one of the interchain disulfide bonds of the antibody is reduced, and optionally, one of the interchain disulfide bonds in the hinge region of the antibody is reduced. In some embodiments, in step (R1) of method II, three of the interchain disulfide bonds of the antibody are reduced, and optionally, two of the interchain disulfide bonds in the Fab region of the antibody and one of the interchain disulfide bonds in the hinge region are reduced.

[0082] In some embodiments, method I and / or method II comprises the following steps:

[0083] (R1) incubating a first reducing agent or a salt thereof with the antibody in a buffer system under a transition metal ion environment to reduce at least one of the interchain disulfide bonds of the antibody;

[0084] (C1) introducing a certain amount of a metal chelator and at least an equimolecular ratio of a first coupling group to react with the reduced thiol group produced in step (R1), calculated on a molar basis of the antibody, wherein the first coupling group is a first capping reagent, a first linker-payload or a first sulfide bridging reagent, and optionally, the first sulfide bridging reagent carries a first linker-payload or a reactive group.

[0085] In some embodiments, in step (C1) of method I, one or both of the first coupling groups are covalently linked to the reduced thiol group generated in step (R1).

[0086] In some embodiments, in step (C1) of method II, three or six of the first coupling groups are covalently linked to the reduced thiol group generated in step (R1).

[0087] The term "disulfide bond" herein refers to a covalent bond having a 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 be formed between the thiol groups of two cysteine ​​residues, respectively, on two polypeptide chains, thereby forming an interchain bridge or interchain bond.

[0088] 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 about 25 amino acid residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently.

[0089] 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 the heavy chain are connected by a disulfide bond. The Fab fragment can be obtained by protease digestion of the full-length antibody. 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, an interchain disulfide bond connects two of the upper heavy chains in the hinge region, or an interchain disulfide bond connects the heavy chain and the light chain in the Fab region.

[0090] The term "antibody" herein refers to any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that binds to a specific antigen. A natural complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR") and the first, second and third constant regions (CH1, CH2 and CH3), while each light chain consists of a variable region ("LCVR") and a constant region (CL). The heavy chains of mammals are divided into α, δ, ε, γ and μ types, while the light chains of mammals are divided into λ or κ types. The antibody is in the shape of a "Y", and the backbone of the Y consists of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y includes the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops, called complementarity determining regions (CDRs) (light (L) chain CDRs include LCDR1, LCDR2 and LCDR3, heavy (H) chain CDRs include HCDR1, HCDR2, HCDR3). The CDR boundaries of an antibody can be defined or identified by the rules of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, MD. (1991)). The three CDRs are located between flanking segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support the hypervariable loops. Each HCVR and LCVR contains four FRs, and the order of CDRs and FRs from amino-terminus to carboxyl-terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively.Several major antibody classes are divided into subclasses, such as IgGl (γl heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgAl (αl heavy chain), or IgA2 (α2 heavy chain).

[0091] In some embodiments, the method comprises step (O1),

[0092] (O1) introducing an oxidizing agent to selectively reoxidize the reduced thiol groups produced in step (R1).

[0093] In some embodiments, the present invention provides a method III for programmatic management of antibody disulfide bond site-specific modification. Method III comprises the following steps:

[0094] (R1) incubating a first reducing agent or a salt thereof with the antibody in a buffer system under a transition metal ion environment to reduce three of the interchain disulfide bonds of the antibody;

[0095] (O1) introducing an oxidizing agent to selectively reoxidize the reduced thiol groups produced in step (R1);

[0096] (C1) introducing a certain amount of a metal chelator and a first coupling group in at least an equimolecular ratio to react with the reduced thiol group in step (O1), calculated based on the molar amount of the antibody.

[0097] In some embodiments, the oxidizing agent in step (O1) reoxidizes the reduced thiol groups in the Fab region of the antibody produced in step (R1), and optionally, in step (O1), four of the reduced thiol groups are reoxidized to form two disulfide bonds.

[0098] In some embodiments, in step (C1) of method III, one or both of the first coupling groups are covalently linked to the remaining thiol groups generated in step (O1).

[0099] In some embodiments, the method comprises step (R2),

[0100] (R2) Incubating a second reducing agent or a salt thereof in a buffer system to reduce one of the interchain disulfide bonds of the antibody in step (O1).

[0101] In some embodiments, the present invention provides a method IV for programmed management of antibody disulfide bond site-specific modification. The method IV comprises the following steps:

[0102] (R1) incubating a first reducing agent or a salt thereof with the antibody in a buffer system under a transition metal ion environment to reduce three of the interchain disulfide bonds of the antibody;

[0103] (O1) introducing an oxidizing agent to selectively reoxidize the reduced thiol groups produced in step (R1);

[0104] (R2) incubating a second reducing agent or a salt thereof in a buffer system to reduce one of the interchain disulfide bonds of the antibody produced in step (O1);

[0105] (C1) introducing a certain amount of a metal chelator and a first coupling group in at least an equimolecular ratio to react with the reduced thiol group generated in step (R2), calculated on a molar basis of the antibody.

[0106] In some embodiments, in step (R2) of method IV, one of the interchain disulfide bonds in the antibody hinge region is reduced.

[0107] In some embodiments, in step (C1) of method IV, two or four of the first coupling groups are covalently linked to the remaining thiol groups generated in step (R2).

[0108] In some embodiments, the method further comprises the following steps:

[0109] (C2) incubating at least an equimolecular ratio of the first coupling group to react with the reduced thiol group produced in step (R1), calculated on a molar basis of the antibody, and then,

[0110] Optionally, a metal chelator and an oxidant are introduced, or

[0111] Optionally, a metal chelator and a first coupling group are introduced.

[0112] In some embodiments, provided herein is a method V for programmed management of site-specific modification of antibody disulfide bonds, the method V comprising the following steps:

[0113] (R1) incubating a first reducing agent or a salt thereof with the antibody in a buffer system under a transition metal ion environment to reduce three of the interchain disulfide bonds of the antibody;

[0114] (C2) incubating at least an equimolecular ratio of the first coupling group to react with the reduced thiol groups generated in step (R1), calculated on a molar basis of the antibody, and then, optionally, introducing a metal chelator and an oxidizing agent.

[0115] In some embodiments, provided herein is a method V for programmed management of site-specific modification of antibody disulfide bonds, the method V comprising the following steps:

[0116] (R1) incubating a first reducing agent or a salt thereof with the antibody in a buffer system under a transition metal ion environment to reduce three of the interchain disulfide bonds of the antibody;

[0117] (C2) incubating at least an equimolecular ratio of the first coupling group to react with the reduced thiol groups generated in step (R1), calculated on a molar basis of the antibody, and then, optionally, introducing a metal chelator and the first coupling group.

[0118] In some embodiments, in step (C2) of method V, the metal chelator can capture excess transition metal ions. Before the introduction of the metal chelator, the first coupling group can react with four of the reduced thiol groups in step (R1). After the introduction of the metal chelator, the first coupling group can react again with two of the remaining reduced thiol groups.

[0119] In some embodiments, in step (C2) of method V, two or four of the first coupling groups are first covalently linked to the remaining thiol groups produced in step (R1). When the metal chelator and the first coupling group are introduced, one or two of the first coupling groups are covalently linked to the remaining thiol groups again.

[0120] In some embodiments, in step (C2) of method V, the first coupling groups are the same or different.

[0121] In some embodiments, when the first sulfide-bridging reagent carries a reactive group, step (C2) comprises the following steps:

[0122] introducing a first thiol bridging agent carrying a reactive group to re-bridge the reduced thiol group generated in step (R1), incubating the first linker-payload in a buffer system to react with the reactive group of the thiol bridging group, and then,

[0123] Optionally, a metal chelator and an oxidant are introduced, or

[0124] Optionally, a metal chelator and a first coupling group are introduced.

[0125] In some embodiments, when the first sulfide-bridging reagent carries a reactive group, step (C1) of methods I, II, III, and IV comprises the following steps:

[0126] A metal chelator and a first sulfide bridging agent carrying a reactive group are introduced to re-bridge the reduced thiol group generated in step (R1), (O1) or (R2), and the first linker-payload is incubated in a buffer system to react with the reactive group of the sulfide bridging group.

[0127] In some embodiments, methods I, II, III, IV, and V further include the following steps:

[0128] (R3) incubating a second reducing agent or a salt thereof in a buffer system to reduce the interchain disulfide bonds of the antibody in step (C1) or (C2), optionally introducing transition metal ions;

[0129] (C3) introducing at least an equimolecular proportion of a second coupling group to react with the reduced thiol group produced in step (R3), calculated on a molar basis of the antibody, optionally introducing a metal chelator, wherein the second coupling group is a second capping reagent, a second linker-payload or a second sulfide bridge reagent, optionally, the second sulfide bridge carries a second linker-payload or a reactive group.

[0130] In some embodiments, when the second sulfide bridging reagent carries a reactive group, step (C3) comprises the following steps:

[0131] A second thiol bridging agent carrying a reactive group is introduced to re-bridge the reduced thiol group generated in step (R3), optionally, a metal chelator is introduced, and then a second linker-payload is incubated in a buffer system to react with the reactive group of the thiol bridging group.

[0132] In some embodiments, if transition metal ions are introduced in step (R3), a metal chelator is introduced in step (C3) to capture excess transition metal ions.

[0133] In some embodiments, in step (R3), one, two, or three of the interchain disulfide bonds of the antibody are reduced.

[0134] In some embodiments, in step (C3), one, two, three, four, or six of the second coupling groups are covalently linked to the reduced thiol group generated in step (R3).

[0135] In some embodiments, in step (R3) of method I and / or method III, three of the interchain disulfide bonds of the antibody are reduced. In some embodiments, in step (C3) of method I and / or method III, three or six of the second coupling groups are covalently linked to the reduced thiol groups generated in step (R3).

[0136] In some embodiments, in step (R3) of method I and / or method III, when a transition metal ion is introduced, one or two of the interchain disulfide bonds of the antibody are reduced. In some embodiments, in step (C3) of method I and / or method III, when a transition metal ion is introduced, one, two or four of the second coupling groups are covalently linked to the reduced thiol groups generated in step (R3).

[0137] In some embodiments, in step (R3) of method II, one of the interchain disulfide bonds of the antibody is reduced. In some embodiments, in step (C3) of method II, one or both of the second coupling groups are covalently linked to the reduced thiol groups generated in step (R3).

[0138] In some embodiments, in step (R3) of method IV and / or method V, two of the interchain disulfide bonds of the antibody are reduced. In some embodiments, in step (C3) of method IV and / or method V, two or four of the second coupling groups are covalently linked to the reduced thiol groups generated in step (R3).

[0139] In some embodiments, in step (R3) of method IV and / or method V, when a transition metal ion is introduced, one of the interchain disulfide bonds of the antibody is reduced. In some embodiments, in step (C3) of method IV and / or method V, when a transition metal ion is introduced, one or both of the second coupling groups are covalently linked to the reduced thiol groups generated in step (R3).

[0140] In some embodiments, when one of the interchain disulfide bonds is reduced in step (R3) in methods I and III, methods I and III further comprise the following steps:

[0141] (R4) incubating a transition metal ion and a second reducing agent or a salt thereof in a buffer system to reduce the interchain disulfide bonds of the antibody in step (C3);

[0142] (C4) introducing a metal chelator and a second coupling group in at least an equimolecular ratio to react with the reduced thiol group generated in step (R4), calculated on a molar basis of the antibody.

[0143] In some embodiments, in step (R4) of method I and / or method III, one of the interchain disulfide bonds of the antibody is reduced. In some embodiments, in step (C4) of method I and / or method III, one or both of the second coupling groups are covalently linked to the reduced thiol groups generated in step (R4).

[0144] In some embodiments, the second coupling group in step (C3) is the same as in step (C4). In some embodiments, the second coupling group in step (C3) is different from that in step (C4).

[0145] In some embodiments, the methods for site-specific modification do not involve enzymatic techniques and glycan modifications.

[0146] In the present invention, in some embodiments, the reducing agent (including the first reducing agent and the second reducing agent) is independently selected from the group consisting of tris(2-carboxyethyl)phosphine (TCEP) or a compound having the following formula (I), or a salt, solvate, stereoisomer thereof,

[0147]

[0148] in

[0149] X, Y and Z are each independently covalently linked to the phosphorus atom via a PC bond, wherein the PC bond is PC(sp 3 ) or PC(sp 2 ); X is formula (III):

[0150]

[0151] L1 is selected from the group consisting of: -CH(R1)-, -C(CH3)(R1), -CH(R1)CH(R2)-,

[0152] -CH(R1)CH(R2)CH(R3)-, an aryl group each independently selectively substituted by a group containing at least a coordinating atom, and a heteroaryl group each independently selectively substituted by a group containing at least a coordinating atom selected from O and S;

[0153] R1, R2 and R3 are each independently H, C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 carboxyalkyl, C1-C3 hydroxylaminoalkyl, C1-C3 N-hydroxylamidealkyl, aryl or heteroaryl; or

[0154] R2 or R3 and L2 form a 5-6 membered optionally substituted ring;

[0155] A is optionally present and is -C(O)-, or -C(O)J-;

[0156] J is an organic group comprising an amino group or an imino group and a carbonyl group, wherein the amino group or the imino group forms an amide group with -C(O), and the carboxyl group is optionally covalently linked to L2;

[0157] L2 is optionally present, L2 serves as a transition metal chelator motif and is -N(R4)(R5) or hydroxyl;

[0158] R4 and R5 are each independently hydrogen, C0-C5 hydroxyalkyl, C1-C5 alkyl, C1-C5 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted 5-6 membered saturated heterocyclyl, optionally substituted aralkyl, optionally substituted arylalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heteroaralkyl, R4 or R5 and R2 or R3 form a 5-6 membered optionally substituted ring;

[0159] R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted aralkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl;

[0160] R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH;

[0161] n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4;

[0162] R4 and R5 are not hydroxyl groups at the same time;

[0163] Y is the same as X,

[0164] Z is the same as X, or

[0165] Y and Z are each independently a 5-6 membered optionally substituted saturated heterocyclic group, a C1-C3 alkyl group, a C1-C3 hydroxyalkyl group, an aryl group, a C1-C3 carboxyalkyl group, a 5-6 membered optionally substituted cycloalkyl group, or

[0166] -C(O)Q is an ester group, an imide group or an amide group,

[0167] X, Y and Z are not -CH2CH2C(O)OH at the same time.

[0168] In the present invention, the term "aryl" refers to an aromatic or heteroaromatic group consisting of one or more rings, containing three to fourteen carbon atoms, preferably six to ten carbon atoms. An exemplary aromatic group is phenyl.

[0169] The term "aryl" also refers to an aromatic group in which one or more H atoms are independently replaced by another group, such as F, CI, Br, I, hydroxy, carboxyl, sulfonyl, amino, methoxy or ethoxy, N-hydroxyformamido, N-hydroxyacetamido, 4-pyridyl, 2-pyridyl,

[0170] The term "C1-C5 alkyl" refers to an aliphatic hydrocarbon group having 1 to 5 carbon atoms in the chain or ring. Exemplary alkyl groups include methyl, ethyl, n-propyl and isopropyl.

[0171] The term "C0-C5 hydroxyalkyl" refers to a hydroxyl group or a C1-C5 alkyl group in which one or several H atoms are substituted by one, two or three hydroxyl groups. Exemplary C1-C5 hydroxyalkyl groups are hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl.

[0172] The term "C1-C5 alkoxy" refers to an oxygen atom attached to a C1-C5 alkyl group. Exemplary C1-C5 alkoxy groups are -OCH3, -OCH2CH 3、 -OCH(CH3)2, -OCH2CH2CH3.

[0173] The term "arylalkoxy" refers to an aromatic group in which one or several H atoms are replaced by alkoxy. Exemplary are phenyl-O-CH2-, phenyl-O-(CH2)2-, phenyl-O-(CH2)3-, phenyl-O-(CH2)4-, phenyl-O-(CH2)5-.

[0174] The term "heteroaryl" refers to an aromatic group in which one or several carbon atoms, preferably one, two, three or four carbon atoms, are replaced by O, N, Si, Se, P or S, preferably by O, S, N. Exemplary heteroaryl groups are imidazolyl, pyridyl, bipyridyl, quinolyl, isoquinolyl.

[0175] The term "heteroaryl" also refers to heteroaromatic groups in which one or several H atoms are independently replaced by other groups such as F, Cl, Br, I, hydroxy, carboxyl, amino, hydroxyalkyl, carboxyalkyl, N-hydroxyamidoalkyl, heteroaryl, etc.

[0176] The term "coordinating atom" refers to an atom containing a lone pair of electrons, examples of which include N, O, S, P, F, Cl, Br, I.

[0177] The term "C1-C5 carboxyalkyl" refers to a C1-C5 alkyl group substituted with one, two or three carboxyl groups. Exemplary C1-C5 carboxyalkyl groups are -COOH, -CH2COOH,

[0178] -CH2CH2COOH, -CH(CH3)COOH.

[0179] The term "C1-C5 hydroxylamine alkyl" refers to a C1-C5 alkyl group substituted with one, two or three hydroxylamine groups. Exemplary C1-C5 hydroxylamine alkyl groups are

[0180] -CH2NHOH, -CH2CH2NHOH.

[0181] The term "C1-C5 N-hydroxyamidoalkyl" refers to a C1-C5 carboxyalkyl group in which one, two or three carboxyl groups form an amide with hydroxylamine. Exemplary C1-C5 N-hydroxyamidoalkyl groups are -C(O)NHOH, -CH2C(O)NHOH, -CH2CH2C(O)NHOH.

[0182] The term "heterocyclyl" refers to an aromatic or non-aromatic C5-C 10A ring wherein one or two of the ring carbon atoms are independently substituted with a heteroatom selected from O, N, P and S. Preferred heteroatoms are O, N and S. Suitable heterocyclic rings are also disclosed in The Handbook of Chemistry and Physics, 76th ed., CRC Press, Inc., 1995-1996, pp. 2-25 to 2-26, the disclosure of which is incorporated herein by reference. Preferred non-aromatic heterocycles include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxadioxolanyl, piperidinyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, dihydrothiopyranyl, azepanyl and condensed systems obtained by condensation with phenyl.

[0183] The term "aralkyl" refers to a linear, branched or cyclic alkyl group attached to at least one aryl group. Preferably, the number of carbon atoms in the chain or ring is 1 to 4. Exemplary aralkyl groups are -CH2C6H5, -CH2CH2C6H5, -CH2CH2CH2C6H5, -CH2(CH3)CH2C6H5, -CH2(CH3)CH2CH2C6H5,

[0184] The term "heteroaralkyl" refers to a linear, branched or cyclic alkyl group attached to at least one heteroaryl group. Preferably, the number of carbon atoms in the chain or ring is 1 to 4. Exemplary heteroaralkyl groups are

[0185] The term "cycloalkyl" refers to a 3-, 4-, 5- or 6-membered saturated or unsaturated non-aromatic carbocyclic ring. Representative cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl. Cycloalkyls may be unsubstituted or substituted with one or more groups including, but not limited to, carboxyl, sulfonyl, amino, hydroxyl, -C(O)NHOH, -CH2C(O)NHOH, -CH2CH2C(O)NHOH, -COOH, -CH2COOH, -CH2CH2COOH, -CH2(CH3)COOH, F, Cl, Br, I.

[0186] The term "halogen" refers to F, Cl, Br or I.

[0187] The term "alkenyl" refers to a straight or branched unsaturated hydrocarbon containing 2 to 12 carbon atoms. An "alkenyl" group contains at least one double bond in the chain. The double bond of an alkenyl group may be uncoupled or coupled to another unsaturated group. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, pentenyl, or hexenyl. An alkenyl group may be unsubstituted or substituted and may be straight or branched.

[0188] The term "cyano" refers to a substituent in which a carbon atom is triple bonded to a nitrogen atom, for example -CN.

[0189] In some embodiments, in the compound of formula (III), L1 is -CH(R1)-, -CH(R1)CH(R2)-, or -CH(R1)CH(R2)CH(R3)-.

[0190] In some embodiments, in the compound of formula (I), R1, R2 and R3 are independently H, methyl, isopropyl, hydroxymethyl, hydroxyethyl, carboxymethyl, carboxyethyl, N-hydroxyacetamido, phenyl, 2-pyridyl, 4-pyridyl or 4-imidazolyl, and R2 and L2 form a 5-6 membered optionally substituted ring.

[0191] In some embodiments, in the compound of formula (I), L1 is -CH(R1)CH(R2)-, R1 is H, and R2 and R4 of L2 form a 5-6 membered optionally substituted ring. In some embodiments, R2 and L2 form In these embodiments of compounds having formula (I), A is -C(O)-, L2 is -N(R4)(R5), and R5 is hydroxy.

[0192] In some embodiments of compounds having formula (I), L1 is -CH(R1)CH(R2)-, R1 is H, and R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl, or 4-imidazolyl.

[0193] In some embodiments of compounds of formula (I), L1 is -CH(R1)CH(R2)-, R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl or 4-imidazolyl, A is -C(O)-, L2 is -N(R4)(R5), R4 is hydrogen, an optionally substituted 5-6 membered saturated heterocyclic group, and R5 is hydroxyl. In some embodiments, R4 is

[0194] In some embodiments of compounds of formula (I), L1 is -CH(R1)CH(R2)-, R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl or 4-imidazolyl, A is -C(O)-, L2 is -N(R4)(R5), R4 and R5 form a 5-6 membered optionally substituted ring. In these examples, L2 is

[0195] In some embodiments of the compound having formula (I), L1 is -CH(R1)CH(R2)-, R1 is methyl, isopropyl, carboxyethyl or N-hydroxyacetamido, and R2 is H.

[0196] In some embodiments of compounds having formula (I), L1 is -CH(R1)CH(R2)-, R1 is methyl, isopropyl, carboxyethyl or N-hydroxyacetamido, R2 is H, A is -C(O)-, L2 is -N(R4)(R5), R4 is hydrogen, and R5 is hydroxyl.

[0197] In some embodiments of compounds having formula (I), L1 is an optionally substituted phenyl group attached to A at the ortho, meta or para position, A is -C(O)-, L2 is -N(R4)(R5) or hydroxy, R4 is hydrogen, and R5 is hydroxy.

[0198] In some embodiments of compounds of formula (I), L1 is phenyl optionally substituted at the ortho, meta or para position by hydroxy, halogen, carboxyl, sulfonyl, amino, methoxy or ethoxy, optionally halogen refers to F, Cl, Br, or I. In these embodiments, A and L2 are absent.

[0199] In some embodiments of compounds of formula (I), L1 is In these examples, A and L2 do not exist.

[0200] In some embodiments of compounds of formula (I), L1 is optionally substituted 4-pyridyl or optionally substituted 4-quinolyl. In some embodiments of compounds of formula (I), L1 is In some embodiments of compounds having Formula (I), A and L2 are absent.

[0201] In some embodiments of compounds having formula (I), L1 is -CH(R1)CH(R2)-, and R1 and R2 are each independently H.

[0202] In some embodiments of compounds having formula (I), A is -C(O)-, L2 is -N(R4)(R5), R4 is hydrogen, and R5 is hydroxy.

[0203] In some embodiments of compounds of formula (I), L2 is -N(R4)(R5), R4 is hydrogen, C1-C5 alkyl, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted 5-6 membered saturated heterocyclyl, optionally substituted aralkyl, optionally substituted aryl, optionally substituted heteroaralkyl, or R4 and R5 form a 5-6 membered optionally substituted ring; R5 is hydroxy.

[0204] In some embodiments of compounds of formula (I), L2 is -N(R4)(R5), R4 is hydrogen, C1-C5 alkyl, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted aralkyl, optionally substituted aryl; R5 is hydroxy.

[0205] In some embodiments of compounds having formula (I), R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, C1-C5 N-hydroxyamidoalkyl, heteroaryl, or heteroaralkyl.

[0206] In some embodiments of compounds having formula (I), R6 is hydrogen, C1-C5 alkyl, C1-C5 hydroxyalkyl, or heteroarylalkyl.

[0207] In some embodiments of compounds of formula (I), R6 is hydrogen, methyl, hydroxymethylamino, benzyl, carboxyethyl, N-hydroxyacetamido, Optionally R6 is hydrogen.

[0208] In some embodiments of compounds of formula (I), R7 is hydroxy, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH, optionally R7 is hydroxyl or C1-C5 alkoxy. In some embodiments, R7 is hydroxyl, methoxy, -NH(CH2CONH)n 3 , optionally R7 is hydroxy or methoxy.

[0209] In the compound of formula (I), n 1 、n 2 and n 3 Independently the numbers 0, 1, 2, 3, 4.

[0210] In some embodiments of compounds of formula (I), R4 is Hydrogen or -(CH2)n 1 (OCH2CH2O)n 2CH(R6)CO(R7); R5 is hydroxyl; R6 is hydrogen, methyl, hydroxymethyl or R7 is hydroxyl or -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0.

[0211] In some embodiments, R4 is Hydrogen or -CH(R6)CO(R7), R5 is hydroxy, R6 is hydrogen, and R7 is hydroxy.

[0212] In some embodiments of compounds having formula (I), L2 is -N(R4)(R5);

[0213] R4 and R5 are each independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or optionally substituted heteroaralkyl;

[0214] R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, C1-C5 N-hydroxyamidealkyl, heteroaryl or heteroaralkyl;

[0215] R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH;

[0216] n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

[0217] In some embodiments of compounds of formula (I), R4 and R5 are each independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or 6-membered heteroarylalkyl;

[0218] R6 is hydrogen;

[0219] R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH;

[0220] n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

[0221] In some embodiments of compounds of formula (I), R4 and R5 are independently -(CH2)n1 (OCH2CH2O)n 2 CH(R6)CO(R7) or

[0222] R6 is hydrogen;

[0223] R7 is hydroxyl or -NH(CH2CONH)n 3 OH;

[0224] n 1 、n 2 and n 3 Each independently is the number 0.

[0225] In some embodiments, R4 and R5 are each independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7),

[0226] R6 is hydrogen,

[0227] R7 is hydroxyl or -NH(CH2CONH)n 3 OH;

[0228] n 1 、n 2 and n 3 Each independently is the number 0.

[0229] In some embodiments of compounds having formula (I), L2 is -N(R4)(R5);

[0230] R4 is hydrogen, C0-C5 hydroxyalkyl, C1-C5 alkyl, optionally substituted C1-C5 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted aralkyl, optionally substituted arylalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heteroaralkyl;

[0231] R5 is hydrogen;

[0232] R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted aralkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl;

[0233] R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH;

[0234] n 1 、n 2 and n3 Each independently is the number 0, 1, 2, 3, 4.

[0235] In some embodiments of compounds of formula (I), R4 is hydrogen, C0-C3 hydroxyalkyl, C1-C3 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), phenyl substituted by carboxyl, hydroxyl, amino, halogen, pyridyl, amino substituted by 2-methylpyridine, benzyl substituted by carboxyl, hydroxyl, amino or halogen, arylalkoxy, Substituted pyridinyl.

[0236] In some embodiments of compounds of formula (I), R4 is hydrogen, hydroxy, methylhydroxy, ethylhydroxy, propylhydroxy, methoxy, ethoxy, and R5 is hydrogen.

[0237] In some embodiments, R4 is hydroxy, methoxy or and R5 is hydrogen.

[0238] In some embodiments of compounds of formula (I), R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7);

[0239] R5 is hydrogen;

[0240] R6 is hydrogen, amino, C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 carboxyalkyl, aryl, aralkyl optionally substituted by hydroxy, halogen, cyano or nitro, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl;

[0241] R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH;

[0242] n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

[0243] In some embodiments, R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7),

[0244] R5 is hydrogen,

[0245] R6 is hydrogen, amino, methyl, hydroxymethyl, benzyl, benzyl substituted by hydroxy, halogen, cyano or nitro, halogen, carboxyethyl, N-hydroxyacetamido,

[0246] R7 is hydroxyl, -NH(CH2CONH)n 3 OH;

[0247] n 1 and n 3 Each independently represents the number 0, 1, 2, 3, 4; n 2 Is the number 0.

[0248] In some embodiments of compounds of formula (I), R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7); R5 is hydrogen; R6 is hydrogen, amino, methyl, hydroxymethyl, benzyl, carboxyethyl, N-hydroxyacetamide,

[0249] R7 is hydroxyl or -NH(CH2CONH)n 3 OH;

[0250] n 1 is the number 2; n 2 is the number 1; n 3 Is the number 0.

[0251] In some embodiments, R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydrogen, R6 is hydrogen; R7 is -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Is the number 0.

[0252] In some embodiments of the compound of formula (I), A is -C(O)J-, J is a peptide residue, including a single amino acid residue, a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, an aminopropionic acid, an aminobutyric acid, an aminovaleric acid, an amino acid, an aminoheptanoic acid, an aminooctanoic acid, or NH2(OCH2CH2O)n 4 CH2COOH,n 4 For numbers 2-10.

[0253] The amino acid is selected from the group consisting of glycine (Gly), alanine (Ala), serine (Ser), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val).

[0254] In some embodiments of compounds having Formula (I), J is the residue of histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine, or asparagine.

[0255] In some embodiments of compounds having formula (I), A is -C(O)J-, J is the residue of histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine or asparagine, L2 is -N(R4)(R5), R4 is hydrogen, and R5 is hydroxyl.

[0256] In some embodiments of compounds having Formula (I), Y and X are the same.

[0257] In some embodiments of compounds having Formula (I), Z and X are the same.

[0258] In some embodiments of compounds of formula (I), Y and Z are each independently

[0259] Q is -NHOH, -NHCH2CH2SO3H, -N(CH2CH2OH)2, -NHCH2COOH, -NHCH2(CH3)COOH, -NH(CH2CH2O)3CH3.

[0260] In the compounds of formula (II), in some embodiments, R1 is H, and R2 is H.

[0261] In some embodiments, the reducing agent (including the first reducing agent and the second reducing agent) is each independently selected from the group consisting of tris(2-carboxyethyl)phosphine (TCEP) or a compound having the following formula (II), or a salt, solvate, stereoisomer thereof,

[0262]

[0263] in,

[0264] R1 is H, -NH2, -C(O)(R3R4), optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl;

[0265] R3 is N, NH or O;

[0266] R4 is H, optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl;

[0267] R2 is H, optionally substituted C1-C5 alkyl, or optionally substituted C1-C5 hydroxyalkyl;

[0268] X is OH, optionally substituted C1-C5 alkoxy or -NR5R6,

[0269] R5 and R6 are each independently H, C0-C5 hydroxyalkyl, optionally substituted C1-C5 alkyl, optionally substituted C2-C8 carboxyalkyl, or optionally substituted C1-C5 alkoxy, optionally substituted heteroarylalkyl, optionally substituted arylalkoxy, optionally substituted arylalkyl, optionally substituted aryl, C1-C5 alkylsulfonyl, -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7),

[0270] R7 C0-C5 hydroxyalkyl, -NHOH,

[0271] R8 is H, optionally substituted aralkyl,

[0272] n 1 and n 2 Each is independently digitized 0, 1, 2, 3, 4,

[0273] Y is the same as X, or Y is an ester or amide of X,

[0274] Z is the same as X or Y, or

[0275] Y and Z are each independently selected from:

[0276]

[0277] X, Y and Z are not the same

[0278] In some embodiments of compounds having formula (II), R1 is H, and R2 is H.

[0279] In some embodiments of compounds having formula (II), X is -OCH3, -OCH2CH3, or -O(CH3)2.

[0280] In some embodiments of compounds of formula (II), X is -NR5R6, R5 is H, and

[0281] R6 is H, C0-C5 hydroxyalkyl, C1-C5 alkoxy, optionally substituted heteroarylalkyl, optionally substituted arylalkoxy, optionally substituted aryl, optionally substituted arylalkyl, C1-C5 alkylsulfonyl or -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7),

[0282] R7 is C0-C3 hydroxyalkyl or -NHOH,

[0283] R8 is H or an optionally substituted aralkyl group,

[0284] n 1 and n 2 Each independently is the number 0.

[0285] In some embodiments of compounds of formula (II), R6 is H, C0-C2 hydroxyalkyl, C1-C3 alkoxy, C1-C3 alkylsulfonyl, bipyridyl, benzyl, arylalkoxy, phenyl optionally substituted with OH, carboxyl or pyridyl, or -CH(R8)CO(R7),

[0286] R7 is OH or -NHOH,

[0287] R8 is H or benzyl optionally substituted by OH, halogen, cyano or nitro.

[0288] In some embodiments of compounds of formula (II), X is -NR5R6, R5 is H, and

[0289] R6 is H, OH, C1 hydroxyalkyl, C2 hydroxyalkyl, C3 hydroxyalkyl, C4 hydroxyalkyl, C5 hydroxyalkyl, C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, heteroarylalkyl optionally substituted with heteroaryl, arylmethoxy, arylethoxy, arylpropyloxy, arylbutoxy, aryl optionally substituted with OH, carboxyl or pyridyl, benzyl, arylethyl, arylpropyl, C1 alkylsulfonyl, C2 alkylsulfonyl, C3 alkylsulfonyl, C5 alkylsulfonyl, C5 alkylsulfonyl, or -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7),

[0290] R7 is OH, C1 hydroxyalkyl, C2 hydroxyalkyl, C3 hydroxyalkyl or -NHOH,

[0291] R8 is H or aralkyl optionally substituted by OH, halogen, cyano or nitro, wherein halogen is selected from F, Cl, Br or I, n 1 and n 2 Each independently is the number 0.

[0292] In some embodiments of compounds of formula (II), X is -NR5R6, R5 is H, and

[0293] R6 is H, OH, -CH2OH, -(CH2)2OH, -CH3, -CH2CH3, -CH2COOH, -(CH2)2COOH, -(CH2)3COOH, -(CH2)4COOH, -(CH2)5COOH, -OCH3, -OCH2CH3, -CH2CONHOH, -OC(C6H5)3, -(CH2)3S(O)2OH,

[0294] In some embodiments of compounds of formula (II), X is -NR5R6, R5 is H, and

[0295] R6 is H, OH, -CH2COOH, -CH2CONHOH, -OC(C6H5)3,

[0296]

[0297] In some embodiments of compounds having formula (II), X is -NR5R6, R5 is H, and R6 is OH, -CH2COOH, -(CH2)2COOH, -(CH2)3COOH, -(CH2)4COOH, -(CH2)5COOH, -OCH3, -OCH2CH3, or -OC(C6H5)3.

[0298] In some embodiments of compounds of formula (II), X is -NR5R6, R5 is OH,

[0299] R6 is C1-C5 alkyl, optionally substituted heteroarylalkyl, optionally substituted arylalkyl, optionally substituted aryl, or -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7),

[0300] R7 is C0-C5 hydroxyalkyl,

[0301] R8 is H,

[0302] n 1 and n 2 Each independently is the number 0, 1, 2, 3, 4.

[0303] In some embodiments of compounds having formula (II), R6 is C1-C3 alkyl, heteroarylalkyl including heteroatom N, optionally substituted benzyl, optionally substituted phenyl, or -CH(R8)CO(R7),

[0304] R7 is C0-C3 hydroxyalkyl,

[0305] R8 is H.

[0306] In some embodiments of compounds having formula (II), R6 is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, heteroarylmethyl, heteroarylethyl, heteroarylpropyl, benzyl, arylethyl, arylpropyl, or -CH(R8)CO(R7),

[0307] R7 is hydroxy, C1 hydroxyalkyl, C2 hydroxyalkyl, C3 hydroxyalkyl, C4 hydroxyalkyl or C5 hydroxyalkyl,

[0308] R8 is H.

[0309] In some embodiments of compounds of formula (II), R6 is -CH3, -CH2COCH3, -CH2COOH,

[0310] In some embodiments of compounds having formula (II), R6 is -CH2COOH.

[0311] In some embodiments of compounds having formula (II), X is -NR5R6,

[0312] R5 and R6 are each independently C1-C5 alkyl, C0-C5 hydroxyalkyl, optionally substituted heteroarylalkyl or -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7),

[0313] R7 is C 0-5 Hydroxyalkyl or -NHOH,

[0314] R8 is H,

[0315] n 1 and n 2 Each independently is the number 0, 1, 2, 3, 4.

[0316] In some embodiments of compounds having formula (II), X is -NR5R6,

[0317] R5 and R6 are each independently C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, OH, C1 hydroxyalkyl, C2 hydroxyalkyl, C3 hydroxyalkyl, C4 hydroxyalkyl, C5 hydroxyalkyl, heteroarylmethyl, heteroarylethyl, heteroarylpropyl or -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7),

[0318] R7 is hydroxy, C1 hydroxyalkyl, C2 hydroxyalkyl, C3 hydroxyalkyl, C4 hydroxyalkyl, C5 hydroxyalkyl or -NHOH,

[0319] R8 is H,

[0320] n 1 and n 2 Each independently is the number 0, 1, 2, 3, 4.

[0321] In some embodiments of compounds of formula (II), X is -NR5R6, R5 and R6 are each independently methyl, ethyl, -(CH2)2OH, -CH2COOH, -CH2CONHOH or

[0322] In some embodiments of compounds of formula (II), Y is Z is

[0323] In some embodiments, the first reducing agent and the second reducing agent are each independently selected from the group consisting of:

[0324]

[0325]

[0326]

[0327]

[0328]

[0329] In some embodiments, the first reducing agent and the second reducing agent are each independently TCEP, tris(3-hydroxypropyl)phosphine (THPP), or dithiothreitol (DTT).

[0330] In some embodiments, the first reducing agent and the second reducing agent are each independently TCEP.

[0332] In some embodiments, the first reducing agent is TCEP.

[0333] In some embodiments, the first reducing agent is

[0334] The second reducing agent is TCEP.

[0335] For antibody drug conjugates, a mixture of ADCs will be produced by a conventional coupling process or a biocoupling process of the present invention. Typically, an antibody molecule belonging to the IgG1 or IgG4 subclass has four interchain disulfide bonds, each bond formed by two -SH groups. The antibody molecule can undergo partial or complete reduction of one or more interchain disulfide bonds to form 2n (n is an integer selected from 1, 2, 3 or 4) reactive -SH groups, and therefore, the number of drugs (or payloads) coupled to a single antibody molecule is 1, 2, 3, 4, 5, 6, 7 or 8. Depending on the number of drugs coupled to a single antibody molecule, different conjugates containing different numbers of drug molecules are named D0, D1, D2, D3, D4, D6, D1+D3, D1+D6, D1+D2, D1+D4, D2+D3, D2+D6, D0+D3, D0+D6, D3+D1, D3+D2, D6+D2, D6+D1, D0+D1, D0+D3, D4+D1 or D4+D2. Thus, the “homogeneity” of an antibody drug conjugate is used to describe the predominant property of one particular type of antibody drug conjugate (i.e., one type selected from D0, D1, D2, D3, D4, D6, D1+D3, D1+D6, D1+D2, D1+D4, D2+D3, D2+D6, D0+D3, D0+D6, D3+D1, D3+D2, D6+D2, D6+D1, D0+D1, D0+D3, D4+D1, or D4+D2 conjugates) in a given antibody drug conjugate mixture.

[0336] The drug-to-antibody ratio (DAR) of an ADC is the average number of drugs connected to each antibody. DAR is a key attribute to measure the quality of an ADC because it can significantly affect the ADC efficacy. DAR distribution (D0, D1, D2, D3, D4, D6, D1+D3, D1+D6, D1+D2, D1+D4, D2+D3, D2+D6, D0+D3, D0+D6, D3+D1, D3+D2, D6+D2, D6+D1, D0+D1, D0+D3, D4+D1 or D4+D2) can reflect the uniformity of the ADC.

[0337] The drug loading is represented by the number of drug moieties per antibody in the molecule of the ADC. For some antibody drug conjugates, the drug loading may be limited by the number of attachment sites on the antibody. For example, when the attachment body is a cysteine ​​thiol, in certain illustrative examples described herein, the 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.

[0338] The term "DO" or "DO-type ADC" herein refers to an ADC in which the average number of drugs coupled to a single antibody molecule is about zero.

[0339] The term "D1" or "D1-type ADC" herein refers to a DAR of about 1, which means that on average about one drug molecule (e.g., 0.5, 1.0, 1.4 molecules) is coupled to a single antibody molecule. The drug molecule can be coupled to the -SH group generated by reducing the disulfide bond between the heavy chain and the light chain or the heavy chain and the heavy chain via a linker. In some embodiments, D1 refers to an ADC in which one of the first sulfide bridge groups carrying the linker-payload rebridges two thiol groups of a single antibody molecule.

[0340] As used herein, the term "D2" or "ADC with D2" refers to: DAR is about 2, which means that on average about two drug molecules (e.g., 1.5, 2.0, 2.4 molecules) are coupled to a single antibody molecule. The drug molecule can be coupled to the -SH group generated by the reduction of the interchain disulfide bond between the heavy chain and the light chain and / or the heavy chain and the heavy chain via a linker. In some embodiments, D2 refers to an ADC in which two of the linker-payloads are coupled to a single antibody molecule.

[0341] The term "D3" or "D3-type ADC" herein refers to a DAR of about 3, which means that on average about three drug molecules (e.g., 2.5, 3.0, 3.4 molecules) are coupled to a single antibody molecule. The drug molecules can be coupled to the -SH groups generated by reducing the interchain disulfide bonds between the heavy and light chains and between the heavy and heavy chains via a linker. In some embodiments, D3 refers to an ADC in which three of the sulfide bridge groups carrying the linker-payload rebridge the six thiol groups of a single antibody molecule.

[0342] The term "D4" or "D4-type ADC" herein refers to an ADC in which an average of about four drug molecules (e.g., 3.5, 4.0, 4.4 molecules) are coupled to one single antibody molecule, wherein the drug molecule can be coupled to the -SH group generated by reducing the interchain disulfide bond. In some embodiments, D4 refers to an ADC in which four of the linker-payloads are coupled to one single antibody molecule.

[0343] As used herein, the term "D6" or "ADC with D6" refers to an ADC in which an average of about six drug molecules (e.g., 5.5, 6.0, 6.4 molecules) are coupled to a single antibody molecule, wherein the drug molecule may be coupled to six -SH groups (generated by reducing three disulfide bonds). In some embodiments, D6 refers to an ADC in which six of the linker-payloads are coupled to a single antibody molecule.

[0344] The term "D8" herein refers to an ADC in which an average of about eight drug molecules (e.g., 7.5, 8.0, 8.4 molecules) are coupled to one single antibody molecule, wherein the drug molecule can be coupled to eight -SH groups generated by reducing four disulfide bonds. In some embodiments, D8 refers to an ADC in which eight of the linker-payloads are coupled to one single antibody molecule.

[0345] The term "about" herein 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 a specific embodiment, the term "about" preceding a numerical value indicates a range of plus or minus 50%, 30%, 15%, 10%, 5%, or 1% of the value.

[0346] In some embodiments, two different linker-payload toxins are coupled to a single antibody to form a dual payload toxin ADC. Depending on the DAR value of each linker-payload toxin, a dual payload toxin ADC may have two DAR values, for example, the first linker-payload toxin is D6 and the second linker-payload toxin is D2. The DAR value of the dual payload toxin ADC in the present disclosure is recorded as "DN+DM", where N represents the average number of the first linker-payload toxin coupled to a single antibody molecule, and M represents the average number of the second linker-payload toxin coupled to a single antibody molecule.

[0347] In some embodiments, "D1+D6" or "D1+D6 type ADC" refers to an ADC in which one of the first sulfide bridging groups carrying a first linker-payload rebridges two thiol groups and six of the second linker-payloads are coupled to a single antibody molecule.

[0348] In some embodiments, "D2+D6" or "D2+D6-type ADC" refers to an ADC in which two of the first linker-payloads and six of the second linker-payloads are coupled to a single antibody molecule.

[0349] In some embodiments, "D0+D2" or "D0+D2 type ADC" 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 first capping agents and two of the second linker-payloads are coupled to a single antibody molecule.

[0350] In some embodiments, the salt refers to an acid addition salt or a base addition salt.

[0351] In some embodiments, the acid addition salt can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, etc.

[0352] In some 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 Group 1 to Group 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 amines, secondary amines and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, etc. Some organic amines include isopropylamine, dibenzylethylenediamine, choline, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.

[0353] In some embodiments, in step (R1) of method I, the molar ratio of the first reducing agent to the transition metal ion is 1:0.4 to 1:250, 1:0.4 to 1:200, 1:1 to 1:70, 1:0.4 to 1:60, 1:0.1 to 1:20, 1:6 to 1:16, 1:0.2 to 1:8, 1:0.5 to 1:8, 1:0.25 to 1:7.5, or 1:0.25 to 1:7.

[0354] In some embodiments, in method I, a first precursor reagent and a transition metal ion having a specific molar ratio selectively reduce one of the four interchain disulfide bonds within the antibody, optionally, reduce one interchain disulfide bond in the hinge region of the antibody.

[0355] In the present invention, the term "transition metal ion" refers to elements of Groups 4 to 12, determined 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 in Group 3 are also generally considered to be transition metals.

[0356] In some embodiments, the transition metal ion is selected from Zn 2+ 、Cd 2+ , Hg 2+ 、Ni 2+ 、Co 2+ , or a combination thereof.

[0357] In some embodiments, the transition metal ion is Zn 2+ .

[0358] 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, the salt of the transition metal ion is chloride, nitrate, sulfate, acetate, iodide, bromide, formates or tetrafluoroborate.

[0359] 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.

[0360] In some embodiments, in step (R1) of method I, when the first reducing agent is TCEP, the molar ratio of the first reducing agent to the transition metal ion is 1:0.4 to 1:200, 1:0.4 to 1:70, 1:1 to 1:16, or 1:2 to 1:16. In some embodiments, in step (R1) of method I, when the first reducing agent is TCEP, the molar ratio of the first reducing agent to the transition metal ion is 1:0.5, 1:2, 1:3, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190 or 1:200.

[0361] In some embodiments, in step (R1) of method I, when the first reducing agent is a compound having formula (I) or (II), the molar ratio of the first reducing agent to the transition metal ion is 1:0.4 to 1:250, 1:0.4 to 1:60, 1:2 to 1:60, 1:4 to 1:60, 1:4 to 1:24, 1:2 to 1:12, or 1:6 to 1:16, optionally the first reducing agent is TCEP, TCEPA or TCEP-NO-trityl. In some embodiments, in step (R1) of method I, when the first reducing agent is a compound having formula (I) or (II), the molar ratio of the first reducing agent to the transition metal ion is 1:1 to 1:180, 1:1 to 1:150, 1:1 to 1:130, 1:1 to 1:100, 1:1 to 1:80, 1:1 to 1:70, 1:60. In some embodiments, in step (R1) of method I, when the first reducing agent is a compound having formula (I) or (II), the molar ratio of the first reducing agent to the transition metal ion is 1:0.5, 1:2, 1:3, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:20, 1:25, 1:30, 1:35, 1:40, 1:4 5. 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:220, 1:240 or 1:250.

[0362] In some embodiments, the molar ratio of the first reducing agent to the antibody in step (R1) is 1:1 to 5:1, 2:1 to 5:1, 4:1 to 5:1, 1:1 to 3:1, 3.8:1 to 4.6:1, 1:1 to 2:1, or 3:1 to 5:1.

[0363] In some embodiments, in step (R1) of method I, the molar ratio of the first reducing agent to the antibody is 3:1 to 0.5:1, optionally, the molar ratio of the first reducing agent to the antibody is 3:1 to 1:1, and more optionally, the molar ratio of the first reducing agent to the antibody is 2:1 to 1:1.

[0364] In some embodiments, in step (R1) of method I, the molar ratio of the first reducing agent to the antibody is 0.6: 1 to 1: 1, 1.5: 1 to 1: 1, 1.2: 1 to 1: 1. In some embodiments, in step (R1) of method I, the molar ratio of the first reducing agent to the antibody is 2.9: 1, 2.8: 1, 2.7: 1, 2.6: 1, 2.5: 1, 2.4: 1, 2.3: 1, 2.2: 1, 2.1: 1, 2: 1, 1.9: 1, 1.8: 1, 1.5: 1, 1.4: 1, 1.2: 1, 1: 1, 0.8: 1 or 0.5: 1.

[0365] In some embodiments, in step (R1) of method I, there is no specific limitation on the concentration of the first reducing agent, as long as the concentrations of the transition metal ion and the antibody are enlarged or reduced in equal proportion. In some embodiments, the concentration of the first reducing agent is 0.01 mM to 0.2 mM, 0.02 mM to 0.15 mM or 0.05 mM to 0.1 mM. In some embodiments, 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.

[0366] In some embodiments, there is no specific limitation on the concentration of the transition metal ion in step (R1) of method I, as long as the concentrations of the first reducing agent and the antibody are increased or decreased in equal proportions.

[0367] In some embodiments, there are specific limitations on the concentration of the antibody in step (R1) of method I, as long as the concentrations of the first reducing agent and the transition metal ion are increased or decreased in equal proportions.

[0368] In some embodiments, the concentrations of the first reducing agent, transition metal ion, and antibody in step (R1) of method I are applicable to the concentrations in step (R1) of methods II, III, IV, and V.

[0369] In some embodiments, in step (R1) of method I, the incubation time is 0° C. to 37° C., 0° C. to 25° C., 0° C. to 15° C., 0° C. to 10° C., or 0° C. to 5° C. In some embodiments, in step (R1) of method I, the incubation time is 4° C., 8° C., 12° C., 15° C., 18° C., 24° C., 30° C., 35° C., or 37° C. In some embodiments, the incubation time in step (R1) is also applicable to the incubation time in step (R1) of methods II, III, IV, and V.

[0370] In some embodiments, in step (R1) of method I, the incubation time is 0.2 hours to 24 hours, and optionally the incubation time is 2 hours to 16 hours. In some embodiments, in step (R1) of method I, the incubation time is 0.5 hours to 24 hours, 0.5 hours to 20 hours, 0.5 hours to 16 hours, 0.5 hours to 12 hours, 0.5 hours to 8 hours or 0.5 hours to 6 hours. In some embodiments, in step (R1) of method I, the incubation time is 0.25 hours, 0.3 hours, 0.5 hours, 0.7 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours.

[0371] In some embodiments, in step (R1) of method I, the molar ratio of the first reducing agent to the antibody is 2: 1 to 3.0: 1, and the incubation time is 0.5 to 9 hours. In some embodiments, in step (R1) of method I, the molar ratio of the first reducing agent to the antibody is 2.8: 1 to 3.0: 1, and the incubation time is 0.5 to 9 hours. In some embodiments, in step (R1) of method I, the molar ratio of the first reducing agent to the antibody is 2.1: 1, 2.2: 1, 2.3: 1, 2.4: 1, 2.5: 1, 2.6: 1, 2.7: 1, 2.8: 1, 2.9: 1 or 3: 1, and the incubation time is 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 9.5 hours or 9.9 hours.

[0372] In some embodiments, in step (R3) in method I, 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 the same as the first reducing agent. In some embodiments, the second reducing agent is different from the first reducing agent. In some embodiments, the second reducing agent is a compound having formula (I) or (II), TCEP, tris (3-hydroxypropyl) phosphine (THPP) or dithiothreitol (DTT). In some embodiments, the second reducing agent is TCEP. In some embodiments, the second reducing agent of method I is also applicable to the reducing agents of methods II, III, IV and V.

[0373] In some embodiments, in step (R3), there is no specific limitation on the concentration of the second reducing agent, as long as the second reducing agent can reduce the interchain disulfide bonds in the antibody. In some embodiments, when the second reducing agent is TCEP or a compound of formula (I) or (II), the molar ratio of the second reducing agent to the antibody in step (R3) is 10:1 to 25:1, 10:1 to 23:1, 1:1 to 20:1, 5:1 to 20:1, 10:1 to 19:1, 10:1 to 18:1, 15:1 to 25:1, 10:1 to 20:1, 15:1 to 20:1, 1:1 to 8:1, 1:1 to 5:1, 1:1 to 3:1, or 1:1 to 2:1.

[0374] In some embodiments, in step (R3), when the second reducing agent is TCEP, TCEP-NO or TCEPA, the molar ratio of the second reducing agent to the antibody in step (R3) is 1:1 to 10:1, 1:1 to 8:1, 1:1 to 5:1, 1:1 to 3:1, or 1:1 to 2:1.

[0375] In some embodiments, in step (R3) of method I, three of the interchain disulfide bonds in the product prepared in step (C1) are reduced in the absence of transition metal ions. In some embodiments, one interchain disulfide bond or two interchain disulfide bonds in the product prepared by step (C1) are reduced using transition metal ions.

[0376] In some embodiments, in step (R3) of method I, in the absence of transition metal ions, 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, 6: 1 to 8: 1. In some embodiments, the molar ratio of the second reducing agent to the antibody is 20: 3. In some embodiments, the molar ratio of the second reducing agent to the antibody of method I is also applicable to the molar ratio in step (R3) of methods III, IV and V.

[0377] In some embodiments, in step (R3) of method I, 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 (R3) of method I, 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 (R3) of method I, the incubation temperature of the second reducing agent is 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 25°C. In some embodiments, the incubation temperature in step (R3) of method I is also applicable to the incubation temperature in step (R2) of method IV and the incubation temperature in step (R3) of methods II, III, IV and V.

[0378] In some embodiments, in step (R3) of method I, the incubation time of the second reducing agent is 0.5 hours to 24 hours, or 5 hours to 20 hours. In some embodiments, in step (R3) of method I, the incubation time of the second reducing agent is 6 hours to 18 hours, 8 hours to 18 hours, 8 hours to 15 hours, or 8 hours to 12 hours. In some embodiments, in step (R3) of method I, the incubation time of the second reducing agent is 8 hours or 12 hours. In some embodiments, the incubation time in step (R3) of method I is also applicable to the incubation time in step (R3) of methods III, IV and V.

[0379] In some embodiments, in step (R3) of method I, a transition metal ion is introduced to selectively reduce two of the interchain disulfide bonds. In some embodiments, in step (R3) of method I, the molar ratio of the second reducing agent to the transition metal ion is 1:0.05 to 1:40, and / or the molar ratio of the second reducing agent to the antibody is 2.5:1 to 20:1, and / or the incubation time is 1 hour to 24 hours. In some embodiments, in step (R3) of method I, 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 (R3) of method I, 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 (R3) of method I, 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 (R3) of method I, the molar ratio of the second reducing agent to the transition metal ion is 1: 0.05 to 1: 40, and / or the molar ratio of the second reducing agent to the antibody is 3: 1 to 15: 1, and the incubation time is 1 hour to 12 hours. In some embodiments, in step (R3) of method I, the molar ratio of the second reducing agent to the transition metal ion is 1:0.05 to 1:40, and / or the molar ratio of the second reducing agent to the antibody is 2.5:1 to 15:1, and the incubation time is 12 hours to 24 hours. In some embodiments, the molar ratio of the second reducing agent to the transition metal ion, the molar ratio of the second reducing agent to the antibody, and the incubation time in step (R3) of method I are also applicable to those in step (R3) of method III.

[0380] In some embodiments, in step (R3) of method I, a transition metal ion is introduced to selectively reduce one of the interchain disulfide bonds. In some embodiments, in step (R3) of method I, the molar ratio of the second reducing agent to the transition metal ion is 1:0.4 to 1:100, and / or the molar ratio of the second reducing agent to the antibody is 0.8:1 to 2.5:1, and / or the incubation time is 0.5 to 24 hours. In some embodiments, in step (R3) of method I, 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 (R3) of method I, the molar ratio of the second reducing agent to the antibody is 0.5: 1, 1: 1, 1.2: 1, 1.4: 1, 1.6: 1, 1.8: 1, 2: 1, 2.2: 1, 2.4: 1, or 2.5: 1. In some embodiments, in step (R3) of method I, the incubation time is 0.5 hours, 1 hours, 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 (R3) of method I, the molar ratio of the second reducing agent to the transition metal ion is 1: 0.5 to 1: 100, and / or the molar ratio of the second reducing agent to the antibody is 0.8: 1 to 2: 1, and the incubation time is 0.5 hours to 24 hours. In some embodiments, in step (R3), 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 2: 1 to 2.5: 1, and the incubation time is 1 hour to 9 hours. In some embodiments, the molar ratio of the second reducing agent to the transition metal ion, the molar ratio of the second reducing agent to the antibody, and the incubation time in step (R3) of method I are also applicable to the incubation time in step (R3) of methods III, IV and V.

[0381] In some embodiments, in step (C1) and step (C3) of method I, the reaction temperature with the reduced thiol group is 4°C to 40°C, 10°C to 40°C, 10°C to 35°C, 10°C to 30°C, 10°C to 25°C, 15°C to 35°C, 20°C to 30°C, 4°C to 37°C, 20°C to 30°C, or 20°C to 25°C. In some embodiments, in step (C1) and in step (C3), the reaction temperature with the reduced thiol group is 24°C. In some embodiments, the reaction temperature with the reduced thiol group in step (C1) and step (C3) of method I is also applicable to the reaction temperature in step (C2) and / or (C3) of method V and steps (C1) and / or (C3) of methods II, III, and IV.

[0382] In some embodiments, in step (C1) and step (C3) of method I, the reaction time with the reduced thiol group is 0.5 to 10 hours, 0.5 to 6 hours, 0.5 to 5 hours, 0.5 to 4 hours, 0.5 to 3 hours, 0.5 to 3 hours, 0.5 to 2 hours, 0.5 to 1 hour. In some embodiments, in step (C1) and step (C3) of method I, the reaction time with the reduced thiol group is 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours. In some embodiments, the reaction time with the reduced thiol group in step (C1) and step (C3) of method I is also applicable to the reaction time in step (C2) and / or (C3) of method V and steps (C1) and / or (C3) of methods II, III and IV.

[0383] In some embodiments, the reaction temperature and time with the reduction of thiol groups in step (C1) and step (C3) are independent.

[0384] In some embodiments, in step (C1) and step (C3) of method I, the reaction temperature with the reactive group is 4°C to 37°C, 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 step (C1) and step (C3) of method I, 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. In some embodiments, the reaction temperature with the reactive group in step (C1) and step (C3) of method I is also applicable to the reaction temperature in step (C2) and / or (C3) of method V and steps (C1) and / or (C3) of methods II, III, and IV.

[0385] In some embodiments, in step (C1) and step (C3) of method I, the reaction time with the reactive group is 2 hours to 12 hours, 2 hours to 10 hours, 4 hours to 10 hours, 6 hours to 10 hours, or 8 hours to 10 hours. In some embodiments, in step (C1) and step (C3) of method I, the reaction time with the reactive group is 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours. In some embodiments, in step (C1) and step (C3) of method I, the reaction time with the reactive group is also applicable to the reaction time in step (C2) and / or (C3) of method V and steps (C1) and / or (C3) of methods II, III and IV.

[0386] In some embodiments, the reaction temperature and time with the reactive group in step (C1) and step (C3) are independent.

[0387] In some embodiments, in step (C1), the first coupling group is in excess based on the amount of the antibody.

[0388] In some embodiments, the molar ratio of the first coupling group to the antibody in step (C1) is 1:1 to 50:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 8:1, 1:1 to 6:1, 1:1 to 5:1, 2:1 to 8:1, or 2:1 to 6:1.

[0389] In some embodiments, in step (C1) of method I, the molar ratio of the first sulphur bridging agent to the antibody is 10: 1 to 1: 1, 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) of method I, the molar ratio of the first sulphur bridging agent to the antibody is 1: 1, 1.5: 1, 2: 1, 3: 1, 3.8: 1, 4: 1, 4.8: 1 or 5: 1.

[0390] In some embodiments, in step (C1) of method I, 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 10: 1 to 1: 1, 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) of method I, the molar ratio of the first linker-payload to the antibody is 5:3.

[0391] In some embodiments, in step (C1) of method I, 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 20: 1, 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) of method I, 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, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, or 20: 1.

[0392] In some embodiments, the molar ratio of the first coupling group to the antibody in step (C1) of method I is also applicable to the molar ratio in step (C1) of method III.

[0393] In some embodiments, in step (C3), the second coupling group is in excess based on the amount of the antibody.

[0394] In some embodiments, the molar ratio of the second coupling group to the antibody in step (C3) is 1:1 to 30:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 8:1, 1:1 to 6:1, 1:1 to 5:1, 2:1 to 8:1, or 2:1 to 6:1.

[0395] In some embodiments, in step (C3) of method I, the molar ratio of the first sulphur bridging agent 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 (C3) of method I, the molar ratio of the second sulphur bridging agent to the antibody is 5: 1, 4.5: 1, 4: 1, 3.8: 1, 3.5: 1 or 3.2: 1.

[0396] In some embodiments, in step (C3) of method I, 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.

[0397] In some embodiments, in step (C3) of method I, 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 1: 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 (C3) of method I, 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 1: 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.

[0398] In some embodiments, the molar ratio of the second coupling group to the antibody in step (C3) of method I is also applicable to the molar ratio in step (C3) of methods III, IV, and V.

[0399] In some embodiments, in step (R4) of method I, a transition metal ion is introduced to selectively reduce one of the interchain disulfide bonds. In some embodiments, in step (R4) of method I, the molar ratio of the second reducing agent to the transition metal ion is 1:0.4 to 1:100, and / or the molar ratio of the second reducing agent to the antibody is 0.8:1 to 2.5:1, and / or the incubation time is 0.5 hours to 24 hours. In some embodiments, in step (R4) of method I, 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 (R4) of method I, the molar ratio of the second reducing agent to the antibody is 0.5: 1, 1: 1, 1.2: 1, 1.4: 1, 1.6: 1, 1.8: 1, 2: 1, 2.2: 1, 2.4: 1, or 2.5: 1. In some embodiments, in step (R4) of method I, the incubation time is 0.5 hours, 1 hours, 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 (R4) of method I, the molar ratio of the second reducing agent to the transition metal ion is 1: 0.5 to 1: 100, and / or the molar ratio of the second reducing agent to the antibody is 0.8: 1 to 2: 1, and the incubation time is 0.5 hours to 24 hours. In some embodiments, in step (R4), 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 2: 1 to 2.5: 1, and the incubation time is 1 hour to 9 hours. In some embodiments, the molar ratio of the second reducing agent to the transition metal ion, the molar ratio of the second reducing agent to the antibody, and the incubation time in step (R4) of method I are also applicable to those in step (R4) of method III.

[0400] In some embodiments, the temperature in step (R4) of method I is the same as the temperature in step (R3) of method I.

[0401] In some embodiments, the parameters in step (C4) of method I are the same as those in step (C1) of method I. In some embodiments, the time and temperature and the molar ratio of the second coupling group to the antibody in step (C4) of method I are the same as those in step (C1) of method I.

[0402] In some embodiments, the second coupling group in step (C4) of method I and the second coupling group in step (C3) of method I are the same or different.

[0403] In some embodiments, the DAR value of the conjugate prepared by method I is 2 (or D2), in which case two first linker-payloads are attached to the antibody. In some embodiments, the DAR value of the conjugate prepared by method I is 1 (or D1), in which case one of the first sulphur bridge reagents carrying the first linker-payload is attached to the antibody. In some embodiments, in method I, steps (R3) and (C3) are used to prepare a dual payload conjugate comprising D2+D3, D2+D6, D2+D4, D2+D2, D1+D3, D1+D6, D1+D4, D1+D2, D0+D3 or D0+D6. In some embodiments, in method I, steps (R3), (C3), (R4) and (C4) are used to prepare D1+D2+D2, D2+D1+D2, D2+D2+D1, D2+D1+D1, D1+D1+D2, D1+D1+D1, D2+D2+D2.

[0404] In some embodiments, in step (R1) of method II, three of the interchain disulfide bonds of the antibody are reduced, optionally, two of the interchain disulfide bonds in the Fab region and one of the interchain disulfide bonds in the hinge region of the antibody are reduced.

[0405] In some embodiments, in step (R1) of method II, the molar ratio of the first reducing agent to the transition metal ion is 1:0.05 to 1:40, 1:0.08 to 1:30, 1:0.1 to 1:30, 1:0.1 to 1:20, 1:0.5 to 8:1 or 1:0.25 to 1:7.5, and the first reducing agent is optionally TCEP or a compound having formula (I) or formula (II).

[0406] In some embodiments, in step (R1) of method II, the molar ratio of the first reducing agent to the transition metal ion is 1:0.1 to 1:15, 1:0.1 to 1:10, 1:0.25 to 1:15, 1:0.25 to 1:12, 1:0.25 to 1:10, 1:0.25 to 1:8, 1:0.25 to 1:7.5, 1:0.25 to 1:7, 1:0.25 to 1:5, 1:0.25 to 1:4 or 1:0.5 to 1:4. In some embodiments, in step (R1) of method II, the molar ratio of the first reducing agent to the transition metal ion is 1:0.08, 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, 1:20, 1:23, 1:2, 1:27, 1:29, 1:32, 1:34, 1:36, 1:38 or 1:40.

[0407] In some embodiments, the molar ratio of the first reducing agent to the transition metal ion in step (R1) of method II is also applicable to the molar ratio in step (R1) of methods III, IV, and V.

[0408] In some embodiments, in step (R1) of method II, the molar ratio of the first reducing agent to the antibody is 2.8:1 to 20:1, 3:1 to 15:1, 3:1 to 6:1, 3.5:1 to 5:1, 4:1 to 10:1, 5:1 to 13:1.

[0409] In some embodiments, in step (R1) of method II, the molar ratio of the first reducing agent to the antibody is 3.5: 1 to 5: 1, 4: 1 to 10: 1, or 5: 1 to 13: 1, 3: 1 to 5: 1, 4: 1 to 5: 1, or 3.8: 1 to 4.6: 1. In some embodiments, in step (R1) of method II, the molar ratio of the first reducing agent to the antibody is 2.8: 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, or 20: 1.

[0410] In some embodiments, the molar ratio of the first reducing agent to the antibody in step (R1) of method II is also applicable to the molar ratio in step (R1) of methods III, IV, and V.

[0411] In certain embodiments, in step (R1) of method II, the incubation time is 1 hour to 24 hours, 14 hours to 24 hours, 16 hours to 20 hours, or 16 hours to 18 hours. In certain embodiments, in step (R1) of method II, the incubation time is 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours. In certain embodiments, the incubation time in step (R1) of method II is also applicable to the incubation time in step (R1) of method III, IV and V.

[0412] In some embodiments, in step (R1) of method II, the molar ratio of the first reducing agent to the antibody is 3: 1 to 6: 1, and the incubation time is 10 hours to 24 hours. In some embodiments, in step (R1) of method II, the molar ratio of the first reducing agent to the antibody is 2.8: 1 to 3: 1, and the incubation time is 10 hours to 24 hours. In some embodiments, in step (R1) of method II, the molar ratio of the first reducing agent to the antibody is 2.8: 1, 2.9: 1 or 3: 1, and the incubation time is 10 hours, 12 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours. In some embodiments, in step (R1) in method II, the molar ratio of the first reducing agent to the 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 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours.

[0413] In some embodiments, the incubation time in the step (R1) in method II is shortened as the molar ratio of the first reducing agent to the antibody increases. In some embodiments, in the step (R1) in method II, the molar ratio of the first reducing agent to the antibody is 6: 1 to 20: 1, and the incubation time is 4 hours to 16 hours. In some embodiments, in the step (R1) in method II, the molar ratio of the first reducing agent to the antibody is 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, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours or 16 hours.

[0414] In some embodiments, in step (R1) in method II, the molar ratio of transition metal ion to antibody is 1:1 to 50:1, 1:1 to 30:1, 1:1 to 20:1, 1:1 to 15:1, 8:1 to 30:1, 12:1 to 30:1, 12:1 to 30:1, 8:1 to 16:1, 4:1 to 30:1, 4:1 to 16:1, 8:1 to 16:1, 1:1 to 10:1, 1:1 to 8:1, 1:1 to 6:1, 1:1 to 5:1, 1:1 to 4:1, 1:1 to 3:1, 1:1 to 2:1, 2:1 to 6:1, 2:1 to 4:1.

[0415] In some embodiments, in step (R3) of method II, one of the interchain disulfide bonds in the product prepared in step (C1) is completely reduced in the absence of transition metal ions.

[0416] In some embodiments, in step (R3) of method II, the molar ratio of the second reducing agent to the antibody is 1: 1 to 20: 1. In some embodiments, in step (R3) of method II, 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.

[0417] In some embodiments, in step (R3) of method II, the incubation temperature of the second reducing agent is 0°C to 37°C, and the incubation time is 0.5 hour to 24 hours.

[0418] In some embodiments, in step (R3) of method II, 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 (R3) of method II, the incubation temperature of the second reducing agent is 25° C.

[0419] In some embodiments, in step (R3) of method II, the incubation time is 1 hour to 20 hours, 5 hours to 20 hours, 6 hours to 18 hours, 8 hours to 18 hours, 8 hours to 15 hours or 8 hours to 12 hours. In some embodiments, in step (R3) of method II, the incubation time is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours or 12 hours.

[0420] In some embodiments, in step (C1) of method II, the molar ratio of the first sulphur bridging agent to the antibody is 3: 1 to 10: 1. In some embodiments, in step (C1) of method II, the molar ratio of the first sulphur bridging agent to the antibody is 3: 1, 3.3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1.

[0421] In some embodiments, in step (C1) of method II, 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 10: 1. In some embodiments, in step (C1) of method II, 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 or 10: 1.

[0422] In some embodiments, in step (C1) of method II, 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 (C1) of method II, 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.

[0423] In some embodiments, in step (C3) of method II, the second coupling group is in excess based on the amount of the antibody.

[0424] In some embodiments, in step (C3) of method II, the molar ratio of the second sulphur bridging agent to the antibody is 1: 1 to 3: 1. In some embodiments, in step (C3) of method II, the molar ratio of the second sulphur bridging agent to the antibody is 1: 1, 1.5: 1, 2: 1 or 3: 1.

[0425] In some embodiments, in step (C3) of method II, when the second linker-payload reacts with the reactive group in the second sulphur bridging reagent, the molar ratio of the second linker-payload to the antibody is 1: 1 to 8: 1. In some embodiments, in step (C3) of method II, when the second linker-payload reacts with the reactive group in the second sulphur 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.

[0426] In some embodiments, in step (C3) of method II, 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 20: 1. In some embodiments, in step (C3) of method II, 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, 16: 1 or 18: 1.

[0427] In some embodiments, the DAR value of the conjugate prepared by method II is 6 (or D6), in which case six of the first linker-payloads are attached to the antibody. In some embodiments, the DAR value of the conjugate prepared by method I is 3 (or D3), in which case three of the first sulphur bridge reagents carrying the first linker-payload are attached to the antibody. In some embodiments, in method II, steps (R3) and (C3) are used to prepare dual payload conjugates, which include D6+D1, D6+D2, D3+D1, D3+D2, D0+D1 and D0+D2.

[0428] In some embodiments, the parameters in step (R1) of method III are the same as those in step (R1) of method II. In some embodiments, the molar ratio of the first reducing agent to the transition metal ion, the molar ratio of the first reducing agent to the antibody, the incubation time and the temperature in step (R1) of method III are the same as those in step (R1) of method II.

[0429] In some embodiments, in step (R1) of method III, the molar ratio of the first reducing agent to the antibody is 4: 1 to 15: 1, and the incubation time is 1 to 16 hours. In some embodiments, in step (R1) of method III, the molar ratio of the first reducing agent to the antibody is 4.5: 1, 5: 1, 5: 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 1 hour. 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, or 16 hours.

[0430] In the present invention, there is no specific limitation on the oxidant in step (O1), as long as the oxidant can reoxidize and reduce thiol groups. In some embodiments, in step (O1), the oxidant is dehydroascorbic acid (DHAA).

[0431] In some embodiments, in step (O1) of method III, the molar ratio of the oxidant to the antibody is 2: 1 to 25: 1, 2: 1 to 20: 1, 4: 1 to 22: 1, 4: 1 to 15: 1, or 6: 1 to 14: 1. In some embodiments, in step (O1) of method III, the molar ratio of the oxidant to the antibody is 1: 1 to 20: 1, 3: 1 to 18: 1, 6: 1 to 18: 1, 8: 1 to 14: 1, 6: 1 to 12: 1, 5: 1 to 20: 1, 4: 1 to 15: 1, or 6: 1 to 14: 1. In some embodiments, in step (O1) of method III, the molar ratio of the oxidant to the antibody in step (O1) is 1:1 to 15:1, 3:1 to 15:1, 6:1 to 15:1, 8:1 to 14:1, 6:1 to 10:1, 8:1 to 12:1, 6:1 to 10:1, 6:1 to 12:1, 3:1 to 8:1, 3:1 to 6:1, 5:1 to 15:1, 5:1 to 10:1, 5:1 to 8:1, 2:1 to 7:1, 4:1 to 9:1, 1:1 to 5:1, 2:1 to 4:1, or 2:1 to 6:1.

[0432] In some embodiments, in step (O1) of method III, the oxidation temperature is 0°C to 37°C, 0°C to 30°C, 0°C to 25°C, 0°C to 20°C, 0°C to 15°C, 0°C to 10°C, or 0°C to 5°C, 5°C to 30°C, 10°C to 30°C, 15°C to 30°C, 20°C to 30°C, 0°C to 25°C, 0°C to 20°C, 10°C to 25°C, 15°C to 30°C, 5°C to 25°C, 10°C to 20°C or 10°C to 15°C.

[0433] In some embodiments, in step (O1) of method III, the oxidation temperature is 10°C to 40°C, 10°C to 35°C, 10°C to 30°C, 10°C to 25°C, 15°C to 35°C, 20°C to 30°C. In some embodiments, in step (O1) of method III, the oxidation temperature is room temperature. As used herein, the term "room temperature" refers to 23°C ± 2°C, 25°C ± 5°C, or 20°C ± 5°C.

[0434] In some embodiments, in step (O1) of method III, the oxidation time is 1 hour to 48 hours, 0.5 hours to 15 hours, 1 hour to 10 hours, 1 hour to 5 hours, 0.5 hours to 5 hours, 0.5 hours to 3 hours, 1 hour to 3 hours, 2 hours to 5 hours, 2 hours to 4 hours or 2 hours to 3 hours.

[0435] In some embodiments, in step (O1) of method III, the oxidation time is 0.5 hour to 4 hours, 0.5 hour to 3 hours, 0.5 hour to 2 hours, 0.5 hour to 1 hour, 1 hour to 4 hours, 1 hour to 3 hours, 1 hour to 2 hours, or 2 hours to 4 hours.

[0436] In some embodiments, the oxidation reaction in step (O1) of method III is carried out at a temperature of 0°C to 37°C, 0°C to 30°C, 15°C to 30°C, or 20°C to 30°C, optionally in the dark, and / or the oxidation time is 1 hour to 48 hours, 1 hour to 5 hours, or 1 hour to 3 hours.

[0437] In some embodiments, in step (O1) of method III, the oxidation temperature is 0°C to 25°C, 0°C to 15°C, 0°C to 10°C, or 0°C to 5°C, and / or the oxidation time is 0.5 hours to 12 hours, 1 hour to 10 hours, 1 hour to 8 hours, 2 hours to 5 hours, or 3 hours to 8 hours.

[0438] In some embodiments, in step (O1) of method III, the oxidation temperature is 15°C to 25°C, and the oxidation time is 1 hour to 3 hours.

[0439] In some embodiments, in step (O1) of method III, the thiol groups in the Fab region are reoxidized. In some embodiments, four thiol groups in the Fab region are reoxidized.

[0440] In some embodiments, the parameters in steps (C1), (R3), (C3), (R4) and (C4) of method III are the same as the parameters in steps (C1), (R3), (C3), (R4) and (C4) of method I.

[0441] In some embodiments, the DAR value of the conjugate prepared by method III is 2 (or D2), in which case two of the first linker-payloads are connected to the antibody. In some embodiments, the DAR value of the conjugate prepared by method III is 1 (or D1), in which case one of the first sulfur bridge reagents carrying the first linker-payload is connected to the antibody. In some embodiments, in method III, steps (R3) and (C3) are used to prepare a dual payload conjugate, which includes D2+D3, D2+D6, D2+D4, D2+D2, D1+D3, D1+D6, D1+D4, D1+D2, D0+D3 or D0+D6. In some embodiments, in method III, steps (R3), (C3), (R4) and (C4) are used to prepare D1+D2+D2, D2+D1+D2, D2+D2+D1, D2+D1+D1, D1+D1+D2, D1+D1+D1, D2+D2+D2.

[0442] In some embodiments, the parameters in steps (R1) and (O1) of method IV are the same as the parameters in steps (R1) and (O1) of method III.

[0443] In some embodiments, a metal chelator is introduced in step (R2) of method IV.

[0444] In some embodiments, in step (R2) of method IV, the molar ratio of metal chelator to antibody is 2:1 to 120:1, 2:1 to 100:1, 2:1 to 80:1, 5:1 to 60:1, 10:1 to 60:1, 20:1 to 60:1, 30:1 to 60:1, 40:1 to 60:1, or 50:1 to 60:1.

[0445] In some embodiments, in step (R2) of method IV, the molar ratio of the second reducing agent to the antibody is 10: 1 to 25: 1, 10: 1 to 23: 1, 10: 1 to 20: 1, 10: 1 to 19: 1, 10: 1 to 18: 1, 15: 1 to 25: 1, 15: 1 to 20: 1, 1: 1 to 8: 1, 1: 1 to 5: 1, 1: 1 to 3: 1, or 1: 1 to 2: 1. In some embodiments, in step (R2) of method IV, the molar ratio of the second reducing agent to the antibody is 1: 1, 1.2: 1, 1.4: 1, 1.6: 1, 1.8: 1, 2: 1, 2.2: 1, 2.4: 1, 2.6: 1, 2.8: 1, or 3: 1.

[0446] In some embodiments, in step (R2) of method IV, the reduction temperature is 0°C to 30°C, and the reduction time is 1 hour to 8 hours.

[0447] In some embodiments, in step (R2) of method IV, 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.

[0448] In some embodiments, in step (R2) of method IV, the reduction time is 1 hour to 8 hours, 1 hour to 7 hours, 1 hour to 6 hours, 1 hour to 5 hours, 2 hours to 5 hours, 2 hours to 4 hours, or 2 hours to 3 hours.

[0449] In some embodiments, in step (R2) of method IV, one of the interchain disulfides of the product prepared by step (O1) of method IV is reduced.

[0450] In some embodiments, in step (R3) of method IV, two 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 of the interchain disulfide bonds in the product prepared by step (C1) is selectively reduced by transition metal ions.

[0451] In some embodiments, the incubation temperature and time in step (R3) of method IV are the same as those in step (R3) of method I.

[0452] In some embodiments, the molar ratio of the second reducing agent to the antibody in step (R3) of method IV is the same as in step (R3) of method I in the absence of transition metal ions.

[0453] In some embodiments, in step (R3) of method IV, a transition metal ion is introduced, one of the interchain disulfide bonds is selectively reduced, and the parameters in step (R3) of method IV are the same as those in step (R3) of method I.

[0454] In some embodiments, the reaction temperature and time in steps (C1) and (C3) of method IV are the same as those in steps (C1) and (C3) of method I.

[0455] In some embodiments, in step (C1) of method IV, the first coupling group is in excess based on the amount of the antibody.

[0456] In some embodiments, in step (C1) of method IV, the molar ratio of the first sulphur bridging agent to the antibody is 2: 1 to 10: 1. In some embodiments, in step (C1) of method IV, the molar ratio of the first sulphur bridging agent to the antibody is 2: 1, 3: 1, 3.3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1.

[0457] In some embodiments, in step (C1) of method IV, 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 2: 1 to 10: 1. In some embodiments, in step (C1) of method IV, the molar ratio of the first linker-payload to the antibody is 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1.

[0458] In some embodiments, in step (C1) of method IV, when the first linker-payload reacts with the reduced thiol group, the molar ratio of the first linker-payload to the antibody is 4: 1 to 20: 1. In some embodiments, in step (C1) of method IV, when the first linker-payload reacts with the reduced thiol group, the molar ratio of the first linker-payload to the antibody is 4: 1, 5: 1, 6: 1, 20: 3, 7: 1, 8: 1, 9: 1, 10: 1, 15: 1 or 20: 1.

[0459] In some embodiments, in step (C3) of method IV, the second coupling group is in excess according to the amount of the antibody. In some embodiments, the molar ratio of the second coupling group to the antibody in step (C3) of method IV is the same as in step (C3) of method I.

[0460] In some embodiments, the DAR value of the conjugate prepared by method IV is 4 (or D4), in which case four of the first linker-payloads are attached to the antibody. In some embodiments, in method IV, steps (R3) and (C3) are used to prepare a dual-payload conjugate comprising D4+D4 or D4+D2.

[0461] In some embodiments, the parameters in step (R1) of method V are the same as those in step (R1) of method II. In some embodiments, the molar ratio of the first reducing agent to the transition metal ion, the molar ratio of the first reducing agent to the antibody, the incubation time and the temperature in step (R1) of method V are the same as those in step (R1) of method II.

[0462] In some embodiments, in step (R1) of method V, the molar ratio of the first reducing agent to the transition metal ion is 1:0.05 to 1:40 or 1:0.5 to 1:10. In some embodiments, in step (R1) of method II, the molar ratio of the first reducing agent to the transition metal ion is 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0463] In some embodiments, in step (R1) of method V, the molar ratio of the first reducing agent to the antibody is 2.8: 1 to 20: 1 or 3.5: 1 to 10: 1. In some embodiments, in step (R1) of method V, the molar ratio of the first reducing agent to the antibody is 3.5: 1, 4: 1, 4.5: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1.

[0464] In some embodiments, the parameters in step (C2) of Method V are the same as those in step (C1) of Method IV.

[0465] In some embodiments, in step (C2) of method V, the reaction temperature is 20° C.-37° C. In some embodiments, in step (C2) of method V, the reaction temperature is 20° C., 24° C., 26° C., 28° C., 30° C., 32° C., 34° C. or 36° C. In some embodiments, increasing the reaction temperature in step (C2) is useful for increasing the content of D4-type ADC.

[0466] In some embodiments, in step (C2) of method V, the molar ratio of the first linker-payload to the antibody is 4: 1 to 30: 1. In some embodiments, in step (C2) of method V, the molar ratio of the first linker-payload to the antibody is 4: 1, 8: 1, 12: 1, 16: 1, 20: 1, 24: 1 or 28: 1. In some embodiments, increasing the molar ratio of the first linker-payload to the antibody in step (C2) helps increase the content of the D4-type ADC.

[0467] In some embodiments, in step (R3) of method V, two of the interchain disulfide bonds in the product prepared by step (C2) are completely reduced in the absence of transition metal ions. In some embodiments, in step (R3) of method V, one of the interchain disulfide bonds in the product prepared by step (C2) is selectively reduced by transition metal ions.

[0468] In some embodiments, the incubation temperature and time in step (R3) of method V are the same as those in step (R3) of method I.

[0469] In some embodiments, the molar ratio of the second reducing agent to the antibody in step (R3) of method V is the same as in step (R3) of method I in the absence of transition metal ions.

[0470] In some embodiments, in step (R3) of method V, a transition metal ion is introduced, one of the interchain disulfide bonds is selectively reduced, and the parameters in step (R3) of method V are the same as those in step (R3) of method I.

[0471] In some embodiments, the parameters in step (C3) of Method V are the same as those in step (C3) of Method IV.

[0472] In some embodiments, the DAR value of the conjugate prepared by method V is 4 (or D4), in which case four of the first linker-payloads are attached to the antibody. In some embodiments, in method V, steps (R3) and (C3) are used to prepare a dual-payload conjugate comprising D4+D4 or D4+D2.

[0473] Without being bound by theory, the reducing agent selectively reduces disulfide bonds in the buffer system. The buffer systems of methods I, II, III, IV and V are independently 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, ADA buffer, PB, acetate buffer, BTP buffer, HEPPSO buffer, POPSO buffer, EPPS buffer or Tris buffer.

[0474] The term "ADA buffer" herein refers to N-(carbamoylmethyl)iminodiacetic acid buffer.

[0475] As used herein, the term "MES buffer" refers to 2-(N-morpholino)ethanesulfonic acid buffer.

[0476] As used herein, the term "Bis-Tris buffer" refers to a bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane buffer.

[0477] The term "PIPES buffer" herein refers to piperazine-1,4-bisethanesulfonic acid buffer.

[0478] The term "MOPS buffer" herein refers to 3-morpholinepropanesulfonic acid buffer.

[0479] The term "BES buffer" herein refers to N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer.

[0480] As used herein, the term "HEPES buffer" refers to 4-hydroxyethylpiperazineethanesulfonic acid buffer.

[0481] As used herein, the term "DIPSO buffer" refers to 3-[bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer.

[0482] The term "MOBS buffer" herein refers to 3-morpholinepropanesulfonic acid buffer.

[0483] The term "MOPSO buffer" herein refers to 3-(N-morpholino)-2-hydroxy-1-propanesulfonic acid buffer.

[0484] The term "TES buffer" herein refers to 2-[tris(hydroxymethyl)methylamino]-1-ethanesulfonic acid buffer.

[0485] The term "ACES buffer" herein refers to N-(carbamoylmethyl)taurine buffer.

[0486] As used herein, the term "TAPSO buffer" refers to 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid buffer.

[0487] The term "PBS" herein refers to phosphate buffered saline.

[0488] The term "ADA buffer" herein refers to N-(carbamoylmethyl)iminodiacetic acid buffer.

[0489] The term "PB buffer" herein refers to phosphate buffered saline.

[0490] The term "BTP buffer" herein refers to Bis-tris propane buffer.

[0491] The term "Heppso buffer" herein refers to N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid buffer.

[0492] The term "POPSO buffer" herein refers to piperazine-N,N'-bis(2-hydroxy-propanesulfonic acid) buffer.

[0493] The term "EPPS buffer" herein refers to 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid buffer.

[0494] The term "Tris buffer" herein refers to tris(hydroxymethyl)aminomethane buffer.

[0495] In some embodiments, the buffer systems of methods I, II, III, and IV are independently Bis-Tris buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, TAPSO buffer or MES buffer.

[0496] In some embodiments, the buffer systems of methods I, II, III, and IV are independently Bis-Tris buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer or TAPSO buffer.

[0497] In some embodiments, the buffer system of method V is selected from the group consisting of Bis-Tris buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, TAPSO buffer, PIPES buffer or MES buffer.

[0498] In some embodiments, the buffer system of methods I, II, III, IV and V is BES buffer.

[0499] In some embodiments, the buffer systems in steps (R1) and (R3) are the same. In some embodiments, the buffer systems in steps (R1) and (R3) are different. In some embodiments, the buffer systems in steps (R1) and (R2) are the same. In some embodiments, the buffer systems in steps (R1) and (R2) are different.

[0500] In some embodiments, the pH of the buffer system is 5.8 to 8.0. In some embodiments, the pH of the buffer system is 6.0 to 7.4. In some embodiments, the pH of the buffer system is 6.4 to 7.4. In some embodiments, the pH of the buffer system is 6.7 to 7.4. In some embodiments, the pH of the buffer system is 6.0, 6.2, 6.5, 6.8, 7.0, 7.2 or 7.4.

[0501] The concentration of the buffer system is independently in the range of 10mM to 100mM (mmol / L), optionally the concentration of the buffer system is 20mM to 100mM, 20mM to 80mM, 20mM to 60mM, 20mM to 40mM, 40mM to 80mM, 40mM to 60mM, 30mM to 80mM, 30mM to 60mM, 50mM to 80mM, or 30mM to 70mM.

[0502] In some embodiments, the concentration of the buffer system in steps (R1) and (R3) is the same. In some embodiments, the concentration of the buffer system in steps (R1) and (R3) is different. In some embodiments, the concentration of the buffer system in steps (R1) and (R2) is the same. In some embodiments, the concentration of the buffer system in steps (R1) and (R2) is different.

[0503] In the present invention, metal chelators can capture excessive transition metal ions. There is no specific limitation on metal chelators, as long as the metal chelators can capture excessive transition metal ions and will not affect the reduction of disulfide bonds in the antibody. In certain embodiments, the metal chelator is selected from the group consisting of the following: 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).

[0504] In some embodiments, the metal chelator is selected from EDTA, NTA and DTPA and sodium salts thereof. In some embodiments, the metal chelator in the method is ethylenediaminetetraacetic acid disodium salt (EDTA-2Na).

[0505] In some embodiments, the molar ratio of metal chelator to antibody in step (C1) of method I, II, III or IV or step (C2) of method V 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.

[0506] In some embodiments, the molar ratio of metal chelator to antibody in step (C3) of method I, II, III, IV or V is 1:1 to 100:1, 1:1 to 60:1, 1:1 to 50:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 8:1, 1:1 to 6:1, 1:1 to 5:1, 2:1 to 8:1, 2:1 to 6:1.

[0507] In some embodiments, the method further comprises a purification step after steps (O1), (C1), (C2) and / or (C3).

[0508] In some embodiments, the product prepared by steps (O1), (C1), (C2) and / or (C3) 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 product enrichment (e.g., D2) can be applied using hydrophobic interaction chromatography (HIC). In some embodiments, the product prepared by steps (O1), (C1), (C2) and / or (C3) is purified by a desalting column, size exclusion chromatography, ultrafiltration, dialysis, etc. In some embodiments, the product prepared by steps (O1), (C1), (C2) and / or (C3) is purified by a desalting column.

[0509] In some embodiments, dialysis, ultrafiltration or gel filtration is used to filter out excess metal chelator and the complex of the metal chelator and the transition metal ion.

[0510] In some embodiments, the method further comprises the following steps:

[0511] Introducing a compound containing at least one thiol group to consume the excess first coupling group in step (C1) and / or (C2) and / or the second coupling group in step (C3)

[0512] There is no specific limitation on the compound consuming excess first coupling group and / or second coupling group, as long as the compound contains at least one thiol group. In some embodiments, the compound is cysteine.

[0513] In some embodiments, a linker-payload comprises a covalently linked linker and payload.

[0514] 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.

[0515] The term "linker" herein refers to a molecule containing at least two substituted groups, one of which can be covalently bonded to a drug molecule and the other of which can be covalently coupled to a reactive group of an antibody or a sulfide-bridging agent.

[0516] 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 include a cleavable linker or a non-cleavable linker. The cleavable linker is optionally a chemically cleavable and enzymatically cleavable linker. Due to high plasma stability and good intracellular cleavage selectivity and efficiency, enzymatically cleavable linkers (such as cathepsin B) are widely selected as cleavable linker candidates in antibody drug conjugates (ADCs). In some embodiments, enzyme-unstable linkers include peptide units (-AAs-), -maleimidocaproyl-(-MC-), -maleimidocaproyl-peptide moiety-(-MC-peptide moiety-), -para-aminobenzyl alcohol-(-PAB-). In some embodiments, the peptide unit is a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.

[0517] In some embodiments, without limitation, the dipeptide may be 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). In some embodiments, the dipeptide may be valine-citrulline-(-Val-Cit-), -valine-lysine-(-Val-Lys-), -valine-arginine-(-Val-Arg-), -phenylalanine-citrulline-(-Phe-Cit-), -phenylalanine-lysine-(-Phe-Lys-), and -phenylalanine-arginine-(-Phe-Arg-). Typical enzyme-labile linkers include -Val-Cit- and -Phe-Lys-, which are recognized by cathepsin B.

[0518] In some embodiments, without limitation, the tripeptide can be 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).

[0519] In some embodiments, without limitation, the tetrapeptide can be glycine-glycine-phenylalanine-glycine (GGFG).

[0520] In some embodiments, when the first linker-payload and / or the second linker-payload reacts 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 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 not limited to, MC-GGFG.

[0521] 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.

[0522] 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.

[0523] 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.

[0524] 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:

[0525]

[0526]

[0527] Wherein, in the linker, n is an integer from 0 to 20, and m is an integer from 0 to 20. In some embodiments, in the linker, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0528] In this method, the linker optionally carries an azide group and / or dibenzocyclooctyne (DBCO), allowing the linker to be attached to the antibody.

[0529] The term "blocking agent" herein includes a first blocking agent and a second blocking agent, and refers to a compound that does not carry a drug and contains at least one substituent that can be covalently coupled to an antibody.

[0530] 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.

[0531] As used herein, the term "payload toxin" refers to any cytotoxic molecule and contains at least one substituent group or partial structure for attachment to a linker structure. The payload can kill cancer cells and / or inhibit the growth, proliferation or metastasis of cancer cells, thereby reducing, alleviating or eliminating one or more symptoms of a disease or condition.

[0532] In some embodiments, the payload is a cytotoxic agent, a cytokine, a nucleic acid, a radionuclide, a chemokine, an immune (co)stimulatory molecule, an immunosuppressive molecule, a kinase, a prodrug converting enzyme, a ribonuclease, a growth factor, a hormone, a coagulation factor, a fibrinolytic protein, a peptide that mimics these, and a fragment, fusion protein or derivative thereof.

[0533] In some embodiments, the payload is a cytotoxic molecule, and at least one substituent group or partial structure allows connection to the linker structure. The payload can kill cancer cells and / or inhibit the growth, proliferation or metastasis of cancer cells, thereby reducing, alleviating or eliminating one or more symptoms of the disease or condition. Payloads include, but are not limited to, topoisomerase inhibitors and tubulin inhibitors.

[0534] 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, capmtothecin, 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.

[0535] In some embodiments, the payload is deludecanoic acid (DXd), cyanine 3 (Cy3), MMAE, MMAD or MMAF. In some embodiments of the present invention, the payload is MMAE, DXd or Cy3.

[0536] The linker-payload is a chemical moiety that is synthesized by connecting the linker to the 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 reactive groups for coupling to antibodies by covalent bonds. In the present invention, drug-maleimide complexes (i.e., maleimide-connected drugs) are used as examples of payloads carrying reactive groups. The most common reactive group that can be combined with thiol groups is maleimide. In addition, organic chlorides, bromides, and iodides are also often used.

[0537] The linker-payload can be any physically active compound, or any compound useful for diagnosing, preventing or treating a disease, such as MC-GGFG-DXd, MC-VC-PAB-MMAE, MC-VC-PAB-MMAD, and MC-VC-PAB-MMAF.

[0538] In some embodiments, the first linker-payload toxin and the second linker-payload toxin are the same. In some embodiments, the first linker-payload toxin and the second linker-payload toxin are different.

[0539] The first sulfur bridging agent and the second sulfur bridging agent independently contain at least two substituent groups that allow re-bridging of the thiol group. In some embodiments, the first sulfur bridging agent and the second sulfur bridging agent are independently selected from, but not limited to, the group consisting of:

[0540]

[0541] In some embodiments, the reactive group contains an azide and / or dibenzocyclooctyne (DBCO).

[0542] In some embodiments, the sulfide bridging agent and the reactive group are linked via an alkyl group or polyethylene glycol (PEG).

[0543] In some embodiments, without limitation, the first sulphur bridging reagent carrying a reactive group and the second sulphur bridging reagent carrying a reactive group are independently selected from the group consisting of:

[0544]

[0545]

[0546] Wherein in the first sulfur bridging reagent carrying a reactive group and the second sulfur bridging reagent carrying a reactive group, n is an integer of 0-20, and optionally n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0547] In some embodiments, the first sulfur bridging agent carrying a reactive group and the second sulfur bridging agent carrying a reactive group are dibromomaleimide-PEG4-N3 having the formula

[0548]

[0549] Under appropriate conditions, the reactive group can react with the linker-payload, and the linker-payload is connected to the sulfide bridge reagent by a covalent bond. As the reactive group or linker-payload toxin changes, the reaction product may change. In the present invention, the products of different reactive groups and linkers-payloads are collectively referred to as sulfide bridge reagents carrying linkers-payloads.

[0550] 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:

[0551] QST

[0552] Wherein Q is selected from the group consisting of:

[0553]

[0554] S is selected from, but not limited to, a cleavable linker or a non-cleavable linker, S is selected from the group consisting of:

[0555]

[0556]

[0557] wherein n is 0-20 and m is 0-20; 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.

[0558] T is the effective load.

[0559] In some embodiments, the first sulfide bridging agent carrying the first linker-payload and the second sulfide bridging agent carrying the second linker-payload are independently selected from the group consisting of:

[0560]

[0561] In some embodiments, the first coupling group is a first linker-payload. In some embodiments, the first coupling group is a first sulphur bridging reagent, optionally a first sulphur bridging reagent carrying a reactive group or a first linker-payload.

[0562] In some embodiments, the second coupling group is a second linker-payload. In some embodiments, the second coupling group is a second sulphur bridging agent, optionally a second sulphur bridging agent carrying a reactive group or a second linker-payload.

[0563] In some embodiments, the first coupling group and the second coupling group independently include maleimide-PEG4-N3-DBCO-Cy3, maleimide-VC-PAB-MMAE, or MC-GGFG-DXd.

[0564] In some embodiments, there is no specific limitation on the antibody. According to the antigen associated with the disease, a person skilled in the art can select a suitable antibody that can be used for the bioconjugation process of the present invention. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a monospecific antibody or a multispecific antibody.

[0565] In some embodiments, the antibody is a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding portion thereof.

[0566] The term "human antibody" 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 human antibody definition explicitly excludes humanized antibodies comprising non-human antigen binding residues.

[0567] 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 includes substantially all or at least one, usually two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of non-human antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies. Optionally, a humanized antibody may include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0568] The term "chimeric antibody" 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 rest of the heavy chain and / or light chain is derived from a different source or species.

[0569] In some embodiments, the antibody means an immunoglobulin, and is a molecule containing an antigen binding site that immunospecifically binds to an antigen. In some embodiments of the invention, the antibody is classified as IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments of the invention, the antibody is classified as IgG.

[0570] In some embodiments, the class of the antibody is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody is IgG1 or IgG4.

[0571] In some embodiments, the antibody is wild-type. The term "wild-type" herein refers to naturally occurring and free of mutations.

[0572] In some embodiments, the antibody is an engineered antibody having two amino acid substitutions of two interchain cysteines that form an interchain disulfide bond in the hinge region.

[0573] In some embodiments, the amino acid substitution is selected from the following substitutions: cysteine ​​to alanine, leucine, arginine, lysine, asparagine, methionine, aspartic acid, phenylalanine, proline, glutamine, serine, glutamic acid, threonine, glycine, tryptophan, histidine, tyrosine, isoleucine or valine, respectively.

[0574] In some embodiments, the amino acid substitution is selected from the following substitutions: cysteine ​​to asparagine, glutamine, serine, threonine or tyrosine, respectively.

[0575] In some embodiments, the amino acid substitution is selected from the following: cysteine ​​to serine.

[0576] In some embodiments, 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.

[0577] 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 regulate 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 regulate glycosylation.

[0578] In some embodiments, the one or more mutations are located in the Fc region. In some instances, the Fc region includes mutations at residue positions L234, L235, or a combination thereof. In some instances, the mutations include L234 and L235. In some instances, the mutations include L234A and L235A. In some cases, the residue positions are referenced to IgG1.

[0579] In some embodiments, the Fc region includes a mutation at residue position L234, L235, D265, N21, K46, L52 or P53 or a combination thereof. In some instances, the mutation includes L234 and L235 combined with a mutation at residue position K46, L52 or P53. In some cases, the residue positions are referenced to IgG1.

[0580] In some embodiments, the Fc region includes mutations at L234, L235 and K46. In some cases, the Fc region includes mutations at L234, L235 and L52. In some cases, the Fc region includes mutations at L234, L235 and P53. In some cases, the Fc region includes mutations at D265 and N21. In some cases, this residue position is with reference to IgG1.

[0581] In some instances, the Fc region includes L234A, L235A, D265A, N21G, K46G, L52R or P53G or a combination thereof. In some instances, the Fc region includes L234A and L235A in combination with K46G, L52R or P53G. In some cases, the Fc region includes L234A, L235A and K46G. In some cases, the Fc region includes L234A, L235A and L52R. In some cases, the Fc region includes L234A, L235A and P53G. In some cases, the Fc region includes D265A and N21G. In some cases, the residue position is with reference to IgG1.

[0582] In some embodiments, the Fc region includes a mutation at residue position L233, L234, D264, N20, K45, L51 or P52. In some instances, the Fc region includes a mutation at L233 and L234 combined with a mutation at residue position K45, L51 or P52. In some cases, the Fc region includes a mutation at L233, L234 and K45. In some cases, the Fc region includes a mutation at L233, L234 and L51. In some cases, the Fc region includes a mutation at L233, L234 and K45. In some cases, the Fc region includes a mutation at L233, L234 and P52. In some instances, the Fc region includes a mutation at D264 and N20. In some cases, positions equivalent to residues L233, L234, D264, N20, K45, L51, or P52 in an IgG1, IgG2, IgG3, or IgG4 framework are contemplated.

[0583] In some embodiments, the Fc region includes L233A, L234A, D264A, N20G, K45G, L51R, or P52G. In some instances, the Fc region includes L233A and L234A. In some instances, the Fc region includes L233A and L234A in combination with K45G, L51R, or P52G. In some cases, the Fc region includes L233A, L234A, and K45G. In some cases, the Fc region includes L233A, L234A, and L51R. In some cases, the Fc region includes L233A, L234A, and K45G. In some cases, the Fc region includes L233A, L234A, and P52G. In some instances, the Fc region includes D264A and N20G. In some cases, the residue position is with reference to IgG1.

[0584] 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, 276(12):1541-155; 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.

[0585] In some embodiments, the antibody of IgG1, IgG2, IgG3 or IgG4 is a human antibody or a humanized antibody. Information of IgG1, IgG2, IgG3 or IgG4 can be obtained at NCBI or UniProt (https: / / www.uniprot.org / ).

[0586] In some embodiments of the present invention, the antibody is a bispecific antibody. In some embodiments of the present invention, the antibody is an IgG1-like bispecific antibody.

[0587] In some embodiments of the present invention, a person 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 a knob-in-holes technology (Ridgway JBB, Presta LG, Paul C. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization (heavy chain heterodimerization antibody CH3 domain "knobs-in-holes" engineering) [J]. "Protein Engineering" (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 multiplespecificities [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 (using CrossMAb technology to generate bispecific and multispecific antibodies) [J]. "Antibodies" (mAbs), 2016, 8 (6), P1010-P1020.) or a combination thereof.

[0588] The term "knob-to-hole structure" herein is used in its broadest sense and covers 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 in the CH3 domains between two antibody heavy chains: i) in the CH3 domain of one heavy chain (the first CH3 domain), one amino acid residue is substituted by 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 by another amino acid residue carrying a smaller side chain, thereby generating a cavity ("hole") within the interface of the second CH3 domain, into which the protrusion ("knob") in the first CH3 domain can be placed.

[0589] 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.

[0590] In some embodiments, the antibody can be a target-specific antibody. In some embodiments of the present invention, without limitation, the antibody can be an anti-HER2 antibody, an anti-FAP antibody, an anti-OX-40 antibody, an anti-41BB antibody, an anti-angiopoietin-2 antibody, an anti-ant-IL-4Rα antibody, an anti-BCMA antibody, an anti-Blys antibody, an anti-BTNO2 antibody, an anti-C5 antibody, an anti-CD122 antibody, an anti-CD13 antibody, an anti-CD133 antibody, an anti-CD137 antibody, an anti-CD138 antibody, an anti-CD16a antibody, an anti- CD19 antibody, anti-CD20 antibody, anti-CD22 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 antibody, anti-GP130 antibody, anti-Her3 antibody, anti-HSG antibody, anti-ICOS antibody, anti-IGF1 antibody, anti-IGF1 / 2 antibody, anti-IGF-1R antibody, anti-IGF2 antibody, anti-IGFR antibody, anti-IL-1 antibody, anti-IL-12 antibody, anti-IL-12p40 antibody, anti-IL-13 antibody, anti-IL-17A antibody, anti-IL-1β antibody, anti-IL-23 antibody, anti-IL-5 antibody, anti-IL-6 antibody, anti-IL-6R antibody, anti-Lag-3 antibody body, anti-LAG3 antibody, anti-MAG antibody, anti-Met antibody, anti-NgR antibody, anti-NogoA antibody, anti-OMGp antibody, anti-OX40 antibody, anti-PD-1 antibody, anti-PDGFR antibody, anti-PDL-1 antibody, anti-PSMA antibody, anti-RGMA antibody, anti-RGMB 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.

[0591] In some embodiments, the antibodies are target specific, targeting HER2 (human epidermal growth factor receptor 2), TROP2 (TACSTD2, tumor-associated calcium signal transducer 2), BCMA (TNFRSF17, TNF receptor superfamily member 17).

[0592] In some embodiments, the antibody is trastuzumab, gosartuzumab, or mabelantuzumab.

[0593] In some embodiments of the present invention, the antibody can be purchased or produced by any method known to those skilled in the art.

[0594] Modified Antibodies

[0595] The present invention provides a modified antibody prepared by the above method.

[0596] In some embodiments, the modified antibody is conjugated to one, two, or three of the conjugating groups.

[0597] In some embodiments, the modified antibody is an antibody drug conjugate (ADC) of the type D1, D2, D3, D4, D6, D1+D3, D1+D6, D1+D2, D1+D4, D2+D3, D2+D6, D0+D3, D0+D6, D3+D1, D3+D2, D6+D2, D6+D1, D0+D1, D0+D3, D4+D1 or D4+D2.

[0598] In some embodiments, the ADC is trastuzumab-[MC-VC-PAB-MMAE]2, gosartuzumab-[MC-VC-PAB-MMAE]2, mabelantuzumab-[MC-VC-PAB-MMAE]2, trastuzumab-[MC-GGFG-DXd]2, trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1, trastuzumab-[MC-VC-PAB-MMAE]6, gosartuzumab-[MC-VC-PAB-MMAE]6, mabelantuzumab-[MC-VC-PAB-MMAE]6, trastuzumab-[MC-VC-PAB-MMAE]6[MC-GGFG-DXd]2, trastuzumab-[MC-VC-PAB-MMAE]6[maleimide-PEG4-N3-DBCO-Cy3]1, trastuzumab-[MC-VC-PAB-MMAE]6, Trastuzumab-[maleimide]3[MC-VC-PAB-MMAE]2, trastuzumab-[maleimide]3[maleimide-PEG4-N3-DBCO-Cy3]1, trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]6, trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[ MC-VC-PAB-MMAE]2, trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4, trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4, trastuzumab-[MC-VC-MMAE]4 or trastuzumab-[MC-VC-MMAE]4[MV-GGFG DXd]2.

[0599] In some embodiments, an antibody with site-specific modification (D6-type ADC) is prepared by method II comprising steps (R1) and (C1), wherein the first coupling group is a first linker-payload.

[0600] In some embodiments, an antibody with site-specific modification (D3-type ADC) is prepared by method II comprising steps (R1) and (C1), wherein the first coupling group is a first sulfide bridging reagent carrying a first linker-payload, or the first coupling group is a sulfide bridging reagent carrying a reactive group that reacts with the first linker-payload.

[0601] In some embodiments, an antibody with site-specific modification (D2-type ADC) is prepared by method I comprising steps (R1) and (C1), wherein the first coupling group is a first linker-payload.

[0602] In some embodiments, an antibody with site-specific modification (D1-type ADC) is prepared by method I comprising steps (R1) and (C1), wherein the first coupling group is a first sulfide bridging agent carrying a first linker-payload, or the first coupling group is a sulfide bridging agent carrying a reactive group that reacts with the first linker-payload.

[0603] In some embodiments, an antibody with site-specific modification (D2-type ADC) is prepared by method III comprising steps (R1), (O1) and (C1), wherein the first coupling group is a first linker-payload.

[0604] In some embodiments, an antibody with site-specific modification (D1-type ADC) is prepared by method III comprising steps (R1), (O1) and (C1), wherein the first coupling group is a first sulfide bridging agent carrying a first linker-payload, or the first coupling group is a sulfide bridging agent carrying a reactive group that reacts with the first linker-payload.

[0605] In some embodiments, an antibody with site-specific modification (D4-type ADC) is prepared by method IV comprising steps (R1), (O1), (R2), and (C1), wherein the first coupling group is a first linker-payload.

[0606] In some embodiments, an antibody with site-specific modification (D4-type ADC) is prepared by method V comprising steps (R1) and (C2), wherein the first coupling group is a first linker-payload.

[0607] In some embodiments, an antibody with site-specific modification (D2+D6 type ADC) is prepared by method I or method III (which also includes steps (R3) and (C3)), wherein the first coupling group and the second coupling group are linker-payload.

[0608] In some embodiments, an antibody with site-specific modification (D2+D3 type ADC) is prepared by method I or method III (which also includes steps (R3) and (C3)), wherein the first coupling group is a first linker-payload, and the second coupling group is a second sulphur bridging agent carrying a second linker-payload, or the second coupling group is a second sulphur bridging agent carrying a reactive group that reacts with the second linker-payload.

[0609] In some embodiments, an antibody with site-specific modification (D1+D6 type ADC) is prepared by method I or method III (which also includes steps (R3) and (C3)), wherein the first coupling group is a first sulphur bridging agent carrying a first linker-payload, or the first coupling group is a first sulphur bridging agent carrying a reactive group that reacts with the first linker-payload, and the second coupling group is a second linker-payload.

[0610] In some embodiments, an antibody with site-specific modification (D1+D3 type ADC) is prepared by method I or method III (which also includes steps (R3) and (C3)), wherein the first coupling group is a first sulfur bridge reagent carrying a first linker-payload, or the first coupling group is a first sulfur bridge reagent carrying a reactive group that reacts with the first linker-payload, and the second coupling group is a second sulfur bridge reagent carrying a second linker-payload, or the second coupling group is a second sulfur bridge reagent carrying a reactive group that reacts with the second linker-payload.

[0611] In some embodiments, an antibody with site-specific modification (D0+D6 type ADC) is prepared by method I or method III (which also includes steps (R3) and (C3)), wherein the first coupling group is a first sulfide bridge reagent or a first capping reagent, and the second coupling group is a second linker-payload.

[0612] In some embodiments, an antibody with site-specific modification (D0+D3 type ADC) is prepared by method I or method III (which also includes steps (R3) and (C3)), wherein the first coupling group is a first sulfide bridging agent or a first capping agent, and the second coupling group is a second sulfide bridging agent carrying a second linker-payload, or the second coupling group is a second sulfide bridging agent carrying a reactive group that reacts with a second linker-payload.

[0613] In some embodiments, an antibody with site-specific modification (D2+D2 type ADC, D2+D4 type ADC) is prepared by method I or method III (which further comprises steps (R3) and (C3) and a transition metal ion is introduced in step (R3)), wherein the first coupling group and the second coupling group are linker-payload.

[0614] In some embodiments, an antibody with site-specific modification (D1+D2, D1+D4 type ADC) is prepared by method I or method III (which also includes steps (R3) and (C3) and introducing a transition metal ion in step (R3)), wherein one coupling group is a first sulfur bridge reagent carrying a first linker-payload, or the first coupling group is a first sulfur bridge reagent carrying a reactive group that reacts with the first linker-payload, and the second coupling group is a second linker-payload.

[0615] In some embodiments, an antibody with site-specific modification (D6+D2 type ADC) is prepared by method II (which also includes steps (R3) and (C3)), wherein the first coupling group and the second coupling group are linker-payload.

[0616] In some embodiments, an antibody with site-specific modification (D6+D1 type ADC) is prepared by method II (which also includes steps (R3) and (C3)), wherein the first coupling group is a first linker-payload, and the second coupling group is a second sulfur bridging reagent carrying a second linker-payload, or the second coupling group is a second sulfur bridging reagent carrying a reactive group that reacts with the second linker-payload.

[0617] In some embodiments, an antibody with site-specific modification (D3+D2 type ADC) is prepared by method II (which also includes steps (R3) and (C3)), wherein the first coupling group is a first sulphur bridging agent carrying a first linker-payload, or the first coupling group is a first sulphur bridging agent carrying a reactive group that reacts with the first linker-payload, and the second coupling group is a second linker-payload.

[0618] In some embodiments, an antibody with site-specific modification (D3+D1 type ADC) is prepared by method II (which also includes steps (R3) and (C3)), wherein the first coupling group is a first sulfur bridge reagent carrying a first linker-payload, or the first coupling group is a first sulfur bridge reagent carrying a reactive group that reacts with the first linker-payload, and the second coupling group is a second sulfur bridge reagent carrying a second linker-payload, or the second coupling group is a second sulfur bridge reagent carrying a reactive group that reacts with the second linker-payload.

[0619] In some embodiments, an antibody with site-specific modification (D0+D2 type ADC) is prepared by method II (which also includes steps (R3) and (C3)), wherein the first coupling group is a first sulfide bridge reagent or a first capping reagent, and the second coupling group is a second linker-payload.

[0620] In some embodiments, an antibody with site-specific modification (D0+D3 type ADC) is prepared by method II (which also includes steps (R3) and (C3)), wherein the first coupling group is a first sulfide bridging agent or a capping agent, and the second coupling group is a second sulfide bridging agent carrying a second linker-payload, or the second coupling group is a second sulfide bridging agent carrying a reactive group that reacts with the second linker-payload.

[0621] In some embodiments, an antibody with site-specific modification (D4+D4 type ADC) is prepared by method IV or method V (which also includes steps (R3) and (C3)), wherein the first coupling group and the second coupling group are linker-payload.

[0622] In some embodiments, an antibody with site-specific modification (D4+D2 type ADC) is prepared by method IV or method V (which further comprises steps (R3) and (C3) and introducing a transition metal ion in step (R3)), wherein the first coupling group and the second coupling group are linker-payload.

[0623] A variety of analytical methods can be used to determine the yield and isomeric mixture of antibodies with site-specific modifications. In some embodiments of the invention, the analytical method is HIC-HPLC (hydrophobic interaction chromatography-high performance liquid chromatography). HIC-HPLC can separate antibodies loaded with different numbers of connexons-payload toxins. The drug loading of the payload toxin can be determined according to the ratio of absorbance, for example, at 250nm and 280nm. For example, if the payload toxin (e.g., drug) can absorb at 250nm, and the antibody absorbs at 280nm, therefore, the ratio of 250 / 280 will increase with the increase of drug loading.

[0624] The method of producing antibodies with site-specific modifications avoids any need for protein engineering or enzyme catalysis, but is based on natural interchain disulfide bonds, requiring only reducing agents and transition metal ions. Therefore, the method disclosed herein has lower complexity, and the homogeneity of the resulting antibodies with site-specific modifications (antibody drug conjugates) is significantly improved.

[0625] In some embodiments, the homogeneity of the D6-type ADC is at least up to 50% of the total weight of the combination of D0, D2, D4, D6 and D8. In some embodiments, the homogeneity of the D6-type ADC is up to 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 91%, 92%, 93%, 94% or 95%.

[0626] In some embodiments, the homogeneity of the D2-type ADC is at least up to 50% of the total weight of the combination of D0, D2, D4, D6 and D8. In some embodiments, the homogeneity of the D2-type ADC is up to 55%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% of the total weight of the combination of D0, D2, D4, D6 and D8.

[0627] In some embodiments, the uniformity of the D1-type ADC is at least 65%. In some embodiments, the uniformity of the D1-type ADC is 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 90%.

[0628] In some embodiments, the D3-type ADC has a uniformity of at least 75%. In some embodiments, the D3-type ADC has a uniformity of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 85%, or 87%.

[0629] In some embodiments, the uniformity of the D2+D6 ADC is at least 65%. In some embodiments, the uniformity of the D2+D6 ADC is 75%, 76%, 77%, 78%, 79%, 80%, 81%, 90%.

[0630] In some embodiments, the uniformity of the D0+D6 ADC is at least 65%. In some embodiments, the uniformity of the D0+D6 ADC is 75%, 76%, 77%, 78%, 79%, 80%, 81%, 84% or 86%.

[0631] In some embodiments, the uniformity of the D6+D2 type ADC is at least 65%. In some embodiments, the uniformity of the ADC comprising D6+D2 is up to 75%, 76%, 77%, 78%, 79%, 80%, 81%, 84%, 86%, or 88%.

[0632] In some embodiments, the uniformity of the D6+D1 type ADC is at least 65%. In some embodiments, the uniformity of the ADC comprising D6+D1 is up to 75%, 76%, 77%, 78%, 79%, 80%, 81%, 84%, 86%, or 90%.

[0633] In some embodiments, the uniformity of the D1+D2 type ADC is at least up to 65%. In some embodiments, the uniformity of the ADC comprising D1+D2 is up to 66%, 68%, 69%, 70%, 73% or 75%.

[0634] In some embodiments, the uniformity of the D1+D4 type ADC is at least 60%. In some embodiments, the uniformity of the ADC comprising D1+D4 is up to 62%, 65%, 67%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 84% or 86%.

[0635] In some embodiments, the uniformity of the D2+D4 type ADC is at least 65%. In some embodiments, the uniformity of the ADC comprising D2+D4 is up to 66%, 68%, 69%, 70%, 73%, 75%, 78%, 80%, 82%, or 84%.

[0636] In some embodiments, the homogeneity of the D4-type ADC is at least up to 70%. In some embodiments, the homogeneity of the ADC comprising D4 is up to 75%, 80%, 85%, 90% or 95%.

[0637] In some embodiments, the uniformity of the D4+D2 type ADC is at least as high as 65%. In some embodiments, the uniformity of the ADC comprising D4+D2 is as high as 68% or 70%.

[0638] The method of the present invention is compatible with current thiol-reactive linker drug technology, with minimal conformational changes and intact Fc function. At the same time, the method is simple to operate and has lower cost. The method provided herein is simple to operate, and it is fully compatible with current thiol-reactive linker drug technology.

[0639] Pharmaceutical composition

[0640] The present invention also provides a pharmaceutical composition comprising the modified antibody prepared by the above method and at least one pharmaceutically acceptable ingredient.

[0641] In some embodiments, the modified antibody is an antibody with a site-specific modification. In some embodiments, the modified antibody is an ADC.

[0642] The term "pharmaceutically acceptable" herein indicates that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients making up the formulation and physiologically compatible with the recipient thereof.

[0643] The term "pharmaceutically acceptable ingredient" as used herein refers to substances useful for the preparation or use of a pharmaceutical composition, and includes, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffers, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegrants, lubricants, wetting agents, sweeteners, flavorings, dyes, and combinations thereof, as known to those skilled in the art (see, for example, Remington The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2013, pp. 1049-1070).

[0644] The pharmaceutical compositions provided herein can be formulated in any manner known in the art, such as, the pharmaceutical compositions provided herein can be formulated in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound to facilitate administration and uniformity of dosage) for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal) administration.

[0645] A pharmaceutical composition is formulated to be compatible with its intended route of administration (eg, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal administration).

[0646] Pharmaceutically acceptable ingredients used in the pharmaceutical compositions disclosed herein may 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 agents or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0647] Suitable components can include, for example, antioxidants, fillers, adhesives, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers (such as sugar and cyclodextrin). 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 the antibody or its Fab provided herein and conjugate, one or more antioxidants (such as methionine) are included to reduce the oxidation of the 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 maximum extent. Therefore, in certain embodiments, a pharmaceutical composition is provided, which includes one or more antibodies or its Fab disclosed herein and one or more antioxidants, such as methionine.

[0648] 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) for mixing with solvents before use, including subcutaneous injection tablets, sterile suspensions for injection, sterile insoluble dry products for combination with solvents before use, and sterile and / or non-pyrogenic emulsions. The solution can be aqueous or non-aqueous.

[0649] In some embodiments, a pH adjuster is present in the pharmaceutical composition, and the pH adjuster is sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, boric acid, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid, etc.

[0650] In some embodiments, there is a stabilizer in the pharmaceutical composition, which is disodium edetate, calcium disodium edetate, dipotassium edetate, diamine edetate, α-lipoic acid, ethylene glycol dimethacrylate, sodium oleate, anhydrous sodium sulfite, sodium ascorbate, deferoxamine, malate, citric acid, succinate, sodium salt of malate, calcium salt, magnesium salt, citric acid, succinate, etc.

[0651] In some embodiments, the pharmaceutical composition is combined with other therapeutic agents. There is no specific limitation on 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 are anticancer agents, anti-autoimmune disease agents, antiemetics, antiallergic agents, etc.

[0652] In some embodiments, the anticancer agent may include, but is not limited to, erlotinib, bortezomib, fulvestrant, sunitinib mesylate imatinib, letrozole, finasunate, platinums (such as oxaliplatin, carboplatin and cisplatin), finasunate, fluorouracil, rapamycin, folinic acid, lapatinib, lonafarnib, sorafenib, gefitinib, capmtothecin, topotecan, bryostatin, adezelesin, anthracyclines, kazelesin, bisoprolol, dolastatin, auristatin, beta-blockers, bisoprolol ... Carcinomycin, 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.).

[0653] The present invention provides use of the modified antibody described herein or the pharmaceutical composition described herein in preparing a therapeutic agent for preventing, diagnosing or treating a disease.

[0654] In some embodiments, the modified antibody is an antibody with a site-specific modification. In some embodiments, the modified antibody is an ADC.

[0655] The term "treatment" as used herein for any disease refers to alleviating or ameliorating the disease (i.e., slowing or stopping 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, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination of the above effects. For tumors, "treatment" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, delaying tumor development, or a combination of these means.

[0656] 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.

[0657] In some embodiments, the disease is a tumor or cancer. In some embodiments, the disease is an autoimmune disease, etc.

[0658] In some embodiments, cancer can include, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphatic malignancies. 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.

[0659] In some embodiments, the pharmaceutical composition is combined with other therapeutic agents. There is no specific limitation on 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 are anticancer agents, anti-autoimmune disease agents, antiemetics, antiallergic agents, etc.

[0660] In some embodiments, anticancer agents include, but are not limited to, erlotinib, bortezomib, fulvestrant, sunitinib mesylate imatinib, letrozole, finasunate, platinums (such as oxaliplatin, carboplatin and cisplatin), finasunate, fluorouracil, rapamycin, folinic acid, lapatinib, lonafarnib, sorafenib, gefitinib, capmtothecin, topotecan, bryostatin, adezelesin, anthracyclines, kazelesin, bisoprolol, dolastatin, auristatin, bisoprolol , 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.).

[0661] In some embodiments, anti-autoimmune disease agents include, but are not limited to, ibuprofen, loxoprofen, naproxen, diclofenac, indomethacin, meloxicam, lornoxicam, nabumetone, celecoxib, acetaminophen, glucocorticoids, azathioprine, cyclophosphamide, and the like.

[0662] In some embodiments, during and after administration of the ADC of the present invention, the patient may feel nauseous. 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, etc.

[0663] In some embodiments, during and after administration of the ADC of the present invention, the patient may experience an allergic reaction. Therefore, antiallergic agents can be used to minimize the risk of allergic reactions. Antiallergic agents include dexamethasone, beclomethasone, hydrocortisone, prednisolone, prednisone, methylprednisolone, hydroxyzine, cyproheptadine, bronchodilators, terbutaline, etc.

[0664] The present invention provides a method for preventing, diagnosing or treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the modified antibody prepared by the above method.

[0665] In some embodiments, the modified antibody is an antibody with a site-specific modification. In some embodiments, the modified antibody is an ADC.

[0666] 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.

[0667] As used herein, the term "therapeutically effective amount" refers to the dosage of the ADC in the present application that causes a biological or medical response in a subject, such as improving symptoms, alleviating the condition, slowing or delaying the progression of a disease, or preventing a disease, etc. 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 according to 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 used. In some embodiments of the present invention, the therapeutically effective amount can vary greatly, 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.

[0668] It will be apparent to those skilled in the art that other suitable modifications and alterations to the invention described herein are 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 for illustrative purposes 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.

[0669] Example

[0670] The invention is further described in the following examples, which are not intended to limit the scope of the invention described in the claims.

[0671] Reagents and manufacturers

[0672] Trastuzumab is commercially available from Roche.

[0673] Gosartuzumab and mabelantuzumab are commercially available from MedChemExpress.

[0674] MC-VC-PAB-MMAE can be purchased from Levena biopharma.

[0675] MC-GGFG-DXd can be purchased from Levena biopharma.

[0676] Desalting columns (type: 40K, 0.5 mL, reference number: 87766, batch number: SJ251704) can be purchased from ThermoScientific.

[0677] DBCO-Cy3 can be purchased from Confluore.

[0678] TCEP is commercially available from Bidepharm.

[0679] Dibromomaleimide, EDTA, and cysteine ​​can be purchased from Aladdin.

[0680] Azido-PEG4-amine can be purchased from Xi'an Kangfuno Biotechnology Co., Ltd.

[0681] The reagents used in the examples are commercially available, including but not limited to 1-hydroxybenzotriazole (HOBT), dimethylacetamide (DMA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIPEA), ethyl acetate (EtOAc), N,N-dimethylformamide (DMF), bicyclic amidine (DBU), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI), trifluoroacetic acid (TFA), dichloromethane (DCM), and tert-butyldiphenylsilyl chloride (TBDPSCl).

[0682] Synthetic Procedure A of Compounds According to the Invention

[0683] Under N2 atmosphere, HATU (190 mg, 0.5 mmol, 1 eq) was added to a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL), followed by DIPEA (2.0 mmol, 4.0 eq). The mixture was stirred for 30 min, and an amine reagent (0.5 mmol, depending on the structure of the compound) was added. The reactants were stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the desired product.

[0684] Synthesis Procedure A-1 of Compounds According to the Invention

[0685] The product prepared by synthesis procedure A was dissolved in DCM (3 mL) and TFA (0.3 mL) was added. The mixture was stirred for 1 h and LCMS showed that the reaction was complete. The mixture was concentrated and the residue was dissolved in distilled water and washed twice with EtOAc. The water layer was freeze-dried to obtain the corresponding product.

[0686] Synthetic Procedure B for Compounds According to the Invention

[0687] Under N2 atmosphere, HOBt (67.5 mg, 0.5 mmol, 1 eq) and EDCI (95.5 mg, 0.5 mmol, 1 eq) were added to a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL), followed by DIPEA (2.0 mmol, 4.0 eq). The mixture was stirred for 30 min, and an amine reagent (0.5 mmol, depending on the structure of the compound) was added. The reactant was stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 chromatographic column to give the corresponding product.

[0688] Synthesis Procedure B-1 of Compounds According to the Invention

[0689] The product prepared by synthesis procedure B was dissolved in DCM (3 mL) and TFA (0.3 mL) was added. The mixture was stirred for 1 h and LCMS showed that the reaction was complete. The mixture was concentrated and the residue was dissolved in distilled water and washed twice with EtOAc. The water layer was freeze-dried to obtain the corresponding product.

[0690] Homogeneity Determination I

[0691] Drug / antibody ratio (DAR) and product distribution were analyzed by HIC-HPLC (Agilent 1200) in combination with a TSK gel butyl-NPR column (4.6 mM IDX 3.5 cm) (available from Tosoh Biosciences) at 30°C with a flow rate of 0.5 mL / min. Solvent A was 1.5 M (NH4)2SO4 and 50 mM potassium phosphate pH 7. Solvent B was 75% v / v 50 mM potassium phosphate (pH 7) and 25% v / v isopropanol. The flushing procedure was as follows:

[0692] 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

[0693] Homogeneity Determination I was carried out using the method of Examples I.1-I.139 and Comparative Examples I.1-I.22.

[0694] Homogeneity determination II:

[0695] HIC-HPLC (Agilent 1200) was used to analyze the distribution of ADC at 25°C 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. 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:

[0696] 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

[0697] The homogeneity determination II was performed using the method of Examples II.1-II.153, Comparative Examples II.1-II.11, Examples III.1-III.107, Comparative Examples III.1-III.5, Examples V.1-V.47 and Comparative Example V.1.

[0698] AKTA using HIC chromatography:

[0699] A polar MC30-HIC butyl column (4.2 mL, ADC purification was performed using an AKTA explorer (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 volume ratio of 1:1, 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 ultracentrifugation.

[0700] Example 1: Synthesis of TCEP-NO-Trtyl and TCEP-NO

[0701]

[0702] Compound 1 O-triethylhydroxylamine (NH2-O-Trt, 176 μmol, 48.5 mg, 1 eq.), EDC (176 μmol, 33.7 mg, 1 eq.) and HOBt (352 μmol, 53.8 mg, 2 eq.) were dissolved in 1.5 mL of (degassed) DMF under an inert atmosphere. The solution was added to TCEP (528 μmol, 150 mg, 3 eq.) dissolved in 1.5 mL of degassed DMF containing DIPEA (704 μmol, 123 μL, 4 eq.) under an inert atmosphere. The reactants were stirred at room temperature for 60 min. Afterwards, DMF was removed in vacuo, and the residue was added to CH3COOH (0.1 N, 5 mL) and EtOAc (2 mL). The resulting mixture was stirred for 5 minutes and filtered to give Compound 2 (TCEP-NO-Trtyl) (61.2 mg, 12.2%) as a white solid. For compound 2 (also known as TCEP-NO-Trtyl or TCEP-19-int1), MS [MH] - =506.2, C 28 H 30 The exact mass of NO6P was calculated to be 507.18. 1 H NMR (400MHz, DMSO-d6): δ7.40-7.15(m,15H),2.48-2.38(m,4H),2.22(s,1H),2.12-1.52(m,7H).

[0703] Without further purification, EtOAc (2 mL), 5% TFA and 5% TIPS were added to compound 2. The reaction was continued for another 2 h. H2O (5 mL) and EtOAc (10 mL) were then introduced. The aqueous phase was further washed twice with EtOAc (10 mL) and concentrated to give TCEP-NO (13 mg). For TCEP-NO, MS [MH] - =263.94,C9H 16 The exact mass of NO6P was calculated to be 265.07. 1 H-NMR (400 MHz, deuterium oxide): δ 2.93-2.85 (m, 4H), 2.73-2.56 (m, 8H).

[0704] Example 2: Synthesis of TCEP-3NO

[0705]

[0706] Compound 1 o-tritylhydroxylamine (NH2-O-Trt, 528 μmol, 145.5 mg, 3 eq.), EDC (528 μmol, 101 mg, 3 eq.) and HOBt (880 μmol, 134.5 mg, 5 eq.) were dissolved in 4 mL of DMF (degassed) under an inert atmosphere. The solution was added to TCEP (176 μmol, 50 mg, 1 eq.) dissolved in 4 mL of degassed DMF containing DIPEA (704 μmol, 123 μl, 4 eq.) under an inert atmosphere. The reaction was stirred at room temperature for 60 min. Afterwards, the DMF was removed in vacuo and the residue was added to CH3COOH (0.1 N, 5 mL) and EtOAc (5 mL). The resulting mixture was stirred for 5 minutes and filtered to give compound 3 in the form of a white solid, to which EtOAc (2 mL), 5% TFA and 5% TIPS were added without further purification. The reaction continued for another 2 h. H2O (5 mL) and EtOAc (10 mL) were then introduced. The aqueous phase was further washed twice with EtOAc (10 mL) and concentrated to give TCEP-NO (32 mg). For TCEP-3NO, MS [M+H] + =295.87, C9H 18 The exact mass calculated for N3O6P is 295.09. 1 H NMR (400 MHz, deuterium oxide): δ 3.08–2.38 (m, 10H), 2.24-2.17 (m, 5.6 Hz, 2H).

[0707] Example 3: Synthesis of TCEP-CO

[0708]

[0709] Compound 4 (176 μmol, 43.2 mg, 1 equivalent), EDC (176 μmol, 33.7 mg, 1 equivalent) and HOBt (352 μmol, 53.8 mg, 2 equivalents) were dissolved in 1.5 mL of DMF (degassed) under an inert atmosphere. Under an inert atmosphere, the solution was added to TCEP (528 μmol, 150 mg, 3 equivalents) dissolved in 1.5 mL of degassed DMF containing DIPEA (704 μmol, 123 μL, 4 equivalents). The reactant was stirred at room temperature for 60 min. Afterwards, DMF was removed in a vacuum, and the residue was added to CHCOOH (0.1 N, 5 mL) and EtOAc (2 mL). The resulting mixture was stirred for 5 minutes and filtered to obtain compound 5 in the form of a white solid, to which EtOAc (2 mL), 5% TFA and 5% TIPS were added without further purification. The reaction continued for another 2 h. Then H2O (5 mL) and EtOAc (10 mL) were introduced. The aqueous phase was further washed twice with EtOAc (10 mL) and concentrated to give TCEP-CO (8 mg). For TCEP-CO, MS [MH] - =363.86, C9H 16 The exact mass of NO6P was calculated to be 365.09. 1 H NMR (400 MHz, deuterium oxide): δ 4.28 (s, 2H), 4.13 (s, 2H), 2.97 (dt, J = 20.0, 6.4 Hz, 2H), 2.83 (dt, J = 18.3, 6.9 Hz, 4H), 2.57-2.48 (m, 6H).

[0710] Example 4: Synthesis of TCEPA

[0711]

[0712] Under an inert atmosphere, triethylamine (176 μmol, 45.6 mg, 1 eq.), EDC (176 μmol, 33.7 mg, 1 eq.) and HOBt (352 μmol, 53.8 mg, 2 eq.) were dissolved in 1.5 mL of (degassed) DMF. Under an inert atmosphere, this solution was added to TCEP (528 μmol, 150 mg, 3 eq.) dissolved in 1.5 mL of degassed DMF containing DIPEA (704 μmol, 123 μL, 4 eq.). The reactant was stirred at room temperature for 60 min. Afterwards, DMF was removed in vacuo, and the residue was added to CHCOOH (0.1 N, 5 mL) and EtOAc (20 mL). The resulting mixture was washed three times with H2O, the organic phase was concentrated, and then precipitated with petroleum ether to obtain TCEPA-Trt in the form of a white solid, to which EtOAc (2 mL), 5% TFA and 5% TIPS were added without further purification. The reaction was continued for another 2 h. H2O (5 mL) and EtOAc (10 mL) were then introduced. The aqueous phase was further washed twice with EtOAc (10 mL) and concentrated to obtain TCEPA (8 mg). MS [M+H] + =250.18,C9H 16 The exact mass of NO5P was calculated to be 249.08. 1 H NMR (400 MHz, deuterium oxide) δ 2.85-2.70 (m, 4H), 2.61-2.43 (m, 6H), 2.15-2.06 (m, 2H).

[0713] Example 5: Synthesis of TCEP-1

[0714]

[0715] 1.TCEP-1-int2

[0716] Under N2 atmosphere, HOBt (1.64 g, 12.0 mmol, 1.2 equiv.) and EDCI (2.32 g, 12 mmol, 1.2 equiv.) were added to a DMF (30 mL) solution of TCEP-1-int1 (3.0 g, 10.0 mmol, 1.0 equiv., Fmoc-glycine, Bidepharm), followed by DIPEA (4.4 mL, 25.2 mmol, 2.5 equiv.). The mixture was stirred for 30 min, and compound 1 triphenylmethylhydroxylamine (2.78 g, 10 mmol, 1.0 equiv., Bidepharm) was added. The reaction mixture was stirred at room temperature for 1 h, and poured into ice water. The precipitate was collected by filtration and washed with water. The filter cake was dried under vacuum to give a crude product TCEP-1-int2 (4.5 g, 80% yield, white solid), which was used directly in the next step without further purification.

[0717] 2.TCEP-1-int3

[0718] DBU (5 mL) was added to a solution of TCEP-1-int2 (4.5 g, 8.1 mmol, 1.0 equivalent) in DMF (25 mL). The resulting mixture was stirred at room temperature for 0.5 h, and LCMS showed that the reaction was complete. The mixture was poured into ice water and extracted with EtOAc, dried in vacuo, and purified with a flash tower (EtOAc / petroleum ether=0-50%) to give the product TCEP-1-int3 (2.0 g, yield 77%, white solid).

[0719] 3.TCEP-1

[0720] TCEP-1 was synthesized in the synthetic procedure B-1, wherein TCEP-1-int3 was an amine reagent, to obtain TCEP-1 (45.1 mg, 28%) as a white solid. MS [MH] - =321.15, C 11 H 19 The exact mass of N2O7P was calculated to be 322.25. 1 H-NMR (400 MHz, deuterium oxide): δ 3.99 (s, 0.64H), 3.87 (s, 1.34H), 2.96–2.81 (m, 6H), 2.63-2.56 (m, 6H).

[0721] Example 6: Synthesis of TCEP-2

[0722]

[0723] TCEP-2 was synthesized according to the synthetic procedure B-1, wherein compound 5 (t-butyl glycine, Bidex Pharmaceuticals) was used as an amine reagent to obtain TCEP-2 (52.3 mg, 34%) in the form of a white solid. MS [MH] - =306.18, C 11 H 18 The exact mass of NO7P was calculated to be 307.24. 1 H NMR (400 MHz, deuterium oxide): δ 3.99 (s, 2H), 3.00–2.76 (m, 6H), 2.60 (dtd, J=14.0, 7.0, 3.8 Hz, 6H).

[0724] Example 7: Synthesis of TCEP-3

[0725]

[0726] TCEP-3 was synthesized in Procedure B, where compound 6 (DL-phenylalanine, Adamas) was used as an amine reagent to obtain TCEP-3 (65.0 mg, 33%) as a white solid. MS [MH] - =396.24, C 18 H 24 The exact mass of NO7P was calculated to be 397.13. 1 H NMR (400 MHz, deuterium oxide): δ 7.43-7.25 (m, 5H), 4.71 (dd, J = 10.3, 4.8 Hz, 1H), 3.31 (dd, J = 13.9, 4.8 Hz, 1H), 2.91 (dd, J = 13.9, 10.3 Hz, 1H), 2.86-2.59 (m, 6H), 2.53-2.27 (m, 6H).

[0727] Example 8: Synthesis of TCEP-4

[0728]

[0729] 1.TCEP-4-int1

[0730] To a solution of ethanolamine (610 mg, 10 mmol, 1.0 eq., Adamas) in DMC (20 mL) was added imidazole (15 mmol, 1.5 eq.), followed by TBDPSCl (10 mmol, 1.0 eq., Adamas) at 0 °C. The mixture was stirred at room temperature for 2 h. TLC showed that the reaction was complete, the reaction mixture was washed with water and brine, the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated to give a crude product, which was used directly in the next step without further purification.

[0731] 2.TCEP-4

[0732] TCEP-4 was synthesized in the synthetic procedure B-1, wherein TCEP-4-int1 was an amine reagent, to obtain TCEP-4 (60.0 mg, 40.8%) as a white solid. MS [MH] - =292.25, C 11 H 20 The exact mass of NO6P was calculated to be 293.10. 1 H NMR (400 MHz, deuterium oxide): δ 4.23 (t, J = 5.4 Hz, 1H), 3.64 (t, J = 5.4 Hz, 1H), 3.48 (t, J = 5.3 Hz, 1H), 3.32 (t, J = 5.5 Hz, 1H), 2.98-2.78 (m, 6H), 2.60 (dp, J = 13.4, 6.8 Hz, 6H).

[0733] Example 9: Synthesis of TCEP-5

[0734]

[0735] TCEP-5 was synthesized in Procedure B, where (2-phenoxy-ethylamine, Bidepharm) was the amine reagent to give TCEP-5 (105.0 mg, 56.8%) as a white solid. MS [MH] - =368.24, C 17 H 24 The exact mass of NO6P was calculated to be 369.13. 1 H NMR (400 MHz, deuterium oxide): δ 7.34 (dd, J = 8.5, 7.2 Hz, 2H), 7.00 (dd, J = 19.2, 7.7 Hz, 3H), 4.14 (t, J = 5.1 Hz, 2H), 3.56 (t, J = 5.1 Hz, 2H), 2.78 (ddt, J = 41.6, 20.1, 6.9 Hz, 6H), 2.60-2.43 (m, 6H).

[0736] Example 10: Synthesis of TCEP-6

[0737]

[0738] 1.TCEP-6-int1

[0739] To a solution of N-methylhydroxylamine hydrochloride (830 mg, 10 mmol, 1.0 eq., Bidepharm) in DMC (20 mL) was added imidazole (15 mmol, 1.5 eq.), followed by TBDPSCl (10 mmol, 1.0 eq., Adamas) at 0 °C. The mixture was stirred at room temperature for 2 h. TLC showed that the reaction was complete, and the reaction mixture was washed with water and brine, and the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated to give a crude product, which was used directly in the next step without further purification.

[0740] 2.TCEP-6

[0741] TCEP-6 was synthesized in Procedure A-1, where TCEP-6-int1 was an amine reagent to obtain TCEP-6 (13.0 mg, 9.3%) as a white solid. MS [M+H] + =280.22, C 10 H 18 The exact mass of NO6P was calculated to be 279.09. 1 H NMR (400 MHz, deuterium oxide): δ 3.15 (s, 3H), 3.01–2.80 (m, 4H), 2.64-2.45 (m, 6H), 2.17-2.08 (m, 2H). Example 11: Synthesis of TCEP-7

[0742]

[0743] TCEP-7 was synthesized in the synthetic procedure B, wherein (aniline, Adamas) was the amine reagent to obtain TCEP-7 (73.0 mg, yield: 45.0%) as a white solid. MS [MH] - =324.21, C 15 H 20 The exact mass of NO5P was calculated to be 325.11. 1 H NMR (400 MHz, deuterium oxide): δ 7.42 (d, J = 4.3 Hz, 4H), 7.25 (p, J = 4.5 Hz, 1H), 2.96 (ddt, J = 32.1, 18.3, 6.9 Hz, 6H), 2.75-2.50 (m, 6H).

[0744] Example 12: Synthesis of TCEP-8

[0745]

[0746] TCEP-8 was synthesized according to the synthetic procedure B, wherein (benzylamine, Adamas) was used as the amine reagent to obtain TCEP-8 (85.6 mg, yield 50.5%) in the form of a white solid. MS [MH]- =338.23, C 16 H 22 The exact mass of NO5P was calculated to be 339.12. 1 H NMR (400 MHz, deuterium oxide): δ 7.43-7.27 (m, 5H), 4.36 (s, 2H), 2.93-2.77 (m, 6H), 2.63-2.45 (m, 6H).

[0747] Example 13: Synthesis of TCEP-9

[0748]

[0749] TCEP-9 was synthesized in Synthesis Procedure A, where (4-aminobenzene-1,2-diol, Bidepharm) was used as an amine reagent to obtain TCEP-9 (63.8 mg, 35.7%) as a white solid. MS [MH] - =356.20, C 15 H 20 The exact mass of NO7P was calculated to be 357.10. 1 H NMR (400 MHz, deuterium oxide): δ 6.97 (d, J = 2.4 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.78 (dd, J = 8.5, 2.5 Hz, 1H), 2.92 (ddt, J = 17.8, 10.0, 7.0 Hz, 6H), 2.61 (dq, J = 13.9, 6.7 Hz, 6H).

[0750] Example 14: Synthesis of TCEP-10

[0751]

[0752] TCEP-10 was synthesized in Synthesis Procedure A, where (5-amino-2-hydroxybenzoic acid, Bidepharm) was used as the amine reagent to obtain TCEP-10 (53.7 mg, 27.9%) as a white solid. MS [MH] - =384.20, C 16 H 20 The exact mass of NO8P was calculated to be 385.09. 1 H NMR (400 MHz, deuterium oxide) δ 7.76 (d, J = 2.7 Hz, 1H), 7.41 (dd, J = 8.9, 2.7 Hz, 1H), 6.89 (d, J = 8.9 Hz, 1H), 3.00-2.83 (m, 6H), 2.61 (dq, J = 13.9, 6.7 Hz, 6H).

[0753] Example 15: Synthesis of TCEP-11

[0754]

[0755] TCEP-11 was synthesized in the synthetic procedure B, wherein (bis(pyridin-2-ylmethyl)amine, Shanghai Jizhi Biochemical Technology Co., Ltd.) was used as an amine reagent to obtain TCEP-11 (10.5 mg, 4.9%) as a brown solid. MS [M+H] + =432.24, C 21 H 26 The exact mass of N3O5P was calculated to be 431.16. 1 H NMR (400 MHz, deuterium oxide) δ 8.82-8.68 (m, 2H), 8.58-8.36 (m, 2H), 8.01-7.81 (m, 4H), 5.34 (s, 1H), 5.29 (s, 1H), 5.04 (d, J = 2.9 Hz, 2H), 4.48 (s, 1H), 3.16 (dt, J = 19.2, 6.5 Hz, 1H), 2.95-2.79 (m, 3H), 2.73-2.48 (m, 5H), 2.20 (ddt, J = 36.1, 11.6, 7.5 Hz, 2H).

[0756] Example 16: Synthesis of TCEP-12

[0757]

[0758]

[0759] TCEP-12 was synthesized in Synthesis Procedure A, where (5-amino-8-hydroxyquinoline, Bidepharm) was used as the amine reagent to obtain TCEP-12 (33.2 mg, 16.9%) as a white solid. MS [MH] - =391.24, C 18 H 21 The exact mass calculated for N2O6P is 392.11. 1 H NMR (400 MHz, deuterium oxide) δ 9.02-8.96 (m, 2H), 8.05-7.98 (m, 1H), 7.84 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 2.90-2.80 (m, 2H), 2.67-2.56 (m, 6H), 2.26-2.17 (m, 4H).

[0760] Example 17: Synthesis of TCEP-15

[0761]

[0762] TCEP-15 was synthesized in Synthesis Procedure B, where (bis(pyridin-2-yl)methylamine, Bidepharm) was the amine reagent to give TCEP-15 (21.7 mg, 10.4%) as a white solid. MS [M+H] + =418.26, C 20 H 24 The exact mass of N3O5P was calculated to be 417.15. 1 H NMR (400 MHz, deuterium oxide) δ 8.69 (td, J = 6.2, 1.6 Hz, 2H), 8.37 (dtd, J = 15.8, 7.9, 1.7 Hz, 2H), 7.93-7.79 (m, 4H), 3.09-2.72 (m, 6H), 2.70-2.52 (m, 6H).

[0763] Example 18: Synthesis of TCEP-18

[0764]

[0765] 1.TCEP-18-int1

[0766] Phenylphosphine (110mg, 1.0mmol, Adamas) was dissolved in acetonitrile (5mL, degassed) in a round-bottom flask dried over a straight flame in N2(g). Potassium hydroxide (10N, 10μL) was added to the mixture, and the resulting solution was cooled to 0°C. Tert-butyl acrylate (0.44mL, 3.0mmol, Adamas) was added. After complete addition of tert-butyl acrylate, the reactant was heated to 50°C and stirred for 8h. The reaction mixture was dissolved in EtOAc (10mL), then washed with brine (2×5mL). The organic matter was dried with Na2SO4, filtered and concentrated. The residue was purified by a flash tower (EtOAc / petroleum ether=0-20% (v / v)) to obtain the product TCEP-18-int1 (254mg, 69.4%) as a transparent liquid.

[0767] 2.TCEP-18

[0768] Under N2 atmosphere, a solution of TCEP-18-int1 (254 mg, 0.69 mmol) in HCl / 1,4-dioxane (4M, Adamas) was stirred at room temperature for 2 h. LCMS showed that the reaction was complete, and the mixture was concentrated to remove 1,4-dioxane. The resulting residue was dissolved in water and freeze-dried to give TCEP-18 (152.7 mg, 88.2%) as a white solid. MS [MH] - =253.19, C 12 H 15The exact mass of O4P was calculated to be 254.07. 1 H NMR (400MHz, DMSO-d6): δ7.74 (dd, J=10.9, 7.3Hz, 2H), 7.62-7.49 (m, 3H), 2.46-2.35 (m, 2H), 2.33-2.00 (m, 6H).

[0769] Example 19: Synthesis of TCEP-19

[0770]

[0771] Under N2 atmosphere, HATU (380 mg, 1.0 mmol, 2.5 eq.) was added to a solution of TCEP-19-int1 (200 mg, 0.39 mmol, 1.0 eq.) in DMF (3 mL) at 0°C, followed by DIPEA (174 μL, 1.0 mmol, 2.5 eq.). The mixture was stirred for 30 min, and glycine tert-butyl ester (1 mmol, Adamas) was added. The reactants were stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to obtain the claimed product. The product was dissolved in DCM (3 mL) and TFA (0.5 mL) was added. The mixture was stirred for 1 h, and LCMS showed that the reaction was complete. The mixture was concentrated, and the residue was dissolved in distilled water (10 mL) and washed with EtOAc (2*5 mL). The aqueous layer was lyophilized to give TCEP-19 (10.2 mg, 6.8%) as a brown solid. MS[M+H] + =380.24, C 13 H 22 The exact mass of N3O8P is calculated to be 379.31. 1 H-NMR (400 MHz, deuterium oxide): δ 3.99 (s, 4H), 2.93-2.84 (m, 4H), 2.76-2.53 (m, 8H).

[0772] Example 20: Synthesis of TCEP-20

[0773]

[0774] Under N2 atmosphere, HATU (380 mg, 1.0 mmol, 2.5 equiv) was added to a solution of TCEP-19-int1 (200 mg, 0.39 mmol, 1.0 equiv) in DMF (3 mL) at 0°C, followed by DIPEA (174 μL, 1.0 mmol, 2.5 equiv). The mixture was stirred for 30 min and sodium 3-aminopropane-1-sulfonate (1 mmol, Adamas) was added. The reactants were stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to obtain the claimed product. The product was dissolved in DCM (3 mL) and TFA (0.5 mL) was added. The mixture was stirred for 1 h and LCMS showed that the reaction was complete. The mixture was concentrated and the residue was dissolved in distilled water (10 mL) and washed with EtOAc (2*5 mL). The aqueous layer was lyophilized to give TCEP-20 (23.7 mg, 11.7%) as a brown solid. MS [MH] - =506.22, C 15 H 30 N3O 10 The exact mass of PS2 was calculated to be 507.51. 1 H NMR (400 MHz, deuterium oxide): δ 3.27 (t, J = 6.8 Hz, 4H), 2.91-2.85 (m, 4H), 2.80-2.71 (m, 4H), 2.69-2.61 (m, 2H), 2.58-2.49 (m, 6H), 1.94-1.85 (m, 4H).

[0775] Example 21: Synthesis of TCEP-21

[0776]

[0777] Under N2 atmosphere, HATU (380 mg, 1.0 mmol, 2.5 equiv) was added to a solution of TCEP-19-int1 (200 mg, 0.39 mmol, 1.0 equiv) in DMF (3 mL) at 0°C, followed by DIPEA (174 μL, 1.0 mmol, 2.5 equiv). The mixture was stirred for 30 min and diethanolamine (1 mmol, Bidepharm) was added. The reactants were stirred at room temperature for 1 h. The reaction mixture was purified by RP-HPLC using a C18 column to obtain the claimed product. The product was dissolved in DCM (3 mL) and TFA (0.5 mL) was added. The mixture was stirred for 1 h and LCMS showed that the reaction was complete. The mixture was concentrated and the residue was dissolved in distilled water (10 mL) and washed with EtOAc (2*5 mL). The aqueous layer was lyophilized to give TCEP-20 (13.8 mg, yield: 7.8) as a white solid. MS[M+H]+ =440.27, C 17 H 34 The exact mass of N3O8P was calculated to be 439.45. 1 H NMR (400 MHz, deuterium oxide): δ 4.50-4.28 (m, 4H), 3.78 (t, J = 5.2 Hz, 4H), 3.38 (t, J = 5.1 Hz, 4H), 3.17 (q, J = 5.2 Hz, 4H), 2.96-2.80 (m, 4H), 2.68-2.51 (m, 8H).

[0778] Example 22: Synthesis of TCEP-23

[0779]

[0780] 1.TCEP-23-int1

[0781] To a solution of 2-(aminooxy)tetrahydro-2H-pyran (1.17 g, 10 mmol, 2.0 equiv., Bidepharm) in DMF (15 mL) was added DIPEA (3.5 mL, 20 mmol, 4 equiv.) followed by 2-(bromomethyl)pyridine hydrobromide (1.3 g, 5.0 mmol, 1.0 equiv., Adamas). The mixture was stirred at 50 °C for 16 h. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (30 mL*3). The organic layer was washed with brine (30 mL), dried over Na2SO4 and filtered. The filtrate was concentrated and purified by flash column to give TCEP-23-int1 (800 mg, 80%) as a colorless oil.

[0782] 2.TCEP-23

[0783] Under N2 atmosphere, HATU (190 mg, 0.5 mmol, 1 eq) was added to a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL), followed by DIPEA (2.0 mmol, 4.0 eq). The mixture was stirred for 30 min, and TCEP-23-int1 (100 mg, 0.5 mmol, 1.0 eq) was added. The reactants were stirred at room temperature for 4 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the desired product. The product was dissolved in HCl / 1,4-dioxane (3 mL). The mixture was stirred for 1 h, and LCMS showed that the reaction was complete. The mixture was concentrated, and the residue was dissolved in distilled water and lyophilized to give TCEP-23 (17.9 mg, 10.0%) as a white solid. MS [M+H] + =357.19, C 15 H21 The exact mass calculated for N2O6P is 356.31. 1 H-NMR (400 MHz, deuterium oxide): δ8.73 (dd, J=6.3, 1.7 Hz, 1H), 8.59 (td, J=7.9, 1.6 Hz, 1H), 8.02 (dd, J=6.4, 3.4 Hz, 2H), 5.21 (s, 2H), 3.21-2.97 (m, 2H), 2.95-2.80 (m, 4H), 2.69-2.56 (m, 6H).

[0784] Example 23: Synthesis of TCEP-24

[0785]

[0786] TCEP-24 was synthesized in the synthetic procedure A, wherein (4-aminobenzoic acid, Bidepharm) was used as the amine reagent to obtain TCEP-24 (21.5 mg, yield: 10.4%) as a white solid. MS [MH] - =412.22, C 17 H 20 The exact mass of NO9P was calculated to be 413.09. 1 H NMR (400 MHz, deuterium oxide): δ 7.87 (d, J = 8.5 Hz, 1H), 7.77 (d, J = 2.1 Hz, 1H), 7.71-7.64 (m, 1H), 3.08-3.00 (m, 2H), 2.96-2.86 (m, 4H), 2.70-2.59 (m, 6H).

[0787] Example 24: Synthesis of TCEP-25

[0788]

[0789] 1.TCEP-25-int1

[0790] Pd / C (150 mg) and two drops of AcOH were added to a MeOH (25 mL) solution of 2-pyridinecarboxaldehyde (1.0 g, 10 mmol, 1.0 equivalents of Adamas) and glycine tert-butyl ester (1.3 g, 10.0 mmol, 1.0 equivalents). The mixture was degassed 3 times and purged with H2, then stirred at room temperature for 16 h under H2 atmosphere. The reaction mixture was filtered through a Celite pad, and the filtrate was concentrated, then purified with a flash tower to give TCEP-25-int1 (1.6 g, 72.0%) as a yellow oil.

[0791] 2.TCEP-25

[0792] Under N2 atmosphere, HATU (190 mg, 0.5 mmol, 1 eq) was added to a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq) in DMF (3 mL), followed by DIPEA (2.0 mmol, 4.0 eq). The mixture was stirred for 30 min, and TCEP-25-int1 (111 mg, 0.5 mmol, 1.0 eq) was added. The reactants were stirred at room temperature for 4 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the desired product. The product was dissolved in HCl / 1,4-dioxane (3 mL). The mixture was stirred for 1 h, and LCMS showed that the reaction was complete. The mixture was concentrated, and the residue was dissolved in distilled water and lyophilized to give TCEP-25 (51.3 mg, 17.2%) as a white solid. MS [M+H] + =399.25, C 17 H 23 The exact mass of N2O7P was calculated to be 398.12. 1 H NMR (400 MHz, deuterium oxide): δ8.60 (dd, J=5.9, 1.6 Hz, 1H), 8.45 (td, J=8.0, 1.6 Hz, 1H), 7.92 (d, J=8.3 Hz, 1H), 7.87 (ddd, J=7.5, 5.9, 1.3 Hz, 1H), 4.88 (s, 2H), 4.39 (s, 2H), 2.84-2.68 (m, 6H), 2.51-2.41 (m, 6H).

[0793] Example 25: Synthesis of TCEP-26

[0794]

[0795] 1.TCEP-26-int1

[0796] Under N2 atmosphere, HATU (4.3 mg, 11.0 mmol, 2.2 equiv) was added to a solution of Fmoc-iminodiacetoacetic acid (1.8 g, 5.0 mmol, 1.0 equiv, Bidepharm) in DMF (30 mL), followed by DIPEA (2.0 mmol, 4.0 equiv). The mixture was stirred for 30 min, and tritylhydroxylamine (3.0 mg, 11.0 mmol, 2.2 equiv) was added. The reactants were stirred at room temperature for 4 h. The reaction mixture was poured into water (200 mL). The precipitate was collected by filtration, and the filter cake was dried under vacuum to give TCEP-23-int1 (4.0 g, 92.0%) as a white solid.

[0797] 2.TCEP-26-int2

[0798] DBU (2 mL) was added to a DMF (10 mL) solution of TCEP-26-int1 (2.0 g, 2.3 mmol, 1.0 equivalent). The mixture was stirred at room temperature for 1 h, then poured into ice water (100 mL), extracted with EtOAc (50 mL * 3). The organic layers were combined, washed with brine, dried over Na2SO4 and filtered. The filtrate was concentrated and purified by a flash tower (EtOAc / petroleum ether = 0-50% (v / v) to obtain TCEP-26-int2 (1.2 g, 80%).

[0799] 3.TCEP-26

[0800] The compound was synthesized in the synthetic procedure A-1, wherein TCEP-26-int2 was an amine reagent, to obtain TCEP-26 (31.5 mg, yield: 21.3%) as a white solid. MS [M+H] + =396.17, C 13 H 22 The exact mass of N3O9P is calculated to be 395.11. 1 H NMR (400 MHz, deuterium oxide) δ 4.13 (s, 2H), 3.97 (s, 2H), 2.85-2.77 (m, 4H), 2.54-2.47 (m, 6H), 2.18-2.08 (m, 2H).

[0801] Example 26: Synthesis of TCEP-28

[0802]

[0803] 1.TCEP-28-int1

[0804] To a DMF solution (15 mL) of o-tritylhydroxylamine (1.4 g, 5.0 mmol, 1.0 equiv) was added DIPEA (1.7 mL, 10 mmol, 2 equiv), followed by tert-butyl bromoacetate (1.0 g, 5.0 mmol, 1.0 equiv, Adamas). The mixture was stirred at 50 ° C for 16 h. The reaction mixture was poured into water (100 mL) and extracted with EtOAc (30 mL * 3). The organic layer was washed with brine (30 mL), dried over Na2SO4 and filtered. The filtrate was concentrated and purified by flash tower to give TCEP-28-int1 (1.4 g, 70%) as a white solid.

[0805] 2.TCEP-28-int2

[0806] To a solution of TCEP-28-int1 (1.4 g, 3.6 mmol, 1.0 equiv) in DCM (15 mL) was added TFA (1.5 mL). The mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated and purified by flash column to give TCEP-28-int2 (380 mg, 71.8%) as a colorless oil.

[0807] 3.TCEP-28

[0808] Under N2 atmosphere, HATU (190 mg, 0.5 mmol, 1 eq.) was added to a solution of TCEP (286.6 mg, 1.0 mmol, 2.0 eq.) in DMF (3 mL), followed by DIPEA (2.0 mmol, 4.0 eq.). The mixture was stirred for 30 min, and TCEP-28-int2 (73.5 mg, 0.5 mmol, 1.0 eq.) was added. The reactants were stirred at room temperature for 2 h. The reaction mixture was purified by RP-HPLC using a C18 column to give the desired product. The product was dissolved in HCl / 1,4-dioxane (3 mL, Adamas). The mixture was stirred for 1 h, and LCMS showed that the reaction was complete. The mixture was concentrated, and the residue was dissolved in distilled water and lyophilized to give TCEP-28 (43.6 mg, 27.1%) as a white solid. MS[MH]-=322.16, C 11 H 18 The exact mass of NO8P was calculated to be 323.08. 1 H NMR (400 MHz, deuterium oxide): δ 4.36 (s, 2H), 2.86-2.77 (m, 6H), 2.56-2.48 (m, 6H).

[0809] Example 27: Synthesis of TCEP-30

[0810]

[0811] TCEP-30 was synthesized in the synthetic procedure A-1, wherein [L-tyrosine tert-butyl ester, Adamas] was used as the amine reagent to obtain TCEP-30 (25.3 mg, 12.25%) as a white solid. MS [M+H] + =414.23, C 18 H 24 The exact mass of NO8P was calculated to be 413.12. 1H NMR (400 MHz, deuterium oxide): δ 7.24-7.14 (m, 2H), 6.91-6.82 (m, 2H), 4.67 (dd, J=10.3, 4.7 Hz, 1H), 3.26 (dd, J=14.0, 4.7 Hz, 1H), 2.92-2.64 (m, 7H), 2.57-2.30 (m, 6H).

[0812] Example 28: Synthesis of TCEP-31

[0813]

[0814] TCEP-31 was synthesized in Synthesis Procedure A, where (DL-3-(4-fluorophenyl)alanine, Bidepharm) was used as the amine reagent to obtain TCEP-31 (27.1 mg, 13.10%) as a white solid. MS [M+H] + =416.01, C 18 H 23 The exact mass of NO7P was calculated to be 415.12. 1 H NMR (400 MHz, deuterium oxide): δ 7.36-7.26 (m, 2H), 7.12 (t, J = 8.8 Hz, 2H), 4.70 (dd, J = 10.1, 4.8 Hz, 1H), 3.31 (dd, J = 14.0, 4.9 Hz, 1H), 2.95 (dd, J = 14.0, 10.1 Hz, 1H), 2.90-2.65 (m, 6H), 2.56-2.40 (m, 6H).

[0815] Example 29: Synthesis of TCEP-32

[0816]

[0817] TCEP-32 was synthesized in Synthesis Procedure A, where (DL-4-cyanophenylalanine, Bidepharm) was used as the amine reagent to obtain TCEP-32 (18.5 mg, 8.76%) as a white solid. MS [M+H] + =423.24, C 19 H 23 The exact mass of N2O7P was calculated to be 422.12. 1 H NMR (400 MHz, deuterium oxide): δ 7.77 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.1 Hz, 2H), 4.77-4.71 (m, 1H), 3.42 (dd, J = 14.0, 5.0 Hz, 1H), 3.05 (dd, J = 14.0, 10.0 Hz, 1H), 2.91-2.70 (m, 6H), 2.68-2.39 (m, 6H).

[0818] Example 30: Synthesis of TCEP-33

[0819]

[0820] TCEP-33 was synthesized in Synthesis Method A, where (DL-4-nitrophenylalanine, Bidepharm) was used as an amine reagent to obtain TCEP-33 (20.7 mg, 9.37%) as a white solid. MS [M+H] + =443.24, C 19 H 23 The exact mass of N2O9P was calculated to be 442.11. 1 H NMR (400 MHz, deuterium oxide): δ 8.22 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 4.84-4.80 (m, 1H), 3.47 (dd, J = 14.0, 4.8 Hz, 1H), 3.11 (dd, J = 13.9, 10.3 Hz, 1H), 2.91-2.73 (m, 6H), 2.58-2.38 (m, 6H).

[0821] Example 31: Synthesis of TCEP-34

[0822]

[0823] TCEP-34 was synthesized in Synthesis Procedure A, where (N-benzylhydroxylamine hydrochloride, Bidepharm) was used as the amine reagent to obtain TCEP-34 (15.7 mg, 8.85%) as a white solid. MS [M+H] + =356.05, C 16 H 22 The exact mass of NO6P was calculated to be 355.12. 1 H NMR (400 MHz, deuterium oxide): δ 7.55-7.34 (m, 5H), 4.84 (s, 2H), 3.14 (dt, J = 19.8, 6.9 Hz, 2H), 2.92 (dt, J = 18.2, 7.0 Hz, 4H), 2.62 (dq, J = 14.5, 7.3 Hz, 6H).

[0824] Example 32: Synthesis of TCEP-35

[0825]

[0826] Synthesis TCEP-35 was synthesized in Synthesis Procedure A, where (N-phenylhydroxylamine, Bidepharm) was used as the amine reagent to obtain TCEP-35 (17.1 mg, 10.0%) as a white solid. MS [M+H] + =341.97, C 15 The exact mass calculated for H2NO6P is 341.10. 1 H NMR (400 MHz, deuterium oxide): δ 7.57-6.88 (m, 5H), 3.27-3.22 (m, 1H), 2.92-2.78 (m, 3H), 2.65-2.53 (m, 6H), 2.29-1.98 (m, 2H).

[0827] Example 33: Synthesis of TCEP-37

[0828]

[0829] 1.TCEP-37-int1

[0830] To a solution of 2,4-dimethoxybenzaldehyde (1.66 g, 10.0 mmol, 1.0 equiv., Adamas) in MeOH (25 mL) was added o-methylhydroxylamine hydrochloride (1.66 g, 20.0 mmol, 2.0 equiv., Bidepharm). The resulting mixture was stirred at room temperature for 16 h, and LCMS showed that the reaction was complete. The mixture was concentrated, and the residue was dissolved in AcOH (20 mL), NaBH3CN was added and stirred at room temperature for 5 h. LCMS showed that the reaction was complete. The reaction mixture was concentrated, and the residue was poured into ice water (200 mL) and extracted with EtOAc (50 mL*3). The organic layers were combined, dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo and purified by flash column (EtOAc / petroleum ether=0-50%) to obtain the product TCEP-37-int1 (N-(2,4-dimethoxybenzyl)-O-methylhydroxylamine, 1.5 g, 76.1%, colorless oil).

[0831] 2.TCEP-37

[0832] TCEP-37 was synthesized in the synthesis procedure A-1, wherein TCEP-73-int1 was used as an amine reagent to obtain TCEP-37 (12.8 mg, 9.14%) as a white solid. MS [M+H] + =280.18, C 10 H 18 The exact mass of NO6P was calculated to be 279.09. 1H NMR (400 MHz, deuterium oxide): δ (s, 3H), 2.89 (dt, J = 18.4, 7.1 Hz, 4H), 2.72 (dd, J = 18.1, 6.6 Hz, 2H), 2.61 (dq, J = 13.9, 6.7 Hz, 6H).

[0833] Example I.1-I.36: Preparation of Antibody-[MC-VC-PAB-MMAE]2 by TCEP or the above reducing agents

[0834] (1) ZnCl2 (0.24 mM) and a reducing agent (0.02 mM) were added to a solution of a monoclonal antibody (0.012 mM, dissolved in MES buffer, pH 6.7, 20 mM), and the reaction mixture was placed at 4°C for 4 hours, 8 hours, or 12 hours;

[0835] (2) Add EDTA (0.6 mM) to capture Zn 2+ ; Introduce MC-VC-PAB-MMAE (0.06 mM) dissolved in DMA, and continue the reaction at 24° C. for 30 minutes when the reducing agent is TCEP, and when the reducing agent is other reducing agents described in Examples I.4-I.36, the reaction time is 1 hour;

[0836] (3) Cysteine ​​(0.08 mM) was added to deplete excess MC-VC-PAB-MMAE; the reaction mixture was purified using a desalting column.

[0837] The type of reducing agent and / or the molar ratio of antibody / reducing agent are adjusted as shown in Table I-1. In Example I.10, the buffer system is MOPS buffer and the pH value is 7.4.

[0838] Comparative Examples I.1-I.12: Preparation of Antibody-[MC-VC-PAB-MMAE]2 by TCEP or the above reducing agents in the absence of transition metal ions

[0839] Comparative Examples I.1-I.3 were prepared similarly to Example I.1, and Comparative Examples I.4-I.12 were prepared similarly to Example I-4, but the concentration of transition metal ions was 0. The parameters of Comparative Examples I.1-I.12 are shown in Table I-1.

[0840] The chromatographic peak areas of Examples I.1-I.36 and Comparative Examples I.1-II.12 are shown in Table I-2, and the homogeneity test results are shown in Table I-1. The chromatograms of E I.1 and E I.4 are shown in Table I-3. Figure 1-2 shown.

[0841] Table I-1

[0842]

[0843]

[0844] In the present invention, "E" is an abbreviation for Example, and "C" is an abbreviation for Comparative Example.

[0845] Table I-2

[0846]

[0847]

[0848] As shown in the results of Table I-1, the coupling process using the same steps without adding transition metal ions in step (1) was used as a negative control (C I.1-C I.12). The present invention successfully demonstrated that the combination of transition metal ions and reducing agents can increase the D2 level in the resulting ADC. By using the process of the present invention to produce antibody drug conjugates, the uniformity of the antibody drug conjugates is greatly improved.

[0849] In addition, as shown in the results of Examples I.4-I.36, compared with the use of Zn-free 2+ Compared with the traditional method of reducing TCEP, the compounds of the present invention can improve the uniformity of D2-type ADC, among which TCEP-6 has the best selective reduction ability, and the D2 content is as high as 94.25%. At the same time, the selective reduction ability of TCEP-NO, TCEP-3NO, TCEP-CO, TCEP-1, TCEP-19, TCEP-20, TECP-21, TCEP-23, TCEP-24, TCEP-26, TCEP-28, TCEP-34, TCEP-35 and TCEP-37 is also very outstanding, and the D2 content is as high as 84%, 87%, or even 90% or 93%.

[0850] Examples I.37-I.63 and Comparative Examples I.13-I.14: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugates Using Different Molar Ratios of ZnCl2 to Reducing Agent

[0851] When the reducing agent is TCEP, the preparation of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is similar to Example I.1, and when the reducing agent is TCEP-NO, the preparation of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is similar to Example I.4, but these adjust the amount of ZnCl2 in step (1). The amount of ZnCl2 and the molar ratio of ZnCl2 to the reducing agent are shown in Table I-3.

[0852] The homogeneity measurement results of Examples I.37-I.63 and Comparative Examples I.13-I.14 are shown in Table I-3, and the chromatographic peak areas are shown in Table I-4.

[0853] Table I-3

[0854]

[0855]

[0856] Table I-4

[0857]

[0858]

[0859] As shown in Table I-3, when the reducing agent is TCEP, the results show that the content of D2 increases with the increase of Zn 2+ The molar ratio of Zn to TCEP increased from 0.4:1 to 12:1. Subsequently, the ratio of D2 content reached a plateau. 2+ When the molar ratio of D2 to TCEP is 250:1, the content of D2 is lower than that of Zn 2+ / TCEP-NO molar ratio (ranging from 0.4:1 to 125:1). These results indicate that Zn 2+ The molar ratio of Zn to TCEP plays a key role in the content of D2 and the selective reduction. 2+ When the molar ratio of D2 to TCEP is 0.4:1 to 200:1, the content of D2 is as high as 60%, 65%, or even 70%, 75%, 80% or 83%.

[0860] When the reducing agent is TCEP-NO, the content of D2 increases by adding transition metal ions. The D2 ratio increases with the increase of Zn 2+ / TCEP-NO molar ratio increases from 0.4 to 6. After that, the D2 ratio reaches a plateau. 2+ When the molar ratio of Zn / TCEP-NO is as high as 200:1 and 250:1, the content of D2 is lower than that of Zn in the range of 2:1 to 125:1. 2+ / TCEP-NO molar ratio. This indicates that transition metal ions, especially Zn 2+ / TCEP-NO ratio plays a key role in determining the D2 ratio and reduction selectivity.

[0861] Example I.64-I.71: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates at different molar ratios of antibody to reducing agent When the reducing agent is TCEP, the preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates is similar to Example I.1. When the reducing agent is not TCEP, the preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates is similar to Example I.4. However, these adjustments are made to the amount of antibody used in step (1) or the incubation time in step (1). The amount of antibody used and the molar ratio of antibody to reducing agent are shown in Table I-5.

[0862] The homogeneity test results for Examples I.64-I.71 are shown in Table I-5, and the chromatographic peak areas are shown in Table I-6.

[0863] Table I-5

[0864]

[0865] Table I-6

[0866]

[0867]

[0868] As shown in Table I-5, using TCEP as a reducing agent, when the molar ratio of antibody / TCEP is 1:1 to 1:3, the content of D2 type ADC is as high as 56%, 70%, or even 80%. When the molar ratio of antibody / TCEP is 1:2 and 1:3, the reduction time is shortened to 1h, and the D2 content is greater than 56%, even reaching 70% and 78%. As shown in Table I-5, in Examples EI.16, E I.19, and E I.23-24, using TCEO-NO as a reducing agent, when the molar ratio of antibody to TCEP-NO is 1:0.9 to 1:3.0, the content of D2 type ADC is as high as 70%, 75%, or even 80%, 85% or 90%. When the molar ratio of antibody / TCEP-NO is 1:2 and 1:2.5, the reduction time is shortened to 1h, and the D2 content is greater than 80%. Examples I.72-I.101 and Comparative Examples I.15-I.22: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugates in Different Buffer Systems

[0869] When the reducing agent is TCEP, the preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is similar to Example 1.1. When the reducing agent is not TCEP, the preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is similar to Example 1.4. However, these adjusted the buffer system (buffer system can be purchased from Macklin), which is shown in Table 1-7.

[0870] The homogeneity measurement results of Examples I.72-I.101 and Comparative Examples I.15-I.22 are shown in Table I-7, and the chromatographic peak areas are shown in Table I-8.

[0871] Table I-7

[0872]

[0873]

[0874] Table I-8

[0875]

[0876]

[0877] As shown in Table 1-7, the buffer system affects the reduction kinetics and selectivity. The results show that the buffer system and its pH value affect the D2 ratio and the uniformity of the conjugate.

[0878] When the reducing agent TCEP is used, MOPS buffer, Bis-Tris buffer, PIPES buffer, BES buffer, MES buffer, HEPES buffer, DIPSO buffer, MOPSO buffer, TES buffer, and ACES buffer help increase the D2 ratio to at least 60%, and even to greater than 65%, 70%, 75%, and 80%.

[0879] When the reducing agent TCEP-NO is used, the D2 ratio is as high as 50% in ADA buffer, PB, DIPSO buffer, MOBS buffer, and ACES buffer. In the buffer systems Bis-Tris buffer, PIPES buffer, MOPS buffer, BES buffer, HEPES buffer, MOPSO buffer, TES buffer, and TAPSO buffer, the D2 ratio is at least 70%.

[0880] Examples I.102-I.123: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate (different reduction times or temperatures in step (1))

[0881] When the reducing agent is TCEP, the preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is similar to Example I.1. When the reducing agent is not TCEP, the preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate is similar to Example I.4. However, these adjust the reduction time or temperature in step (1), as shown in Table I-9.

[0882] The homogeneity test results of Examples I.102-I.123 are shown in Table I-9, and the chromatographic peak areas are shown in Table I-10.

[0883] Table I-9

[0884]

[0885] Table I-10

[0886]

[0887] As shown in Examples I.1 and I.102-I.104, when TCEP is used as a reducing agent, when the reducing agent temperature in step (1) is 4°C to 37°C, the content of D2 is as high as 75%, or even 80%. As shown in Examples I.105-I.111, when TCEP is used as a reducing agent, when the reducing agent time in step (1) is 1 hour to 8 hours, the content of D2 is as high as 75%, 80%, or even 83%. When the reduction time in step (1) is 1 hour to 4 hours, the content of D2 increases and reaches a plateau after 4 hours.

[0888] As shown in the results of Examples I.112-I.123, using TCEP-NO as a reducing agent, when the reduction temperature is 4-37°C and the reduction time is 0.25 hours to 37 hours, the content of D2 type ADC is as high as 80%. The content of D2 increases as the reduction time of step (1) increases from 0.25 hours to 1 hour, and reaches a steady state after 1 hour, indicating that the reaction kinetics is very fast. Example I.124: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate by engineering antibodies The preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates is similar to the preparation of ADC in Example I.4, but using engineered antibodies.

[0889] The engineered antibody is a mutant of trastuzumab with a cysteine ​​to serine mutation replacing the disulfide bond between the heavy and light chains (ordered from Biointron).

[0890] The homogeneity test results and chromatographic peak areas of Example I.124 are shown in Table I-11.

[0891] Table I-11

[0892]

[0893] As shown in Table I-11, the D2 content prepared from the engineered antibody was as high as 96%. These results indicate that this method is also applicable to antibodies with simple mutations and may have better reduction selectivity in some mutant antibodies.

[0894] Example I.125-I-126: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate (D1 type ADC)

[0895] 1. Synthesis of dibromomaleimide-PEG4-N3

[0896]

[0897] 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%).

[0898] A solution of azido-PEG4-amine (105 mg, 0.4 mmol, 1 eq) in dichloromethane (2 mL) was added to a stirred solution of product 1 (139 mg, 0.4 mmol, 1 eq) in dichloromethane (2 mL).

[0899] 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. Concentrated in vacuo, then purified by column chromatography (100% EtOAc as mobile phase) to give dibromomaleimide-PEG4-N3 (150 mg, 0.3 mmol, 75%) as a pale yellow oil.

[0900] 2. Preparation of Trastuzumab-Maleimide-[PEG4-N3-DBCO-Cy3]1

[0901] (1) Incubate the reducing agent TCEP / TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in MES (20 mM, pH 6.7) in the presence of ZnCl2 (0.24 mM), and place the reaction mixture at 4 °C for 4 h;

[0902] (2) introducing EDTA (0.6 mM) and dibromomaleimide-PEG4-N3 (0.013 mM) to react with the reduced thiol group from step (1) at 24° C. for 3 hours, and then recovering the product using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1;

[0903] (3) Trastuzumab-[maleimide-PEG4-N3]1 and DBCO-Cy3 (0.02 mM) were incubated in MES (20 mM, pH 6.7) at 25°C for 8 h, and then trastuzumab-maleimide-PEG4-N3-DBCO-Cy3 was recovered using a desalting column.

[0904] The results of the chromatographic peak area and uniformity determinations of Examples I.125-I.126 are shown in Table I-12.

[0905] Table I-12

[0906]

[0907] As shown in Table I-12, the results show that maleimide-PEG4-N3-DBCO-Cy3 is successfully linked to trastuzumab. In addition, when TCEP is used, the content of D1 type ADC is generally as high as 77.71%, and when TCEP-NO is used, the D1 ratio is as high as 83.61%.

[0908] Example I.127: Preparation of trastuzumab-[maleimide]1[MC-GGFG-DXd]6 (D0+D6 type ADC)

[0909] (1) In MES (20 mM, pH 6.7) in the presence of ZnCl2 (0.24 mM), the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) were incubated at 4 °C for 4 h;

[0910] (2) introducing EDTA (0.6 mM) and dibromomaleimide (0.013 mM) to react with the reduced thiol group from step (1) at 24° C. for 3 hours, and then recovering the product using a desalting column to obtain trastuzumab-[maleimide] 1;

[0911] (3) incubating trastuzumab-[maleimide] 1 and TCEP (0.08 mM) in MES (20 mM, pH 6.7) at 25 °C for 12 h;

[0912] (4) MC-GGFG-DXd (0.14 mM) was introduced into the solution from step (3), and the reaction mixture was placed at 24°C for 1 hour, and then trastuzumab-[maleimide]1[MC-GGFG-DXd]6 was recovered using a desalting column.

[0913] Example I.128: Preparation of trastuzumab-[maleimide] 1[MC-VC-PAB-MMAE] 6 (D0+D6 type ADC) The method is similar to Example I.127. The difference is that the reducing agent in step (1) of Example I.128 is TCEP, and the second linker-payload in step (4) of Example I.128 is MC-VC-PAB-MMAE.

[0914] The homogeneity test results and chromatographic peak areas of Examples I.127-I.128 are shown in Tables I-13 and I-14.

[0915] Table I-13

[0916] serial number Attached photos reducing agent D0(％) / / E I.127-Step (2) 5 TCEP-NO 100 E I.128-Step (2) 7A TCEP 100 / / serial number Attached photos reducing agent D0+D4(%) D0+D6(%) D8(％) E I.127-Step (4) 6 TCEP-NO 4.17 84.68 11.16 E I.128-Step (4) 7B TCEP 19.33 73.50 7.17

[0917] Table I-14

[0918]

[0919] The results showed that the dual-payload conjugates were successfully synthesized. In addition, when TCEP-NO was used, the uniformity of D0+D6 type ADC was generally as high as 84.68%, and when TCEP was used, the content of D0+D6 type ADC was generally as high as 73.50%. The results showed that the method of the present invention can modify antibodies to have site specificity and prepare different types of ADCs while improving uniformity.

[0920] Example I.129: Preparation of dual payload conjugate trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]6 (D2+D6 type ADC)

[0921] (1) In MES (20 mM, pH 6.7) in the presence of ZnCl2 (0.24 mM), the first reducing agent TCE (0.02 mM) and trastuzumab (0.012 mM) were incubated at 4 °C for 4 h;

[0922] (2) introducing EDTA (0.6 mM) and excess MC-VC-PAB-MMAE (0.06 mM) to react with the reduced thiol groups from step (1), the reaction temperature was 24° C. and the reaction time was 1 hour, and then the product was recovered using a desalting column to obtain trastuzumab-[MC-VC-PAB-MMAE]2;

[0923] (3) incubating trastuzumab-[MC-VC-PAB-MMAE]2 and TCEP (0.08 mM) in MES (20 mM, pH 6.7) at 25 °C for 8 h;

[0924] (4) MC-GGFG-DXd (0.14 mM) was introduced into the solution from step (3), and the reaction mixture was placed at 24°C for 1 hour, and then the reducing agent trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]6 was recovered using a desalting column. Example I.130: Preparation of dual payload conjugate trastuzumab-[MC-MMAF]2[MC-GGFG-DXd]6 (D2+D6 type ADC)

[0925] The method is similar to Example 1.129, except that the reducing agent in step (1) is TCEP-NO and the linker-payload in step (2) is MC-MMAF.

[0926] The homogeneity test results and chromatographic peak areas of Examples I.129-I.130 are shown in Tables I-15 and I-16.

[0927] Table I-15

[0928]

[0929] Table I-16

[0930]

[0931] As shown in Table I-15, the results show that when TCEP is used, the content of D2+D6 ADC is generally as high as 72.43%, while when TCEP-NO is used, the content of D2+D6 ADC is generally as high as 81.31%, which indicates that the process of this method is conducive to the site-specific modification of D2+D6 antibodies and can improve uniformity.

[0932] Examples I.131-I.132: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]2 (D2+D2 type ADC)

[0933] (1) incubating the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in BES (20 mM, pH 7.0) in the presence of an effective amount of ZnCl2 (0.12 mM) at a temperature of 4°C and a time of 4 h;

[0934] (2) introducing EDTA (0.6 mM) and MC-VC-PAB-MMAE (0.048 mM) to react with the reduced thiol group from step (1), the reaction temperature was 24 ° C and the reaction time was 1 h, and then the product was recovered using a desalting column to obtain trastuzumab-[MC-VC-PAB-MMAE]2;

[0935] (3) incubating ZnCl2 (1.2 mM), the second reducing agent TCEP-3 (0.0144 mM) / TCEP-6 (0.0216 mM) and the product from step (2) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 4 h;

[0936] (4) Introducing EDTA (3 mM) to capture Zn 2+ , and introduce MC-GGFG-DXd (0.1 mM) to react with the reduced thiol groups from step (3), the reaction temperature is 24° C. and the reaction time is 1 h;

[0937] (5) Purify the reaction mixture using a desalting column.

[0938] The homogeneity test results and chromatographic peak areas of Examples I.131-I.132 are shown in Tables I-17 and I-18.

[0939] Table I-17

[0940]

[0941]

[0942] Table I-18

[0943]

[0944] In step (3) of Examples I.131 and I.132, one of the interchain disulfide bonds in the D2-type ADC is reduced. As shown in Table I-17, the results show that the content of D2+D2-type ADC is usually as high as 68% or 70%, which indicates that the process of this method is conducive to site-specific modification of D2+D2-type antibodies and can improve uniformity.

[0945] Example I.133: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2 (D1+D2 type ADC)

[0946] (1) Incubating the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in BES (20 mM, pH 7.0) in the presence of an effective amount of ZnCl2 (0.24 mM) at a temperature of 4 °C and a time of 4 h;

[0947] (2) introducing EDTA (0.6 mM) and dibromomaleimide-PEG4-N3 (0.013 mM) to react with the reduced thiol group obtained in step (1), the reaction temperature is 25 ° C and the reaction time is 1 h, and then using a desalting column to recover the product to obtain trastuzumab-[maleimide-PEG4-N3]2;

[0948] (3) Incubate trastuzumab-[maleimide-PEG4-N3]1 from step (2) and DBCO-Cy3 (0.02 mM) in MES (20 mM, pH 6.7) at a reaction temperature of 25°C and a reaction time of 6 h, and then recover trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 using a desalting column.

[0949] (4) incubating ZnCl2 (1.2 mM), a second reducing agent TCEP-3 (0.0144 mM) and the product from step (3) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 4 h;

[0950] (5) Introducing EDTA (3 mM) to capture Zn 2+ , and introducing MC-VC-PAB-MMAE (0.1 mM) to react with the reduced thiol groups from step (4), the reaction temperature was 25° C. and the reaction time was 1 h;

[0951] (6) Purify the reaction mixture using a desalting column.

[0952] The homogeneity test results and chromatographic peak areas of Example I.133 are shown in Tables I-19 and I-20.

[0953] Table I-19

[0954]

[0955] Table I-20

[0956]

[0957] As shown in Table I-19, the results indicate that the D1+D2 ADC content is typically as high as 80% or 83%, which indicates that the process of this method is beneficial to the site-specific modification of D1+D2 antibodies and can improve uniformity.

[0958] Examples I.134-I.135: Preparation of trastuzumab-[maleimide]1[MC-VC-PAB-MMAE]4 (D0+D4 type ADC)

[0959] (1) incubating the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in MES (20 mM, pH 6.7) in the presence of an effective amount of ZnCl2 (0.24 mM) at a temperature of 4°C and a time of 4 h;

[0960] (2) introducing EDTA (0.6 mM) and dibromomaleimide (0.013 mM) to react with the reduced thiol group from step (1), the reaction temperature was 24° C. and the reaction time was 3 hours, and then the product was recovered using a desalting column to obtain trastuzumab-[maleimide] 1;

[0961] (3) incubating ZnCl2 (0.36 mM), the second reducing agent TCEP-3 (0.036 mM) / TCEP-6 (0.048 mM) and the product from step (2) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 16 h;

[0962] (4) Introducing EDTA (0.6 mM) to capture Zn 2+ , and introducing MC-VC-PAB-MMAE (0.1 mM) to react with the reduced thiol groups from step (4), the reaction temperature was 24° C. and the reaction time was 1 h;

[0963] (5) Purify the reaction mixture using a desalting column.

[0964] The homogeneity test results and chromatographic peak areas of Examples I.134 and I.135 are shown in Tables I-21 and I-22.

[0965] Table I-21

[0966] serial number Attached photos reducing agent D0(％) / / E I.134-Step (2) 13A TCEP-NO 100 / / serial number Attached photos / D0+D2(%) D0+D4(%) D0+D6(%) E I.134-Step (5) 13B TCEP-3 18.96 61.27 19.77 E I.135-Step (5) 13C TCEP-6 21.23 61.85 16.92

[0967] Table I-22

[0968]

[0969] In step (3) of Examples I.134-I.135, two of the interchain disulfide bonds in the D2-type ADC are reduced. As shown in Table I-21, the results show that the D0+D4-type ADC content is generally as high as 55% or 61%, which indicates that the process of this method is conducive to site-specific modification of D0+D4-type antibodies and can improve uniformity.

[0970] Examples I.136-I.137: Preparation of trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4 (D2+D4 type ADC)

[0971] (1) Incubating the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in BES (20 mM, pH 7.0) in the presence of an effective amount of ZnCl2 (0.24 mM) at a temperature of 4 °C and a time of 4 h;

[0972] (2) introducing EDTA (0.6 mM) and MC-GGFG-DXd (0.072 mM) to react with the reduced thiol group from step (1), the reaction temperature was 24 ° C and the reaction time was 1 h, and then the product was recovered using a desalting column to obtain trastuzumab-[MC-GGFG-DXd]2;

[0973] (3) incubating ZnCl2 (0.36 mM), the second reducing agent TCEP-3 (0.036 mM) / TCEP-6 (0.048 mM) and the product from step (2) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 16 h;

[0974] (4) Introducing EDTA (0.6 mM) to capture Zn 2+ , and introducing MC-VC-PAB-MMAE (0.1 mM) to react with the reduced thiol groups from step (4), the reaction temperature was 24° C. and the reaction time was 1 h;

[0975] (5) Purify the reaction mixture using a desalting column.

[0976] The homogeneity test results and chromatographic peak areas of Examples I.136 and I.137 are shown in Tables I-23 and I-24.

[0977] Table I-23

[0978]

[0979] Table I-24

[0980]

[0981] As shown in Table I-23, the results indicate that the content of D2+D4 type ADC is usually as high as 70%, 75%, or even 78% or 80%, which indicates that the process of this method is conducive to the site-specific modification of D2+D4 type antibodies and can improve uniformity.

[0982] Example I.138: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 (D1+D4 type ADC)

[0983] (1) Incubating the first reducing agent TCEP-NO (0.02 mM) and trastuzumab (0.012 mM) in BES (20 mM, pH 7.0) in the presence of an effective amount of ZnCl2 (0.24 mM) at a temperature of 4 °C and a time of 4 h;

[0984] (2) introducing EDTA (0.6 mM) and dibromomaleimide-PEG4-N3 (0.013 mM) to react with the reduced thiol group from step (1), the reaction temperature is 25 ° C and the reaction time is 6 h, and then the product is recovered using a desalting column to obtain trastuzumab-maleimide-PEG4-N3;

[0985] (3) Trastuzumab-maleimide-PEG4-N3 and DBCO-Cy3 (0.02 mM) were incubated in MES (20 mM, pH 6.7) at 25 °C for 6 h, and then trastuzumab-maleimide-PEG4-N3-DBCO-Cy3 was recovered using a desalting column.

[0986] (4) incubating ZnCl2 (0.36 mM), a second reducing agent TCEP-3 (0.0408 mM) and the product from step (3) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 16 h;

[0987] (5) Introducing EDTA (0.6 mM) to capture Zn 2+ , and introducing MC-VC-PAB-MMAE (0.1 mM) to react with the reduced thiol groups from step (4), the reaction temperature was 24° C. and the reaction time was 1 h;

[0988] (6) Purify the reaction mixture using a desalting column.

[0989] The homogeneity test results and chromatographic peak areas of Example I.138 are shown in Tables I-25 and I-26.

[0990] Table I-25

[0991] serial number Attached photos reducing agent D2(％) D1+D2(%) D4(％) D1+D4(%) E I.138 15 TCEP-3 3.09 23.96 2.45 70.49

[0992] Table I-26

[0993]

[0994] As shown in Table I-25, the results indicate that the content of D1+D4 type ADC is usually as high as 60%, 65%, or even 70%, which indicates that the process of this method is conducive to the site-specific modification of D1+D4 type antibodies and can improve uniformity.

[0995] Example I.139: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]2[MC-MMAF] (D2+D2+D2 type ADC)

[0996] (1) incubating a first reducing agent TCEP-NO (0.02304 mM) and trastuzumab (0.012 mM) in BES (20 mM, pH 7.0) in the presence of an effective amount of ZnCl2 (0.36 mM) at 4°C for 3 h; (2) introducing EDTA (0.72 mM) and MC-VC-PAB-MMAE (0.06 mM) to react with the reduced thiol groups from step (1) at 24°C for 1 h, and then recovering the product using a desalting column to obtain trastuzumab-[MC-VC-PAB-MMAE]2;

[0997] (3) incubating ZnCl2 (1.2 mM), the second reducing agent TCEP-3 (0.02472 mM) and the product from step (2) in BES buffer (pH 7.0.20 mM), and placing the reaction mixture at 4°C for 2.5 h;

[0998] (4) Introduction of EDTA (2.4 mM) to capture Zn 2+ , and introducing MC-GGFG-DXd (0.048 mM) to react with the reduced thiol groups produced in step (3), the reaction temperature was 24° C. and the reaction time was 1 hour, and the reaction mixture was purified using a desalting column and AKTA and HIC chromatography;

[0999] (5) incubating ZnCl2 (1.2 mM), the second reducing agent TCEP-3 (0.0168 mM) and the product from step (4) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 6 h;

[1000] (6) Introduction of EDTA (2.4 mM) to capture Zn 2+ , and MC-MMAF (0.048 mM) was introduced to react with the reduced thiol groups produced in step (5), the reaction temperature was 24° C. and the reaction time was 1 hour; the reaction mixture was purified using a desalting column.

[1001] The homogeneity test results and chromatographic peak areas of Example I.139 are shown in Tables I-27 and I-28.

[1002] Table I-27

[1003]

[1004] Table I-28

[1005]

[1006]

[1007] As shown in Table I-27, the results indicate that the D2+D2+D2 type ADC content is typically as high as 75% or 80%, which indicates that the process of this method is beneficial to the site-specific modification of the D2+D2+D2 type antibodies and can improve uniformity.

[1008] Example II.1: Preparation of Antibody-[MC-VC-PAB-MMAE]6 (D6-type ADC)

[1009] (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 to mix, and then the reaction mixture was incubated at 4°C for 18 h;

[1010] (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;

[1011] (3) Purify the reaction mixture using a desalting column.

[1012] Examples II.2-II.3: Preparation of Antibody-[MC-VC-PAB-MMAE]6 (D6-type ADC)

[1013] The method of Examples II.2-II.3 is similar to that of Example II.1, except that the antibody is gosartuzumab / mabelantuzumab, the buffer system is MES buffer and Zn 2+ The molar ratio of TCEP is 0.87:1.

[1014] Example II.4.: Preparation of Antibody-[MC-VC-PAB-MMAE]6 (D6-type ADC)

[1015] (1) ZnCl2 (0.024 mM) and the reducing agent TCEP-NO-Trtyl (0.048 mM) were added to a solution of the monoclonal antibody trastuzumab (0.012 mM, dissolved in BES buffer, pH 7.0, 20 mM), and the reaction mixture was placed at 4°C for 18 hours;

[1016] (2) EDTA-2Na (0.6 mM) and MC-VC-PAB-MMAE (0.096 mM) in DMA were introduced and the reaction was continued at room temperature for 1 hour;

[1017] (3) Purify the reaction mixture using a desalting column.

[1018] Examples II.5-II.29. and Comparative Example II.1: Preparation of Antibody-[MC-VC-PAB-MMAE]6 (D6-type ADC)

[1019] The methods of Examples II.5-II.29 and Comparative Example II.1 are the same as those of Example II.4, except that the types of reducing agent and antibody, the molar ratio of reducing agent to antibody and / or Zn 2+ The molar ratio of the reducing agent to the reducing agent is shown in Table II-1. Meanwhile, in Examples II.27-II.29, the incubation time in step (1) is 16 hours.

[1020] Table II-1

[1021]

[1022]

[1023] The homogeneity test results and chromatographic peak areas of Examples II.1-II.29 and Comparative Example II.1 are shown in Table II-2 and Table II-3. The chromatograms of EII.1 and EII.4 are shown in Table II-3. Figure 16-17 shown.

[1024] Table II-2

[1025]

[1026]

[1027] Table II.3

[1028]

[1029] As shown in the table above, the linker-payload (MC-VC-PAB-MMAE) was successfully linked to the antibody, indicating that the ADCs of Examples II.1-II.29 prepared by the reducing agent in the present invention were successfully synthesized.

[1030] In addition, as shown in the results of Examples [II.4-II.29], compared with the use of Zn-free 2+ Compared with the traditional method of reducing TCEP, the compounds of the present invention can improve the uniformity of D6-type ADC, among which TCEP-3 has the best selective reduction ability and the D6 content is as high as 95.52%. At the same time, TCEP-1, TCEP-2, TCEP-10, TCEP-25, TCEP-26, TCEP-28, TCEP-30, TCEP-31 and TCEP-33 also have good selective reduction ability and the D6 content is as high as 90%.

[1031] According to the results of Examples II.1-II.3 and II.25-II.26, the reducing agent in the present invention is suitable for preparing ADCs with different antibodies.

[1032] Examples II.30-II.60 Preparation of antibody-[MC-VC-PAB-MMAE]6 conjugates (the molar ratio of reducing agent to antibody and / or reduction time in step (1) are different)

[1033] When the reducing agent is TCEP, the method is similar to Example II.1, and when the reducing agent is not TCEP, the method is similar to Example II.4. The difference is the concentration of ZnCl2 and the reducing agent in step (1) and / or the reduction time in step (1), as shown in Table II-4. Meanwhile, the antibody in Examples II.31-II.32 is Mabelantuzumab.

[1034] Table II-4

[1035]

[1036] The homogeneity test results and chromatographic peak areas of Examples II.30-II.60 are shown in Table II-5 and Table II-6.

[1037] Table II-5

[1038]

[1039] Table II-6

[1040]

[1041]

[1042] As shown in the results in Table II-5, the linker-payload (MC-VC-PAB-MMAE) was successfully linked to the antibody, indicating that the ADCs of Examples II.30-II.60 prepared by TCEP, TCEPA or TCEP-NO were successfully synthesized.

[1043] As shown in Examples II.30-II.39, using TCEP as a reducing agent, when the molar ratio of TCEP to antibody is 3:1 to 13:1, the content of D6 is as high as 55%, 80%, and even 85% and 87%. When the molar ratio of TCEP to antibody is 8:1 to 13:1, the reduction time in step (1) is shortened to 6h. When the molar ratio of TCEP to antibody is 8:1 to 12:1 and the reduction time in step (1) is 6 hours, the content of D6 is as high as 87% or 89%. The results show that the molar ratio of TCEP / antibody plays an important role in determining the content and selective reduction of D6.

[1044] According to the results of Examples II.40-II.60, using TCEP-NO or TCEPA as a reducing agent, when the molar ratio of ZnCl2 to the monoclonal antibody is 2 and the molar ratio of the reducing agent to the antibody is 2.8: 1 to 4.6: 1, the highest proportion of D6 is 84.71%. And, when the molar ratio of ZnCl2 to the monoclonal antibody is 1 and the molar ratio of the reducing agent to the antibody is 3.8: 1 to 13: 1, D6 is at least 40%. The resulting ADC with a higher level of D6 indicates that the molar ratio of the reducing agent to the antibody in the range of 2.8 to 13 is beneficial to improve the uniformity of the ADC.

[1045] According to Examples II.52-II.60, when the molar ratio of the reducing agent to the antibody is 5:1 to 13:1, the reduction time in step (1) is shortened to 6 hours. When the molar ratio of the reducing agent to the antibody is 6:1 to 13:1, and the reduction time in step (1) is 6 hours, the content of D6 is as high as 65%, 70%, or even as high as 75% or 80%.

[1046] Examples II.61-II.87 and Comparative Examples II.2-II.9: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugates (Zn 2+ different molar ratios with TCEP and / or different reduction times in step (1))

[1047] When the reducing agent is TCEP, the method is similar to Example II.1, and when the reducing agent is not TCEP, the method is similar to Example II.4. The difference lies in the concentration of ZnCl2, the concentration of TCEP and / or the reduction time in step (1). The parameters in step (1) are shown in Table II-7.

[1048] The method of Comparative Example II.2 is the same as that of Example II.1, Comparative Example II.3 is the same as that of Example II.34, Comparative Example II.4 is the same as that of Example II.36, Comparative Example II.5 is the same as that of Example II.38, Comparative Example II.6 is the same as that of Example II.4, Comparative Example II.7 is the same as that of Example II.55, Comparative Example II.8 is the same as that of Example II.57, and Comparative Example II.9 is the same as that of Example II.59, except that the concentration of ZnCl2 in step (1) is 0 and the reducing agent in Comparative Example II.6 is TCEPA.

[1049] Table II-7

[1050]

[1051]

[1052] mAb is the abbreviation for monoclonal antibody.

[1053] The uniformity measurement results and chromatographic peak areas of Examples II.61-II.87 and Comparative Examples II.2-II.9 are shown in Table II-8 and Table II-9.

[1054] Table II-8

[1055]

[1056]

[1057] Table II-9

[1058]

[1059]

[1060] As shown in Table II-8, the linker-payload (MC-VC-PAB-MMAE) was successfully linked to trastuzumab, indicating that the TCEP / TCEPA / TCEP-NO and Zn 2+ The ADCs prepared in Examples II.61-II.87 were successfully synthesized.

[1061] According to the results in Table II-8, the proportion of D6 in Comparative Examples II.2-II.9 is only 11%, 17%, 20% or 31%, but the proportion of D6 in Examples II.61-II.87 is at least 60%. 2+ It is very important to improve the uniformity of ADC with D6. 2+ When the molar ratio of the reducing agent is within the range of 0.05 to 30, it is beneficial to improve the uniformity of ADC.

[1062] Examples II.88-II.110 and Comparative Examples II.10-II.11: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates by using different buffer systems

[1063] When the reducing agent is TCEP, the method is similar to Example II.1, and when the reducing agent is not TCEP, the method is similar to Example II.79. The difference lies in the molar ratio of TCEPA to antibody and / or the buffer system, as shown in Table II-10.

[1064] Table II-10

[1065]

[1066]

[1067] The uniformity measurement results and chromatographic peak areas of Examples II.88-II.110 and Comparative Examples II.10-II.11 are shown in Table II-11 and Table II-12.

[1068] Table II-11

[1069] N0. reducing agent D0(％) D2(％) D4(％) D6(％) D8(％) E II.88 TCEP 0 5 11 79 5 E II.89 TCEP 0 11 31 56 2 E II.90 TCEP 0 2 7 83 8 E II.91 TCEP 0 4 7 84 5 E II.92 TCEP 0 1 4 82 13 E II.93 TCEP 0 0 2 87 11 E II.94 TCEP 0 0 3 88 9 E II.95 TCEP 0 0 4 87 9 E II.96 TCEP 0 2 5 86 7 E II.97 TCEP 0 0 5 87 8 E II.98 TCEP 0 1 4 88 7 C II.10 TCEP 2 36 46 16 0 C II.11 TCEP 0 8 33 47 12 E II.99 TCEPA 0 2 5 85 8 E II.100 TCEPA 0 0 4 87 9 E II.101 TCEPA 0 0 4 88 8 E II.102 TCEPA 0 3 5 85 7 E II.103 TCEPA 0 0 7 86 7 E II.104 TCEPA 0.96 3.89 4.05 84.65 6.46 E II.105 TCEPA 1.09 11.27 14.10 71.45 2.09 E II.106 TCEPA 0 1.22 4.83 86.55 7.40 E II.107 TCEPA 1.04 4.67 5.54 83.54 5.21 E II.108 TCEPA 0 3.70 3.90 88.11 4.28 E II.109 TCEPA 1.41 8.83 20.36 66.55 2.85 E II.110 TCEPA 0 7.59 17.42 52.10 22.89

[1070] Table II-12

[1071]

[1072] As shown in the results in Table II-11, the linker-payload (MC-VC-PAB-MMAE) was successfully linked to trastuzumab, indicating that the ADC in different buffers had been successfully synthesized.

[1073] According to the results of Table II-11, it is shown that the type of buffer system will affect the content of D6 by affecting the reduction kinetics and selectivity. When TCEP is used, the buffer system of Examples II.88-II.98 can be used to increase the content of D6. When TCEPA is used, the proportion of D6 in Examples II.99-II.110 exceeds 50%. In addition, in Examples II.99-II.108, the proportion of D6 exceeds 70%, and the highest proportion of D6 is 88%.

[1074] Examples II.111-II.119: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates at different pH values

[1075] When the reducing agent is TCEP, the method is similar to Example II.1, and when the reducing agent is not TCEP, the method is similar to Example II.79. The difference lies in the pH value of the buffer system, the molar ratio of TCEPA to the antibody and / or the type of the buffer system, as shown in Table II-13.

[1076] Table II-13

[1077]

[1078]

[1079] The uniformity test results and chromatographic peak areas of Examples II.111-II.119 are shown in Table II-14 and Table II-15.

[1080] Table II-14

[1081] serial number reducing agent D0 (wt%) D2 (wt%) D4 (wt%) D6 (wt%) D8 (wt%) E II.111 TCEP 0 4 10 79 7 E II.112 TCEP 0 0 7 89 4 E II.113 TCEP 0 0 6 90 4 E II.114 TCEP 0 0 5 90 5 E II.115 TCEPA 0 6.21 8.05 80.67 5.07 E II.116 TCEPA 0 5.34 13.90 77.02 3.75 E II.117 TCEPA 0 2.73 14.61 79.28 3.38 E II.118 TCEPA 1.43 14.67 25.41 58.49 0 E II.119 TCEPA 0 13.45 24.28 60.53 1.74

[1082] Table II-15

[1083]

[1084] As shown in the results in Table II-14, the linker-payload (MC-VC-PAB-MMAE) was successfully linked to trastuzumab, indicating that ADCs with buffer pH values ​​ranging from 5.8 to 7.4 were successfully synthesized.

[1085] Examples II.120-II.126: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates in buffer systems of different concentrations

[1086] When the reducing agent is TCEP, the method is similar to Example II.1, and when the reducing agent is TCEPA, the method is similar to Example II.79. The difference lies in the concentration of the buffer system and / or the molar ratio of TCEPA to the antibody, as shown in Table II-16.

[1087] Table II-16

[1088]

[1089] The uniformity test results and chromatographic peak areas of Examples II.120-II.126 are shown in Table II-17 and Table II-18.

[1090] Table II-17

[1091]

[1092]

[1093] Table II-18

[1094]

[1095] As shown in Table II-17, the results show that the concentration of the buffer system of Examples II.120-II.126 helps to increase the content of D6. Examples II.127-II.143: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (different reduction time and / or reducing agent temperature in step (1))

[1096] When the reducing agent is TCEP, the method is similar to Example II.1, and when the reducing agent is TCEPA, the method is similar to Example II.79. The difference lies in the reduction time and / or reducing agent temperature, reducing agent, molar ratio of reducing agent to antibody and / or molar ratio of ZnCl2 to antibody in step (1), see Table II-19 for details.

[1097] Table II-19

[1098]

[1099] The homogeneity test results and chromatographic peak areas of Examples II.127-II.143 are shown in Table II-20 and Table II-21.

[1100] Table II-20

[1101]

[1102]

[1103] Table II-21

[1104]

[1105] As shown in Table II.20, using TCEP, 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 to 10 hours, the content of D6 is as high as 86%, even reaching 90%, which indicates that by increasing the molar ratio of TCEP to antibody, the method shown here reduces the reduction time cost. At the same time, when the reducing agent temperature in step (1) is 0°C to 25°C, the content of D6 is as high as 80%, 85%, and even 88%.

[1106] As shown in Examples II.135-II.140, the results show that when the molar ratio of the reducing agent to the antibody is 3.5:1, 9:1 or 10:1 and the reducing agent time in step (1) is 4 hours to 22 hours, the content of D6 is as high as 75%, even reaching 80%, 85% or 88%, which also shows that when the molar ratio of the reducing agent to the antibody is increased, the method shown in this article reduces the reduction time cost. At the same time, the results show that when the reducing agent temperature in step (1) is 4°C to 25°C, the content of D6 is as high as 75%, 85%, and even 87%.

[1107] Example II.144: Preparation of trastuzumab-[bismaleimide-DBCO]3 conjugate

[1108] 1. Synthesis of bismaleimide-DBCO (thio bridge)

[1109]

[1110] Intermediate 2: DBU (30.7 g, 0.2 mol, 4.0 equiv) was added to a mixture of 1 (6.5 g, 50.0 mmol, 1.0 equiv) in DMF (100 mL). The mixture was stirred at 80 ° C for 1 h, and 2-bromoacetic acid benzyl ester (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 tower (EtOAc / petroleum ether=0-40%) to give product 2 (4.5 g, 21.0%) as a white solid.

[1111] Intermediate 3: DBU (3.22 g, 21.16 mol, 2.0 equiv) was added to a mixture of intermediate 2 (4.5 g, 10.58 mmol, 1.0 equiv) in DMF (50 mL). 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 tower (EtOAc / petroleum ether=0-40%) to give product 3 (4.8 g, 79.8%) as a white solid.

[1112] 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 equiv) in THF (50 mL). The mixture was degassed 3 times and purged with H2. Under 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 the product 4 (3.0 g, 91.5%) as a white solid.

[1113] Intermediate 5: To a mixture of intermediate 4 (50 mg, 1.00 mmol, 1.0 eq.) in DCM (5 mL) was added HATU (0.92 g, 2.4 mmol, 2.4 eq.) and DIPEA (0.65 g, 5.0 mmol, 5.0 eq.). The mixture was stirred at room temperature for 0.5 h, and 4a (0.51 g, 2.0 mmol, 2.0 eq.) was added. The resulting mixture was stirred at room temperature for 2 h. LCMS showed that intermediate 4 was completely consumed. The mixture was quenched by adding HCl (0.5 M, 2 mL), purified by RP column (water / MeCN=10%-70%), and the eluent was lyophilized to give the product 5 (0.51 g, 80.3%) as a white solid.

[1114] Intermediate 6: TFA (100 uL) was added to a mixture of intermediate 5 (100 mg, 0.16 mmol, 1.0 equiv) in DCM (2 mL). 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.

[1115] 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, the residue was purified by preparative HPLC (water / MeCN=10-50%), and the eluent 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. 1H 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).

[1116] 2. Preparation of Trastuzumab-[Bismaleimide-DBCO]3

[1117] (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 to mix, and then the reaction mixture was incubated at 4°C for 18 h;

[1118] (2) EDTA (0.6 mM) and bismaleimide-DBCO (0.045 mM) were introduced to react with the reduced thiol groups produced in step (1) at room temperature for 1 hour, and then trastuzumab-[bismaleimide-DBCO]3 was recovered using a desalting column.

[1119] Example II.145: Preparation of trastuzumab-[bismaleimide-DBCO]3 conjugate

[1120] (1) ZnCl2 (0.024 mM) and the reducing agent TCEPA (0.048 mM) were added to a solution of the monoclonal antibody trastuzumab (0.012 mM, in BES buffer, pH 7.0, 20 mM), and the reaction mixture was allowed to stand at 4°C for 18 hours;

[1121] (2) EDTA-2Na (0.6 mM) and bismaleimide-DBCO (0.045 mM) dissolved in DMA were introduced and the reaction was continued at room temperature for 1 hour, and then trastuzumab-[bismaleimide-DBCO]3 was recovered using a desalting column.

[1122] The uniformity test results and chromatographic peak areas of Examples II.144-II.145 are shown in Table II-22 and Table II-23.

[1123] Table II-22

[1124] serial number Attached photos D0(％) D1(％) D3(％) D4(％) E II.144 18 0 0 86.55 13.45 E II 145 19 0 0 82.93 17.07

[1125] Table II-23

[1126]

[1127] As shown in the table above, the results show that the content of trastuzumab-[bismaleimide-DBCO]3 is usually as high as 82% or 86%, which indicates that the process of this method is conducive to site-specific modification of D3-type antibodies and can improve uniformity.

[1128] Example II.146: Preparation of trastuzumab-[MC-GGFG-DXd]6[MC-VC-PAB-MMAE]2 (D6+D2 type ADC) (1) ZnCl2 (0.048 mM) and reducing agent TCEP (0.048 mM) were added to a BES (pH 7.0, 20 mM) buffer solution of monoclonal antibody trastuzumab (0.012 mM), and the reaction mixture was placed at 4°C for 18 h;

[1129] (2) Introduce EDTA-2Na (0.6 mM) and MC-GGFG-DXd (0.1 mM) dissolved in DMA,

[1130] (1) and continue the reaction at room temperature for 1 hour;

[1131] (2) The obtained product and the second reducing agent TCEP (0.02 mM) were incubated at room temperature for 3 h; the second linker-payload MC-VC-PAB-MMAE (0.05 mM) was added and the reaction was continued at room temperature for 2 h.

[1132] (3) Purify the obtained product using a desalting column

[1133] Example II.147: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6[MC-GGFG-DXd]2 (ADC with D6+D2)

[1134] (1) ZnCl2 (0.024 mM) and reducing agent TCEPA (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;

[1135] (2) EDTA-2Na (0.6 mM) and MC-VC-PAB-MMAE (0.08 mM) in DMA were introduced and the reaction was continued at room temperature for 1 hour;

[1136] (3) The product obtained in step (2) was incubated with the second reducing agent TCEP (0.02 mM) at 37°C for 3 hours; the second linker-payload MC-GGFG-DXd (0.05 mM) was added and the reaction was continued at room temperature for 2 hours.

[1137] (4) Purify the obtained product using a desalting column

[1138] The uniformity test results and chromatographic peak areas of Examples II.146-II.147 are shown in Table II-24 and Table II-25.

[1139] Table II-24

[1140] serial number Attached photos D2(％) D4(％) D6(％) D8(％) E II.146-Step (2) 20A 0 2.24 88.82 8.93 E II.147-Step (2) 21A 4.48 4.97 86.49 4.06 serial number Attached photos / D4+D2(%) D6+D2(%) D8(％) E II.146-Step (4) 20B / 3.58 87.10 9.32 serial number Attached photos D2(％) D4(％) D6+D2(%) D8(％) E II.147-Step (4) 21B 3.67 3.88 84.81 7.65

[1141] Table II-25

[1142]

[1143] As shown in the table above, the results indicate that the D6+D2 ADC content is typically as high as 84.81% or 87.10%, which indicates that the process of this method is beneficial to the site-specific modification of the D6+D2 antibody and can improve the uniformity.

[1144] Example II.148: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6[maleimide-PEG4-N3-DBCO-Cy3]1 (D6+D1 type ADC)

[1145] (1) ZnCl2 (0.0408 mM) and TCEP (0.024 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;

[1146] (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;

[1147] (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 was continued at room temperature for 2 h; then the second linker-payload DBCO-Cy3 (0.05 mM) was added, and the reaction was continued at room temperature for 4 h.

[1148] (4) Purify the obtained product using a desalting column.

[1149] Example II.149: Preparation of trastuzumab-[MC-GGFG-DXd]6[maleimide-PEG4-N3-DBCO-Cy3]1 (ADC with D6+D1)

[1150] (1) ZnCl2 (0.024 mM) and reducing agent TCEPA (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;

[1151] (2) EDTA-2Na (0.6 mM) and MC-GGFG-DXd (0.08 mM) in DMA were introduced and the reaction was continued at room temperature for 1 hour, and then the product was recovered using a desalting column;

[1152] (3) Incubate the product obtained in step (2) and the second reducing agent TCEP (0.02 mM) at room temperature for 2 hours; add the thio-bridging reagent [dibromomaleimide-PEG4-N3] (0.012 mM) and continue the reaction at room temperature for 2 hours; then add the second linker-payload toxin [DBCO-Cy3] (0.05 mM) and continue the reaction at room temperature for 4 hours.

[1153] (4) Purify the obtained product using a desalting column.

[1154] The uniformity test results and chromatographic peak areas of Examples II.148-II.149 are shown in Table II-26 and Table II-27.

[1155] Table II-26

[1156] serial number Attached photos D2(％) D4(％) D6(％) D8(％) E II.148-Step (2) 22A 4.48 4.97 86.49 4.06 E II.149-Step (2) 23A 0 4.58 92.20 3.23 serial number Attached photos D4+D1(%) D6+D1(%) D8(％) / E II.148-Step (4) 22B / 89.89 10.11 / E II.149-Step (4) 23B 4.26 92.25 3.50 /

[1157] Table II-27

[1158]

[1159] As shown in the table above, the results indicate that the D6+D1 ADC content is typically as high as 89.89% or 92.25%, which indicates that the process of this method is beneficial to the site-specific modification of the D6+D1 antibody and can improve uniformity.

[1160] Example II.150: Preparation of trastuzumab-[maleimide]6[MC-VC-PAB-MMAE]2 (D0+D2 type ADC)

[1161] (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 to mix, and then the reaction mixture was incubated at 4°C for 18 h;

[1162] (2) introducing EDTA (0.6 mM) and (2-aminoethyl)maleimide (0.1 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-[maleimide] 6;

[1163] (3) Trastuzumab-[maleimide]6 and 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 was continued at room temperature for 2 h.

[1164] (4) Purify the obtained product using a desalting column.

[1165] Example II.151: Preparation of trastuzumab-[maleimide]6[MC-VC-PAB-MMAE]2 (D0+D2 type ADC)

[1166] This method is similar to Example II.150, except that the reducing agent in step (1) is TCEPA, and the incubation temperature of trastuzumab-[maleimide]6 and the reducing agent TCEP in step (3) is 37°C.

[1167] The uniformity test results and chromatographic peak areas of Examples II.150-II.151 are shown in Table II-28 and Table II-29.

[1168] Table II-28

[1169] serial number Attached photos D0(％) / / / E II.150-Step (2) 24A 100 / / / E II.151-Step (2) 25A 100 / / / serial number Attached photos D0 D0+D1(%) D0+D2(%) D0+D3(%) E II.150-Step (4) 24B 11.66 6.31 70.92 11.11 E II.151-Step (4) 25B 24.30 5.18 70.53 0

[1170] Table II-29

[1171]

[1172] As shown in the table above, the results indicate that the content of ADC with D0+D2 is generally as high as 70%, which indicates that this method is beneficial for site-specific modification of antibodies with D0+D2 and improves uniformity.

[1173] Example II.152: Preparation of trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1 (D0+D1 type ADC)

[1174] (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 to mix, and then the reaction mixture was incubated at 4°C for 18 h;

[1175] (2) introducing EDTA (0.6 mM) and (2-aminoethyl)maleimide (0.1 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-[maleimide] 6;

[1176] (3) Trastuzumab-[maleimide]2 and the second reducing agent TCEP (0.02 mM) were incubated at room temperature for 18 h. The second sulfur bridge reagent having a reactive group dibromomaleimide-PEG4-N3 (0.013 mM) reacted with the reduced thiol group at a temperature of 24 °C and a reaction time of 3 h;

[1177] (4) The obtained 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.

[1178] Example II.153: Preparation of trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1 (D0+D1 type ADC)

[1179] This method is similar to Example II.152, except that the reducing agent in step (1) is TCEPA, and the incubation temperature of trastuzumab-[maleimide]2 and TCEP in step (3) is 37°C.

[1180] The uniformity test results and chromatographic peak areas of Examples II.152-II.153 are shown in Table II-30 and Table II-31.

[1181] Table II-30

[1182]

[1183]

[1184] Table II-31

[1185]

[1186] As shown in the table above, the results indicate that the D0+D1 type ADC content is typically as high as 80% or 92.96%, which indicates that the process of this method is beneficial to the site-specific modification of the D0+D1 type antibodies and can improve the uniformity.

[1187] Example III.1: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate (D2-type ADC)

[1188] (1) ZnCl2 (0.012 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 18 hours;

[1189] (2) DHAA (0.096) was added and vortexed to mix. The mixture was then incubated in the dark. The reaction conditions were as shown in Table 8 below;

[1190] (3) Purify using a desalting column to remove excess DHAA, if necessary;

[1191] (4) EDTA (0.06 mM) and MC-VC-PAB-MMAE (0.048 mM) in DMA were added, and the mixture was incubated at room temperature for 1 hour.

[1192] (5) Purify the reaction mixture using a desalting column.

[1193] Example III.2-III.53: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate (D2-type ADC)

[1194] The method is similar to Example III.1, except for the reducing agent and / or parameters in step (1) and step (2), which are shown in Table III-1 and Table III-2. Meanwhile, in Examples III.40-III.53, the oxidation time in step (2) is 2 hours.

[1195] Table III-1

[1196]

[1197]

[1198] Table III-2

[1199]

[1200] Comparative Examples III.1-III.3: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugate (Zn 2+Different molar ratio with TCEP)

[1201] The method of Comparative Example III.1 is the same as that of Example III.29, Comparative Example III.2 is the same as that of Example III.32, and Comparative Example III.3 is the same as that of Example III.35, except that the concentration of ZnCl2 in step (1) is 0.

[1202] The uniformity measurement results and chromatographic peak areas of Examples III.1-III.53 and Comparative Examples III.1-III.3 are shown in Table III-3 to Table II-6.

[1203] Table III-3

[1204]

[1205]

[1206] Table III 4

[1207]

[1208]

[1209] Table III-5

[1210]

[1211] Table III-6

[1212]

[1213]

[1214] As shown in the results in Table III-3-III-6, the linker-payload (MC-VC-PAB-MMAE) was successfully linked to trastuzumab, indicating that the D2-type ADC was successfully synthesized.

[1215] As shown in Examples III.25-III.53, when the molar ratio of TCEP to antibody is 4:1 to 10:1, the content of D6 is as high as 60%, 70%, 80%, and even 85% and 90%. At the same time, the reduction time in step (1) is shortened to 4 hours or 1 hour, which reduces the reduction time cost. The results also show that the molar ratio of DHAA to antibody plays an important role in determining the content and selective oxidation of D2. When the molar ratio of DHAA to antibody is 4:1 to 24:1, the content of D2 is as high as 60%, 70%, 80%, 85%, and even 90% or 95%.

[1216] Zn 2+The molar ratio of Zn to antibody also affects the content and selective oxidation of D2. 2+ When the concentration of Zn is 0, the content of D2 is as low as 7.87%. 2+ When the molar ratio of the D2-antibody is 1:1 or 2:1, it helps to improve the uniformity of the D2-type ADC.

[1217] When purification was performed after oxidation, the proportion of D6 was as high as 95%, indicating that the purification operation can significantly improve the homogeneity of ADCs with D2.

[1218] These results clearly demonstrate that the process of this approach is beneficial for significantly improving the homogeneity of D2-type conjugates.

[1219] Examples III.54-III.60: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate (different oxidation temperatures and / or times in step (2))

[1220] The method is similar to Example III.1, except for the oxidation temperature, time, type of reducing agent and / or molar ratio of reducing agent to antibody in step (2), see Table III-7 for details.

[1221] The uniformity measurement results and chromatographic peak areas of Examples III.54-III.60 are shown in Table III-7-Table II-8.

[1222] Table III-7

[1223]

[1224]

[1225] Table III-8

[1226]

[1227] As shown in the above table, the results show that when the oxidation temperature in step (2) is 4°C to 37°C, the content of D2 is as high as 70%, even reaching 90%, and the oxidation time in step (2) is 1 hour to 48 hours.

[1228] Examples III.61-III.92 and Comparative Examples III.4-III.5: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugates by Using Different Buffer Systems

[1229] The methods of the examples and comparative examples are similar to those of Example III.1, except for the buffer system or reducing agent, which are shown in Table III-9.

[1230] Meanwhile, in Examples III.61-III.92 and Comparative Examples 4-5, the oxidation time in step (2) was 2 hours. The molar ratio of the reducing agent to the antibody in Examples III.77-III.92 and Comparative Example III.5 was 3.5:1.

[1231] The uniformity measurement results and chromatographic peak areas of Examples III.61-III.92 are shown in Table III-9-Table III-10.

[1232] Table III-9

[1233]

[1234]

[1235] Table III-10

[1236]

[1237] As shown in the table above, the results show that the type and pH of the buffer system will affect the content of D2 by affecting the reduction kinetics and selectivity. The buffer system of Examples III.61-III.92 can be used to increase the content of D2, and the pH value of the buffer system is 5.8 to 7.4.

[1238] Example III.93: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1 (D1-type ADC)

[1239] (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 to mix, and then the reaction mixture was incubated at 4°C for 18 h;

[1240] (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;

[1241] (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;

[1242] (4) In BES buffer (20 mM, pH 7.0), trastuzumab-[maleimide-PEG4-N3]1 and DBCO-MMAE (0.02 mM) were incubated at 25°C for 8 h.

[1243] (5) The reaction mixture was purified by HIC chromatography using a desalting column and AKTA.

[1244] Example III.94: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 (D1-type ADC)

[1245] This method is similar to Example III.93. The difference is that the reducing agent is TCEPA, the concentration of DHAA in step (2) is 0.096 mM and the first linker-payload is DBCO-Cy3. Meanwhile, in step (5) of Example III.94, the reaction mixture is purified using a desalting column.

[1246] The uniformity measurement results and chromatographic peak areas of Examples III.93-III.94 are shown in Table III-11.

[1247] Table III-11

[1248]

[1249] As shown in the table above, the results indicate that the D1-type ADC content is typically as high as 83.81% or 97.58%, which indicates that the process of this method is beneficial to the site-specific modification of the D1-type antibody and can improve the uniformity.

[1250] Example III.95: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1[MC-GGFG-DXd]6 (D1+D6 type ADC)

[1251] (1) Trastuzumab-[maleimide-PEG4-N3-DBCO-MMAE]1 (0.008 mM) prepared in Example III.93 and TCEP (0.08 mM) in BES buffer (20 mM, pH 7.0) were introduced at a reaction temperature of 37° C. and a reaction time of 16 hours;

[1252] (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.

[1253] Example III.96: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]6 (D1+D6 type ADC)

[1254] (1) Trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 (0.008 mM) prepared in Example III.94 and TCEPA (0.08 mM) in BES buffer (20 mM, pH 7.0) were introduced at a reaction temperature of 24°C and a reaction time of 24 hours;

[1255] (2) MC-VC-PAB-MMAE (0.14 mM) was introduced into the solution from step (1), and the reaction mixture was allowed to stand at 24°C for 1 hour, and then trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]6 was recovered using a desalting column.

[1256] The uniformity measurement results and chromatographic peak areas of Examples III.95-III.96 are shown in Table III-12 and Table III-13.

[1257] Table III-12

[1258] serial number Attached photos D1+D2(%) D1+D4(%) D1+D6(%) D8％ E III.95 30 5.66 5.98 82.42 5.94 serial number Attached photos D6(％) D1+D6(%) D2+D6(%) / E III.96 31 7.79 88.02 4.20 /

[1259] Table III-13

[1260]

[1261] As shown in the table above, the results indicate that the D1+D6 ADC content is typically as high as 82.42% or 88.02%, which indicates that the process of this method is beneficial to the site-specific modification of the D1+D6 antibody and can improve uniformity.

[1262] Example III.97: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2 (D1+D2 type ADC)

[1263] (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 to mix, and then the reaction mixture was incubated at 4°C for 18 h;

[1264] (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;

[1265] (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;

[1266] (4) incubating trastuzumab-[maleimide-PEG4-N3]1 (0.013 mM) at a reaction temperature of 25°C for 1 h, and then incubating DBCO-Cy3 (0.02 mM) in BES buffer (20 mM, pH 7.0) at a reaction temperature of 25°C for 8 h;

[1267] (5) purifying the reaction mixture using a desalting column;

[1268] (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;

[1269] (7) Introducing EDTA (3 mM) to capture Zn 2+ , and introducing MC-VC-PAB-MMAE (0.048 mM) to react with the reduced thiol groups from step (6), the reaction temperature was 25 ° C and the reaction time was 2 h;

[1270] (8) Purify the reaction mixture using a desalting column.

[1271] Example III.98: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2 (D1+D2 type ADC)

[1272] (1) Incubate ZnCl2 (0.8 mM), the second reducing agent TCEP-6 (0.0144 mM) and trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 (0.008 mM) prepared in Example III.94 in BES buffer (pH 7.0, 20 mM), and place the reaction mixture at 4°C for 4 hours;

[1273] (2) Introduction of EDTA (3 mM) to capture Zn 2+ , and introducing MC-VC-PAB-MMAE (0.048 mM) to react with the reduced thiol groups produced in step (2), the reaction temperature is 25° C., and the reaction time is 2 hours;

[1274] (3) Purify the reaction mixture using a desalting column.

[1275] The uniformity measurement results and chromatographic peak areas of Examples III.97-III.98 are shown in Table III-14 and Table III-15.

[1276] Table III-14

[1277] serial number Attached photos D1(％) D2(％) D1+D2(%) D4(％) D1+D4(%) E III.97 32 2.84 2.39 71.97 2.49 20.31 E III.98 33 3.72 7.92 73.80 4.06 10.51

[1278] Table III-15

[1279]

[1280] One of the interchain disulfide bonds in the D1-type ADC was reduced by using step (6) in Example III.97 and step (1) in Example III.98. As shown in the table above, the results show that the content of D1+D2-type ADC is usually as high as 71.97% or 73.8%, which indicates that the process of this method is conducive to the site-specific modification of D1+D2-type antibodies and can improve uniformity.

[1281] Examples III.99-III.100: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 (D1+D4 type ADC)

[1282] (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 to mix, and then the reaction mixture was incubated at 4°C for 18 h;

[1283] (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;

[1284] (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), the reaction temperature is 25 ° C and the reaction time is 2 h, and then the product is recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1;

[1285] (4) incubating ZnCl2 (Example III.99: 0.024 mM; Example III.100: 0.36 mM), the second reducing agent TCEP (0.024 mM) and the product from step (3) (0.008 mM), and placing the reaction mixture at 4°C for 16 h;

[1286] (5) Introducing EDTA (3 mM) to capture Zn 2+ , and introducing 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;

[1287] (6) Purify the reaction mixture using a desalting column.

[1288] Examples III.101-III.102: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 (D1+D4 type ADC)

[1289] (1) TCEPA (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 to mix, and then the reaction mixture was incubated at 4°C for 14 h;

[1290] (2) adding DHAA (0.096 mM) to selectively reoxidize the reduced thiol groups mainly located in the Fab region generated in step (1) at 25° C. for 2 hours;

[1291] (3) introducing EDTA (0.6 mM) and dibromomaleimide-PEG4-N3 (0.013 mM) (i.e., the first thio-bridging reagent) to react with the reduced thiol group produced in step (3), the reaction temperature is 25° C., and the reaction time is 1.5 hours, and then the product is recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1;

[1292] (4) incubating trastuzumab-[maleimide-PEG4-N3]1 (0.013 mM) at 25°C for 8 hours, and then introducing DBCO-Cy3 (0.02 mM) in BES buffer (20 mM, pH 7.0) at 25°C for 8 hours;

[1293] (5) purifying the reaction mixture using a desalting column;

[1294] (6) incubating ZnCl2 (0.8 mM), a second reducing agent TCEP-3 (Example III.101: 0.0384 mM) or TCEP-6 (Example III.102: 0.0336 mM) and the product from step (5) in BES buffer (pH 7.0, 20 mM) and placing the reaction mixture at 4°C for 16 hours;

[1295] (7) Introduction of EDAT (3 mM) to capture Zn 2+ , and introducing MC-VC-PAB-MMAE (0.08 mM) to react with the reduced thiol groups produced in step (6), the reaction temperature is 25° C., and the reaction time is 2 hours;

[1296] (8) Purify the reaction mixture using a desalting column.

[1297] The uniformity measurement results and chromatographic peak areas of Examples III.99-III.102 are shown in Table III-16 and Table III-17.

[1298] Table III-16

[1299]

[1300] Table III-17

[1301]

[1302] Two of the interchain disulfide bonds in the D1-type ADC were reduced by using step (4) in Example 99 and by using step (6) in Example 101. As shown in the table above, the results confirmed that the content of the D1+D4-type ADC was generally as high as 80%, indicating that the process of this method is conducive to site-specific modification of the D1+D4-type antibody and can improve uniformity.

[1303] Example III.103: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]4 conjugate (D2+D4 type ADC)

[1304] (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 to mix, and then the reaction mixture was incubated at 4°C for 16 h;

[1305] (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;

[1306] (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 in step (2), and the reaction was incubated at room temperature for 1 h;

[1307] (4) purifying the reaction mixture using a desalting column;

[1308] (5) incubating ZnCl2 (0.552 mM), a second reducing agent TCEP (1.2 mM) and the product from step (4) in BES buffer (pH 7.0, 20 mM), and placing the reaction mixture at 4 ° C for 2.5 h;

[1309] (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;

[1310] (7) Purify the reaction mixture using a desalting column.

[1311] Example III.104: Preparation of trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4 (D2+D4 type ADC)

[1312] (1) TCEPA (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 to mix, and then the reaction mixture was incubated at 4°C for 16 h;

[1313] (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;

[1314] (3) EDTA (0.6 mM) and MC-GGFG-DXd (0.048 mM) in DMA were introduced to react with the retained thiol groups in step (2) and incubated at room temperature for 1.5 hours;

[1315] (4) purifying the reaction mixture using a desalting column;

[1316] (5) incubating ZnCl2 (0.8 mM), a second reducing agent TCEP-3 (0.072 mM) and the product from step (4) in BES buffer (pH 7.0, 20 mM), and allowing the reaction mixture to stand at 4°C for 16 hours;

[1317] (6) Introduction of EDAT (3 mM) to capture Zn 2+ , and introducing MC-VC-PAB-MMAE (0.08 mM) to react with the reduced thiol groups from step (5), the reaction temperature was 25 ° C and the reaction time was 2 h;

[1318] (7) Purify the reaction mixture using a desalting column.

[1319] Example III.105-106: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]4 conjugate (D2+D4 type ADC)

[1320] (1) TCEPA (0.042 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 to mix, and then the reaction mixture was incubated at 4°C for 16 h;

[1321] (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;

[1322] (3) EDTA (0.12 mM) and MC-VC-PAB-MMAE (0.048 mM) dissolved in DMA were introduced to react with the remaining thiol groups in step (2), and the reaction was incubated at room temperature for 1 h;

[1323] (4) purifying the reaction mixture using a desalting column;

[1324] (5) incubating ZnCl2 (Example III.105: 0.36 mM; Example III.106: 0.024 mM), a second reducing agent TCEP-6 (Example III.105: 0.042 mM; Example III.106: (0.054 mM) in BES buffer (pH 7.0, 20 mM) and the product from step (4), and allowing the reaction mixture to stand at 3 ° C for 1 hour;

[1325] (6) Introduction of EDAT (1.2 mM) to capture Zn 2+ , and MC-GGFG-DXd (0.096 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;

[1326] (7) Purify the reaction mixture using a desalting column.

[1327] The uniformity measurement results and chromatographic peak areas of Examples III.103-III.106 are shown in Table III-18 and Table III-19.

[1328] Table III-18

[1329]

[1330] Table III-19

[1331]

[1332] As shown in the table above, the results indicate that the D2+D4 ADC content is typically as high as 80% or 90%, which indicates that the process of this method is beneficial for site-specific modification of D2+D4 antibodies and can improve uniformity.

[1333] As shown in Examples III.105-III.106, the results show that when the molar ratio of the transition metal ion to the reducing agent in step (5) increases, the selective reducing ability of the reducing agent in step (5) increases.

[1334] Example V.1: Preparation of trastuzumab-[MC-VC-PAB-MMAE]4 conjugate (ADC with D4)

[1335] (1) ZnCl2 (0.0096 mM) and TCEP-3 (0.048 mM) were added to a solution of monoclonal antibody trastuzumab (0.012 mM, dissolved in BES buffer, pH 7.0, 20 mM), and the reaction mixture was placed at 4°C for 16 hours;

[1336] (2) MC-VC-PAB-MMAE (0.072 mM) dissolved in DMA was introduced and the reaction was continued at room temperature for 2 h;

[1337] (3) Purify the reaction mixture using a desalting column.

[1338] Examples V.2-V.12: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]4 Conjugate (ADC with D4) The method is similar to Example V.1, except that the concentration of ZnCl2 is as shown in Table V-1.

[1339] The homogeneity test results and chromatographic peak areas of Examples V.1-V.12 are shown in Table V-1 and Table V-2. The chromatogram of Example V.1 is shown in Fig.39 shown.

[1340] Table V-1

[1341]

[1342] Table V-2

[1343]

[1344] As shown in the results in the table above, the linker-payload (MC-VC-PAB-MMAE) was successfully linked to trastuzumab, indicating that the D4-type ADC was successfully synthesized.

[1345] According to the results of Table V-1, D4 is at least 85% when the molar ratio of ZnCl2 to the monoclonal antibody is 0.8:1 to 4:1 and the molar ratio of the reducing agent to ZnCl2 is 1:0.2 to 1:1. In addition, when the molar ratio of ZnCl2 to the monoclonal antibody is 2.3:1, the highest proportion of D4 is 91.02%.

[1346] Example V.13: Preparation of trastuzumab-[MC-VC-PAB-MMAE]4 conjugate (ADC with D4)

[1347] (1) ZnCl2 (0.0276 mM) and TCEP-3 (0.054 mM) were added to a solution of monoclonal antibody trastuzumab (0.012 mM, dissolved in Bis-Tris buffer, pH 6.7, 20 mM), and the reaction mixture was placed at 4°C for 16 hours;

[1348] (2) MC-VC-PAB-MMAE (0.084 mM) dissolved in DMA was introduced and the reaction was continued at room temperature for 2 h;

[1349] (3) Purify the reaction mixture using a desalting column.

[1350] Examples V.14-V.28 and Comparative Example V.1: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]4 Conjugate (D4 ADC) Using Different Buffer Systems

[1351] This method is similar to Example V.13. The difference is that the buffer system is as shown in Table V-3. Meanwhile, in Examples V.25-V.27, the reaction time in step (1) is 3.5 hours, and the concentration of MC-VC-PAB-MMAE is 0.072 mM.

[1352] The homogeneity measurement results and chromatographic peak areas of Examples V.13-V.28 and Comparative Example V.1 are shown in Table V-3 and Table V-4.

[1353] Table V-3

[1354] serial number Buffer system D0(％) D2(％) D4(％) D6(％) D8(％) E V.13 Bis-Tris, pH 6.7 2.01 4.26 90.61 3.12 0 E V.14 PIPES, pH 6.7 4.29 9.15 83.92 2.65 0 E V.15 HEPES, pH 7.0 0 3.56 93.14 3.30 0 E V.16 MOPS, pH 7.0 1.92 6.82 87.86 3.40 0 E V.17 MOBS, pH 7.4 0 3.42 92.92 3.66 0 E V.18 DIPSO, pH 7.4 0 3.98 93.12 2.90 0 E V.19 MOPSO, pH 7.4 0 3.96 92.90 3.14 0 E V.20 TES, pH 7.4 0 4.77 92.84 2.39 0 E V.21 TAPSO, pH 7.4 0 4.24 93.71 2.05 0 E V.22 ACES, pH 7.4 0 4.53 92.87 2.60 0 E V.23 BES, pH 7.0 1.22 4.46 92.35 1.97 0 E V.24 MES, pH 7.0 2.15 5.87 88.03 3.95 0 E V.25 MES, pH 5.8 9.72 8.33 70.27 9.22 2.46 E V.26 MES, pH 6.4 8.70 8.36 76.53 5.29 1.11 E V.27 MES, pH 6.7 4.83 4.91 83.90 4.80 1.57 E V.28 PB, PH 6.7 3.71 16.18 76.60 3.50 0 C V.1 ADA, PH 6.7 0 0 0 0 0

[1355] Table V-4

[1356]

[1357] As shown in the table above, when the buffer system is an ADA system, the reaction is largely unsuccessful. When the buffer system is a Bis-Tris buffer, a PIPES buffer, a MOPS buffer, a BES buffer, a HEPES buffer, a MOPSO buffer, a MOBS buffer, a TES buffer, a DIPSO buffer, a MES buffer, an ACES buffer or a TAPSO buffer, the content of D4 is greater than 80%.

[1358] Exam...

Claims

1. A method for programmed management of antibody disulfide bond site-specific modification, comprising the steps of: (R1) contacting a first reducing agent or a salt thereof with an antibody in a buffer system in the presence of a transition metal ion to reduce at least one of the interchain disulfide bonds of the antibody.

2. The method according to claim 1, characterized in that In step (R1), one of the interchain disulfide bonds of the antibody is reduced, optionally, one of the interchain disulfide bonds in the hinge region of the antibody is reduced.

3. The method according to claim 1, characterized in that In step (R1), three of the interchain disulfide bonds of the antibody are reduced, and optionally, two of the interchain disulfide bonds in the Fab region and one of the interchain disulfide bonds in the hinge region of the antibody are reduced.

4. The method according to claim 1, characterized in that: The method comprises step (O1), (O1) introducing an oxidizing agent to selectively reoxidize the reduced thiol groups produced in step (R1).

5. The method according to claim 4, characterized in that In step (O1), the oxidizing agent reoxidizes the reduced thiol groups in the Fab region of the antibody, optionally, four of the reduced thiol groups are reoxidized.

6. The method according to claim 4, characterized in that The method comprises step (R2), (R2) incubating a second reducing agent or a salt thereof in the buffer system to reduce one of the interchain disulfide bonds of the antibody produced in step (O1).

7. The method according to any one of claims 1 to 6, characterized in that The method further comprises the following steps: (C1) introducing a certain amount of a metal chelator and at least an equimolecular ratio of a first coupling group to react with the reduced thiol group produced in step (R1), (O1) or (R2), calculated on a molar basis of the antibody, wherein the first coupling group is a first capping reagent, a first linker-payload or a first sulfide bridging reagent, and optionally, the first sulfide bridging reagent carries the first linker-payload or reactive group.

8. The method according to claim 3, characterized in that The method further comprises the following steps: (C2) incubating at least an equimolecular ratio of the first coupling group to react with the reduced thiol group generated in step (R1), calculated on a molar basis of the antibody; then, Optionally, a metal chelator and an oxidant are introduced, or Optionally, a metal chelator and a first coupling group are introduced.

9. The method according to any one of claims 7 or 8, characterized in that The method further comprises the following steps: (R3) incubating the second reducing agent or a salt thereof in the buffer system to reduce the interchain disulfide bonds of the antibody produced in step (C1) or (C2), and optionally, introducing the transition metal ions; (C3) introducing at least an equimolecular ratio of a second coupling group to react with the reduced thiol group produced in step (R3), calculated on a molar basis of the antibody, optionally introducing the metal chelator, wherein the second coupling group is a second capping agent, a second linker-payload or a second sulfide bridging agent, optionally the second sulfide bridging agent carries the second linker-payload or a reactive group.

10. The method according to claim 9, characterized in that In step (R3), one, two or three of the interchain disulfide bonds of the antibody are reduced.

11. The method according to claim 7, characterized in that In step (C1), one, two, three or six of the first coupling groups are covalently linked to the reduced thiol group produced in step (R1).

12. The method according to claim 7, characterized in that In step (C1), one or two of the first coupling groups are covalently linked to the remaining thiol groups produced in step (O1).

13. The method according to claim 7, characterized in that In step (C1), two or four of the first coupling groups are covalently linked to the reduced thiol groups generated in step (R2).

14. The method according to claim 8, characterized in that In step (C2), two, three, four or six of the first coupling groups are covalently linked to the reduced thiol group generated in step (R1).

15. The method according to claim 9, characterized in that In step (C3), one, two, three, four or six of said second coupling groups are covalently linked to said reduced thiol group produced in step (R3).

16. The method according to claim 1 or 6, characterized in that: The reducing agent, including the first reducing agent and the second reducing agent, is independently selected from the group consisting of tris(2-carboxyethyl)phosphine (TCEP), or a compound having formula (I) or a salt, solvate, stereoisomer thereof; wherein In formula (I), X, Y and Z are each independently covalently linked to the phosphorus atom via a PC bond, wherein the PC bond is PC(sp 3 ) or PC(sp 2 ); X is formula (III): L1 is selected from -CH(R1)-, -C(CH3)(R1), -CH(R1)CH(R2)-, -CH(R1)CH(R2)CH(R3)-, an aryl group optionally and independently substituted by a group containing at least one coordinating atom selected from N, O and S, and a heteroaryl group optionally and independently substituted by a group containing at least one coordinating atom selected from O and S; R1, R2 and R3 are each independently H, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, C1-C5 hydroxyaminoalkyl, C1-C5 N-hydroxyamidealkyl, aryl or heteroaryl; or R2 or R3 and L2 form a 5-6 membered optionally substituted ring; A is optionally present and is -C(O)-, or -C(O)J-; J is an organic group comprising an amino group or an imino group and a carbonyl group, wherein the amino group or the imino group forms an amide group with -C(O), and the carboxyl group is optionally covalently linked to L2; L2 is optionally present, L2 is a transition metal chelator motif and is -N(R4)(R5) or hydroxyl; R4 and R5 are each independently hydrogen, C0-C5 hydroxyalkyl, C1-C5 alkyl, C1-C5 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted 5-6 membered saturated heterocyclyl, optionally substituted aralkyl, optionally substituted arylalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heteroaralkyl, R4 or R5 and R2 or R3 form a 5-6 membered optionally substituted ring; R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted aralkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4; R4 and R5 are not hydroxyl groups at the same time; Y is the same as X, Z is the same as X, or Y and Z are each independently a 5-6 membered optionally substituted saturated heterocyclic group, a C1-C5 alkyl group, a C1-C5 hydroxyalkyl group, an aryl group, C1-C5 carboxyalkyl, 5-6 membered optionally substituted cycloalkyl, or -C(O)Q is an ester group, an imide group or an amide group, X, Y and Z are not -CH2CH2C(O)OH at the same time.

17. The method according to claim 16, characterized in that In formula (I), L1 is an optionally substituted phenyl group attached to A in the ortho, meta or para position, A is -C(O)-; L2 is -N(R4)(R5) or hydroxyl; R4 is hydrogen and R5 is hydroxyl.

18. The method according to claim 17, characterized in that In formula (I), L1 is a phenyl group substituted by a hydroxyl group or a carboxyl group at the ortho position or the meta position, and the phenyl group is connected to A at the para position.

19. The method according to claim 16, characterized in that In formula (I), L1 is phenyl optionally substituted in the ortho, meta or para position by hydroxy, halogen, carboxyl, sulfonyl, amino, methoxy or ethoxy, A and L2 do not exist.

20. The method according to claim 16, characterized in that In formula (I), L1 is optionally substituted 4-pyridyl or optionally substituted 4-quinolyl, A and L2 do not exist.

21. The method according to claim 20, characterized in that In formula (I), L1 is 22. The method according to claim 16, characterized in that In formula (I), L1 is -CH(R1)CH(R2)-, R1 and R2 are each independently H, methyl, isopropyl, hydroxymethyl, hydroxyethyl, carboxymethyl, carboxyethyl, N-hydroxyacetamido, phenyl, 2-pyridyl, 4-pyridyl or 4-imidazolyl, and R2 and L2 form a 5-6 membered optionally substituted ring.

23. The method according to claim 22, characterized in that In formula (I), L1 is -CH(R1)CH(R2)-; R1 is H, and R2 and R4 of L2 form a 5-6 membered optionally substituted ring; L2 is -N(R4)(R5), and R5 is hydroxyl.

24. The method according to claim 22, characterized in that In formula (I), L1 is -CH(R1)CH(R2)-, R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl or 4-imidazolyl.

25. The method according to claim 16, characterized in that In formula (I), L1 is -CH(R1)CH(R2)-, R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl or 4-imidazolyl, A is -C(O)-; L2 is -N(R4)(R5); R4 is hydrogen, an optionally substituted 5-6 membered saturated heterocyclic group, R5 is hydroxyl.

26. The method according to claim 25, characterized in that In formula (I), R4 is 27. The method according to claim 16, characterized in that In formula (I), L1 is -CH(R1)CH(R2)-, R1 is H, R2 is methyl, hydroxymethyl, hydroxyethyl, carboxyethyl, phenyl, N-hydroxyacetamido, 2-pyridyl, 4-pyridyl or 4-imidazolyl, A is -C(O)-; L2 is -N(R4)(R5); R4 and R5 form a 5-6 membered optionally substituted ring.

28. The method according to claim 27, characterized in that In formula (I), L2 is 29. The method according to claim 16, characterized in that In formula (I), L1 is -CH(R1)CH(R2)-, R1 is methyl, isopropyl, carboxyethyl or N-hydroxyacetamide, R2 is H.

30. The method according to claim 16, characterized in that In formula (I), L1 is -CH(R1)CH(R2)-, R1 is methyl, isopropyl, carboxyethyl or N-hydroxyacetamide, R2 is H, A is -C(O)-; L2 is -N(R4)(R5); R4 is hydrogen, and R5 is hydroxyl.

31. The method according to claim 16, characterized in that In formula (I), L1 is -CH(R1)CH(R2)-, R1 and R2 are each independently H.

32. The method according to claim 16 or 31, characterized in that In formula (I), L2 is -N(R4)(R5); R4 is hydrogen, C1-C5 alkyl, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted 5-6 membered saturated heterocyclyl, optionally substituted aralkyl, optionally substituted aryl, optionally substituted heteroaralkyl, or R4 and R5 form a 5-6 membered optionally substituted ring; R5 is a hydroxyl group, R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, C1-C5 N-hydroxyamidealkyl, heteroaryl or heteroaralkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

33. The method according to claim 32, characterized in that In formula (I), R4 is hydrogen, methyl, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), a 5-6 membered saturated heterocyclic group including a heteroatom N or O, benzyl, benzyl substituted by hydroxy on the phenyl ring, phenyl optionally substituted by hydroxy, halogen or carboxyl, heteroarylalkyl including a heteroatom N, or R4 and R5 form a 5-6 membered ring; R5 is a hydroxyl group, R6 is hydrogen, C1-C5 alkyl, C1-C5 hydroxyalkyl, or heteroarylalkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

34. The method according to claim 32, characterized in that In formula (I), R4 is Hydrogen or <h2 style=";text-align:left;direction:ltr">-(CH2)n<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> (OCH2CH2O)n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> CH(R6)CO(R7), R5 is a hydroxyl group, R6 is hydrogen, methyl, hydroxymethyl or R7 is hydroxyl or -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0.

35. The method according to claim 31, characterized in that In formula (I), L2 is -N(R4)(R5); R4 and R5 are each independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or optionally substituted heteroarylalkyl, R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, C1-C5 N-hydroxyamidealkyl, heteroaryl or heteroaralkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

36. The method according to claim 35, characterized in that In formula (I), R4 and R5 are each independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or 6-membered heteroarylalkyl, R6 is hydrogen, R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

37. The method according to claim 36, characterized in that In formula (I), R4 and R5 are each independently -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7) or R6 is hydrogen, R7 is hydroxyl or -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0.

38. The method according to claim 31, characterized in that In formula (I), L2 is -N(R4)(R5); R4 is hydrogen, C0-C5 hydroxyalkyl, C1-C5 alkyl, optionally substituted C1-C5 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), optionally substituted aralkyl, optionally substituted arylalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heteroaralkyl; R5 is hydrogen, R6 is hydrogen, amino, C1-C5 alkyl, C1-C5 hydroxyalkyl, C1-C5 carboxyalkyl, aryl, optionally substituted aralkyl, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

39. The method according to claim 38, characterized in that In formula (I), R4 is hydrogen, C0-C3 hydroxyalkyl, C1-C3 alkoxy, -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), phenyl substituted by carboxyl, hydroxyl, amino, halogen, pyridyl, amino substituted by 2-methylpyridine, benzyl substituted by carboxyl, hydroxyl, amino or halogen, arylalkoxy, pyridyl, bipyridyl, substituted by carboxyl R5 is hydrogen, R6 is hydrogen, amino, C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 carboxyalkyl, aryl, aralkyl optionally substituted by hydroxy, halogen, cyano or nitro, C1-C5 N-hydroxyamidoalkyl, heteroaryl or heteroaralkyl; R7 is hydroxyl, C1-C5 alkoxy, -NH(CH2CONH)n 3 OH; n 1 、n 2 and n 3 Each independently is the number 0, 1, 2, 3, 4.

40. The method according to claim 39, characterized in that In formula (I), R4 is hydrogen, hydroxy, ethylhydroxy, methoxy, R5 is hydrogen.

41. The method according to claim 38, wherein in formula (I), R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydrogen, R6 is hydrogen, amino, methyl, hydroxymethyl, benzyl, carboxyethyl, benzyl substituted by -OH, F, -CN or -NO2, N-hydroxyacetamido, R7 is hydroxyl, -NH(CH2CONH)n 3 OH; n 1 and n 3 Each of them is independently digitized 0, 1, 2, 3, 4, n 2 Is the number 0.

42. The method according to claim 38, characterized in that In formula (I), R4 is -(CH2)n 1 (OCH2CH2O)n 2 CH(R6)CO(R7), R5 is hydrogen, R6 is hydrogen, amino, methyl, hydroxymethyl, benzyl, carboxyethyl, N-hydroxyacetamido, heteroaryl or heteroaralkyl; R7 is hydroxyl, -NH(CH2CONH)n 3 OH; n 1 is the number 0 or 2, n 2 is a number 0 or 1, n 3 Is the number 0.

43. The method according to claim 16, characterized in that In formula (I), J is a peptide residue, including a single amino acid residue, a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, aminopropionic acid, aminobutyric acid, aminovaleric acid, amino acid, aminoheptanoic acid, aminooctanoic acid, or NH2(OCH2CH2O)n 4 CH2COOH,n 4 For numbers 2-10, The amino acid is selected from the group consisting of glycine (Gly), alanine (Ala), serine (Ser), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val).

44. The method according to claim 43, characterized in that In formula (I), J is the residue of histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine or asparagine.

45. The method according to claim 43 or 44, characterized in that In formula (I), A is -C(O)J-, J is a residue of histidine, serine, alanine, glycine, phenylalanine, asparagine, tyrosine or asparagine, L2 is -N(R4)(R5), R4 is hydrogen, and R5 is hydroxyl.

46. ​​The method according to claim 16, characterized in that In formula (I), Y and Z are each independently Q is -NHOH, -NHCH2CH2SO3H, -N(CH2CH2OH)2, -NHCH2COOH, -NHCH2(CH3)COOH, -NH(CH2CH2O)3CH3.

47. The method according to claim 16, characterized in that The reducing agent is selected from the group consisting of:

48. The method according to claim 7 or 9, characterized in that The first sulphur bridging reagent and the second sulphur bridging reagent each independently contain at least two substituent groups to allow re-bridging of the thiol group.

49. The method according to claim 48, characterized in that The first sulphur bridging agent and the second sulphur bridging agent are each independently selected from the group consisting of:

50. The method according to claim 48, characterized in that The reactive groups each independently include an azide group and / or a dibenzocyclooctyne (DBCO).

51. The method according to claim 2, characterized in that In step (R1), the molar ratio of the first reducing agent to the transition metal ion is 1:0.4 to 1:250, 1:0.4 to 1:200, 1:1 to 1:70, 1:0.4 to 1:60, 1:0.1 to 1:20, 1:6 to 1:16, 1:0.2 to 1:8, 1:0.5 to 1:8, 1:0.25 to 1:7.5, or 1:0.25 to 1:

7.

52. The method according to claim 51, characterized in that When the first reducing agent is TCEP, the molar ratio of the first reducing agent to the transition metal ion in step (R1) is 1:0.4 to 1:200, 1:0.4 to 1:70, 1:1 to 1:16, or 1:2 to 1:

16.

53. The method according to claim 51, characterized in that When the first reducing agent is a compound having formula (I), optionally, the first reducing agent is TCEP-NO, TCEP-3NO, or TCEP-CO, the molar ratio of the first reducing agent to the transition metal ion in step (R1) is 1:0.4 to 1:250, 1:0.4 to 1:60, 1:2 to 1:60, 1:4 to 1:60, 1:4 to 1:24, 1:2 to 1:12, or 1:6 to 1:

16.

54. The method according to claim 2, characterized in that The molar ratio of the first reducing agent to the antibody is 3:1 to 0.5:1, optionally, the molar ratio of the first reducing agent to the antibody is 3:1 to 1:1, more optionally, the molar ratio of the first reducing agent to the antibody is 2:1 to 1:

1.

55. The method according to claim 2, characterized in that The incubation time in step (R1) is 0.2 hours to 24 hours. Optionally, the incubation time in step (R1) is 2 hours to 16 hours.

56. The method according to any one of claims 51 to 55, characterized in that The molar ratio of the first reducing agent to the antibody is 2.8:1 to 3.0:1, and the incubation time is 0.5 hour to 9 hours.

57. The method according to claim 3, characterized in that In step (R1), the molar ratio of the first reducing agent to the transition metal ion is 1:0.05 to 1:40, 1:0.08 to 1:30, 1:0.1 to 1:30, 1:0.1 to 1:20, 1:0.5 to 8:1 or 1:0.25 to 1:7.5, optionally, the first reducing agent is TCEP or the compound having formula (I).

58. The method according to claim 3, characterized in that In step (R1), the molar ratio of the first reducing agent to the antibody is 2.8:1 to 20:1, 3:1 to 15:1, 3:1 to 6:1, 3.5:1 to 5:1, 4:1 to 10:1, 5:1 to 13:

1.

59. The method according to claim 8, characterized in that In step (R1), the molar ratio of the first reducing agent to the transition metal ion is 1:0.05 to 1:40 or 1:0.5 to 1:

10. Optionally, the first reducing agent is TCEP or the compound having formula (I).

60. The method according to claim 8, characterized in that The molar ratio of the first reducing agent to the antibody in step (R1) is 2.8:1 to 20:

1. Optionally, the molar ratio of the first reducing agent to the antibody in step (R1) is 3.5:1 to 10:

1.

61. The method according to claim 3 or 8, characterized in that The incubation time in step (R1) is 1 hour to 24 hours, 14 hours to 24 hours, 16 hours to 20 hours, or 16 hours to 18 hours.

62. The method according to any one of claims 3 to 8, characterized in that In step (R1), the molar ratio of the first reducing agent to the antibody is 2.8 to 3.0, and the incubation time is 10 hours to 24 hours.

63. The method according to claim 3 or 8, characterized in that In step (R1), the molar ratio of the first reducing agent to the antibody is 6:1 to 20:1, and the incubation time is 4 hours to 16 hours.

64. The method according to claim 4, characterized in that In step (R1), the molar ratio of the first reducing agent to the antibody is 4:1 to 15:1, and the incubation time is 1 hour to 16 hours.

65. The method according to claim 1, characterized in that The incubation temperature in step (R1) is 0°C to 37°C, 0°C to 25°C, 0°C to 15°C, 0°C to 10°C, or 0°C to 5°C.

66. The method according to claim 1, characterized in that The transition metal ion is Zn 2+ 、Cd 2+ , Hg 2+ 、Ni 2+ 、Co 2+ or a combination thereof, optionally, the transition metal ion is Zn 2+ .

67. The method according to claim 1, 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, ADA buffer, PB buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, TAPSO buffer, PBS, acetate buffer, BTP buffer, HEPPSO buffer, POPSO buffer, EPPS buffer or Tris buffer. Optionally, the buffer system is selected from the group consisting of Bis-Tris buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, TAPSO buffer or MES buffer.

68. The method according to claim 8, characterized in that The buffer system is selected from the group consisting of Bis-Tris buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer, TAPSO buffer, PIPES buffer or MES buffer.

69. The method according to claim 67 or 68, characterized in that The concentration of the buffer system is 10 mM to 100 mM, 20 mM to 80 mM, 20 mM to 40 mM, 20 mM to 60 mM, 40 mM to 80 mM, or 40 mM to 60 mM.

70. The method according to claim 67 or 68, characterized in that The pH value of the buffer system is 5.5 to 8. Optionally, the pH value of the buffer system is 6.4 to 7.4, or 6.7 to 7.

4.

71. The method according to claim 4, characterized in that The oxidant in step (O1) is dehydroascorbic acid (DHAA).

72. The method according to claim 4 or 71, characterized in that In step (O1), the molar ratio of the oxidant to the antibody is 2:1 to 25:1, 2:1 to 20:1, 4:1 to 22:1, 4:1 to 15:1, or 6:1 to 14:

1.

73. The method according to claim 4, characterized in that The oxidation reaction in step (O1) is carried out at a temperature of 0°C to 37°C, 0°C to 30°C, 15°C to 30°C, or 20°C to 30°C, optionally in the dark, and / or the oxidation time is 1 hour to 48 hours, 1 hour to 5 hours, or 1 hour to 3 hours.

74. The method according to claim 73, characterized in that The oxidation temperature in step (O1) is 0°C to 25°C, 0°C to 15°C, 0°C to 10°C, or 0°C to 5°C, and / or the oxidation time is 1 hour to 8 hours, 2 hours to 5 hours, or 3 hours to 8 hours.

75. The method according to claim 6 or 9, characterized in that In step (R2) or (R3), the second reducing agent and the antibody are incubated at a temperature of 0°C to 37°C, 0°C to 30°C, 15°C to 30°C, or 20°C to 30°C, and / or the incubation time is 0.2 hours to 24 hours, 1 hour to 10 hours, 5 hours to 10 hours, 1 hour to 5 hours, or 1 hour to 3 hours.

76. The method according to claim 6, characterized in that In step (R2), the molar ratio of the second reducing agent to the antibody is 1:1 to 3:1 or 1:1 to 2:

1.

77. The method according to claim 9, characterized in that In step (R3), the molar ratio of the second reducing agent to the transition metal ion is 1:0.05 to 1:40, and / or the molar ratio of the second reducing agent to the antibody is 2.5:1 to 20:1, and / or the incubation time is 1 hour to 24 hours.

78. The method according to claim 9, characterized in that In step (R3), the molar ratio of the second reducing agent to the transition metal ion is 1:0.4 to 1:100, and / or the molar ratio of the second reducing agent to the antibody is 0.8:1 to 2.5:1, and / or the incubation time is 0.5 hour to 24 hours.

79. The method according to claim 78, characterized in that The method further comprises the following steps: (R4) incubating the transition metal ion and the second reducing agent or a salt thereof in a buffer system to reduce the interchain disulfide bonds of the antibody produced in step (C3); (C4) introducing the metal chelator and the second coupling group in at least an equimolecular ratio to react with the reduced thiol group generated in step (R4), calculated on a molar basis of the antibody.

80. The method according to claim 79, characterized in that In step (R4), the molar ratio of the third reducing agent to the transition metal ion is 1:0.4 to 1:100, and / or the molar ratio of the third reducing agent to the antibody is 0.8:1 to 2.5:1, and / or the incubation time is 0.5 hour to 24 hours.

81. The method according to claim 7, characterized in that The metal chelating agent is ethylenediaminetetraacetic acid disodium salt (EDTA-2Na).

82. The method according to claim 7, characterized in that When the first sulphur bridging reagent carries the reactive group, the step (C1) comprises the following steps: The metal chelator and the first sulfide bridging reagent carrying a reactive group are introduced to re-bridge the reduced thiol group generated in step (R1), (O1) or (R2), and then the first linker-payload is incubated in the buffer system to react with the reactive group of the sulfide bridging group.

83. The method according to claim 8, characterized in that When the first sulphur bridging reagent carries the reactive group, the step (C2) comprises the following steps: introducing the first thiol bridging reagent carrying the reactive group to re-bridge the reduced thiol group generated in step (R1), incubating the first linker-payload in the buffer system to react with the reactive group of the thiol bridging group; then, Optionally, the metal chelating agent and the oxidizing agent are introduced, or Optionally, the metal chelator and the first coupling group are introduced.

84. The method according to claim 9, characterized in that When the second sulfide bridging reagent carries the reactive group, the step (C3) comprises the following steps: The second thiol bridging reagent carrying a reactive group is introduced to re-bridge the reduced thiol group generated in step (R3), and optionally, the metal chelator is introduced, and then the second linker-payload is incubated in the buffer system to react with the reactive group of the thiol bridging group.

85. The method according to claims 4, 7-9, characterized in that The method further comprises a purification step after steps (O1), (C1), (C2) and / or (C3).

86. The method according to claim 1, characterized in that The antibody is a monoclonal antibody, a polyclonal antibody, a monospecific antibody or a multispecific antibody. Optionally, the antibody is IgG1 or IgG4.

87. The method according to claim 1, characterized in that The antibody is an engineered antibody having two amino acid substitutions of two interchain cysteines to form an interchain disulfide bond in the hinge region, optionally, the amino acid substitutions are selected from the following substitutions: cysteine ​​to alanine, to leucine, to arginine, to lysine, to asparagine, to methionine, to aspartic acid, to phenylalanine, to proline, to glutamine, to serine, to glutamic acid, to threonine, to glycine, to tryptophan, to histidine, to tyrosine, to isoleucine or to valine, more optionally, the amino acid substitution is selected from the following substitutions: cysteine ​​to serine.

88. The method according to claim 7 or 9, 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.

89. The method according to claim 7 or 9, characterized in that The payload of the linker-payload is selected from any one of the substituent groups containing at least one allowing the payload to be connected to the linker, optionally, the payload is a cytotoxic agent, a cytokine, a nucleic acid, a radionuclide, a chemokine, an immune (co)stimulatory molecule, an immunosuppressive molecule, a kinase, a prodrug converting enzyme, a ribonuclease, a growth factor, a hormone, a coagulation factor, a fibrinolytic protein, a peptide and a fragment thereof, a fusion protein or a derivative thereof that mimics these.

90. A modified antibody prepared by the method of any one of claims 1-89.

91. The modified antibody according to claim 90, characterized in that The modified antibody is conjugated to one, two or three conjugation groups.

92. The modified antibody according to claim 90 or 91, characterized in that The modified antibody is a D1, D2, D3, D4, D6, D1+D3, D1+D6, D1+D2, D1+D4, D2+D3, D2+D6, D0+D3, D0+D6, D3+D1, D3+D2, D6+D2, D6+D1, D0+D1, D0+D3, D4+D1 or D4+D2 type antibody drug conjugate (ADC).

93. A pharmaceutical composition comprising a modified antibody prepared by the method of any one of claims 1-89 and at least one pharmaceutically acceptable ingredient.

94. Use of the modified antibody prepared by the method of any one of claims 1-89 or the pharmaceutical composition of claim 93 in the preparation of a therapeutic agent for preventing, diagnosing or treating a disease.

95. A method for preventing or treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a modified antibody prepared by the method of any one of claims 1-89.

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