Novel mercaptan reducing agent as well as preparation method and application thereof

Abstract

The present disclosure relates to a novel thiol reducing agent of formula (I), a process for their preparation and their use in antibody modification.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims priority to PCT application number PCT / CN2022 / 113992 filed on August 22, 2022, PCT application number PCT / CN2022 / 119999 filed on September 20, 2022, PCT application number PCT / CN2022 / 119955 filed on September 20, 2022, PCT application number PCT / CN2022 / 131521 filed on November 11, 2022, and PCT application number 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 novel thiol reducing agent and a preparation method and use thereof. The thiol reducing agent can be used for antibody modification. 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] Antibody-drug conjugate (ADC) is an innovative biopharmaceutical product that connects monoclonal antibodies to small molecule drugs through suitable linkers to deliver potent cytotoxic drugs to cells expressing antigens. ADC ideally combines the specificity of antibodies and the high potency of cytotoxic drugs, thereby enhancing their targeted cytotoxic activity.

[0006] In general, conjugation of antibodies to cytotoxic agents usually involves direct conjugation to exposed residues (including lysine) on therapeutic IgG (immunoglobulin G) antibodies, or reduction of interchain disulfide bonds to expose cysteine ​​residues. In addition, other newly developed methods have been developed to introduce conjugation sites into mAbs, such as site-specific glycan conjugation, cysteine ​​engineering, introduction of unnatural amino acids, and addition of short peptide tags to drug-linkers. There are typically 80 lysine residues on antibodies; however, less than 10 residues can be chemically conjugated. Cysteine ​​conjugation can achieve the reduction of up to 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 Aug;9(8):872.).

[0007] The drug-to-antibody ratio (DAR), or the number of drug molecules conjugated to a single antibody, is important in determining the potency of an ADC. DAR values ​​vary widely and depend on other ADC variables. DAR values ​​also depend on the conjugation site and the light or heavy chain used for conjugation. DAR values ​​can affect drug efficacy, as low drug loadings lead to reduced potency, while high drug loadings affect toxicity and pharmacokinetics (“Introduction to Antibody-Drug Conjugates.” Antibodies (Basel). 2021 Dec;10(4):42.). Establishing a reliable conjugation process has always been challenging, as traditional non-site-specific conjugation and the distribution of the conjugate are largely affected by factors such as pH, concentration, salt ion concentration, and cosolvents.

[0008] Many methods have been developed to improve the uniformity of ADCs. For example, Genentech's THIOMAB technology was developed based on improving the uniformity of ADCs through antibody engineering, by introducing cysteine ​​in the primary sequence of the antibody to achieve site-specific coupling to improve the uniformity of the product ("Cysteine-Based Coupling: Challenges and Solutions." Bioconjug Chem. 2021 Aug 18;32(8):1525-1534.).

[0009] US20210040145 discloses a short peptide Tub-tag consisting of 14 amino acids, which can be applied to the C-terminus of any POI and can catalyze the addition of various tyrosine derivatives. Tub-tag technology uses tubulin tyrosine ligase to connect functionalized components to the C-terminus of the antibody, thereby uniformly generating DAR2-type antibody conjugates.

[0010] However, these technologies require protein engineering and / or enzyme catalysis, and thus have some disadvantages, such as low antibody expression levels, risk of immunogenicity, complex purification processes, and / or high costs.

[0011] After the homogeneity is improved, antibody-drug conjugates can obtain benefits such as better stability and lower immunogenicity, and further bring benefits in treatment, such as better efficacy and lower toxicity. Therefore, the development of new reducing agents and preparation processes for high-homogeneity ADCs is an urgent need and a long-term goal. Summary of the invention

[0012] For the above purpose, the present invention provides a reducing agent having the following formula (I):

[0013]

[0014] or a salt, solvate or stereoisomer thereof, characterized in that

[0015] R1 is H, -NH2, -C(O)(R3R4), optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl;

[0016] R3 is N, NH or O;

[0017] R4 is H, optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl;

[0018] R2 is H, optionally substituted C1-C5 alkyl, or optionally substituted C1-C5 hydroxyalkyl;

[0019] X is OH, optionally substituted C1-C5 alkoxy or -NR5R6,

[0020] R5 and R6 are each independently H, C0-C5 hydroxyalkyl, optionally substituted C1-C5 alkyl, optionally substituted C2-C8 carboxyalkyl, optionally substituted C1-C5 alkoxy, optionally substituted heteroarylalkyl, optionally substituted arylalkoxy, optionally substituted arylalkyl, optionally substituted aryl, C1-C5 alkylsulfonyl, or -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7),

[0021] R7 is C0-C5 hydroxyalkyl, -NHOH,

[0022] R8 is H, optionally substituted aralkyl,

[0023] n 1 and n 2 Each is independently digitized 0, 1, 2, 3, 4,

[0024] Y is the same as X, or Y is an ester or amide of X,

[0025] Z is the same as X or Y, or

[0026] Y and Z are each independently selected from:

[0027]

[0028] X, Y and Z are not the same

[0029] In one aspect, the present invention provides a composition comprising the above-mentioned reducing agent and a transition metal ion. The transition metal ion is Zn 2+ 、Cd 2+ , Hg 2+ 、Ni 2+ 、Co 2+ or a combination thereof.

[0030] In one aspect, the present invention provides a method for preparing the above-mentioned reducing agent, characterized in that at least one X' is connected to the compound of formula II by introducing a condensation reagent under an inert atmosphere,

[0031]

[0032] R1 is H, -NH2, -C(O)(R3R4), optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl;

[0033] R3 is N, NH or O;

[0034] R4 is H, optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl;

[0035] R2 is H, optionally substituted C1-C5 alkyl, or optionally substituted C1-C5 hydroxyalkyl;

[0036] X' is an optionally substituted C1-C5 alkyl alcohol or NR5R6,

[0037] R5 and R6 are each independently H, C0-C5 hydroxyalkyl, optionally substituted C1-C5 alkyl, optionally substituted C2-C8 carboxyalkyl, optionally substituted C1-C5 alkoxy, optionally substituted heteroarylalkyl, optionally substituted arylalkoxy, optionally substituted arylalkyl, optionally substituted aryl, C1-C5 alkylsulfonyl, or -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7),

[0038] R7 is C0-C5 hydroxyalkyl, -NHOH,

[0039] R8 is H, optionally substituted aralkyl,

[0040] n 1 and n 2 independently the numbers 0, 1, 2, 3, 4,

[0041] Y is the same as X, or Y is an ester or amide of X,

[0042] Z is the same as X or Y, or

[0043] Y and Z are each independently selected from:

[0044]

[0045] In one aspect, provided herein is a use of the above reducing agent or the above composition in reducing antibody interchain disulfide bonds.

[0046] In one aspect, the present invention provides a method for preparing an antibody having a site-specific modification, comprising the following steps:

[0047] (A1) In a buffer system, the above-mentioned reducing agent (as the first reducing agent) and transition metal ions are co-incubated with the antibody to selectively reduce the interchain disulfide bonds in the antibody, thereby obtaining an antibody with reduced thiol groups.

[0048] In some embodiments, two interchain disulfide bonds in the Fab region of the antibody and one interchain disulfide bond in the hinge region of the antibody are reduced.

[0049] In some embodiments, the method further comprises the following steps:

[0050] (A2) introducing an oxidizing agent to selectively reoxidize the reduced thiol groups produced in step (A1), and optionally, reoxidize the reduced thiol groups in the Fab region of the antibody.

[0051] In some embodiments, the method further comprises the following steps:

[0052] (A3) Incubating a second reducing agent in a buffer system to selectively reduce the interchain disulfide bonds in step (A2), and optionally, reducing the interchain disulfide bonds in the hinge region of the antibody.

[0053] In some embodiments, the method further comprises the following steps,

[0054] (B1) introducing a metal chelator and a first load unit to react with the reduced thiol group produced in step (A1), step (A2) or step (A3); wherein the first load unit serves as a capping agent and optionally a linker payload toxin or a first thio-bridging agent; and, as the case may be, the first thio-bridging agent may carry a linker payload toxin or a reactive group.

[0055] In some embodiments, the method further comprises the following steps:

[0056] (B2) incubating a second reducing agent in a buffer system to reduce the interchain disulfide bonds in step (B1), and optionally introducing transition metal ions; and

[0057] (B3) introducing a second load unit to react with the reduced thiol group produced in step (B2), and optionally introducing a metal chelator; wherein the second load unit is a second linker-payload or a second thio-bridging reagent; optionally, the second thio-bridging reagent may carry a second linker-payload toxin or a reactive group.

[0058] In one aspect, provided herein is a modified antibody prepared by the above method.

[0059] In some embodiments, the modified antibody is an antibody with a site-specific modification; as the case may be, the modified antibody may include an ADC with D2, an ADC with D4, an ADC with D1, an ADC with D6, an ADC with D3, an ADC with D1+D6, an ADC with D1+D2, an ADC with D1+D4, an ADC with D2+D4, an ADC with D6+D2, an ADC with D6+D1, an ADC with D3+D1, an ADC with D3+D2, an ADC with D0+D6, or an ADC with D0+D2.

[0060] In one aspect, provided herein is a pharmaceutical composition comprising an antibody (prepared by the above method and having site-specific modification) and at least one pharmaceutically acceptable ingredient.

[0061] In one aspect, provided herein is the use of an antibody (prepared by the above method and having site-specific modification) or the above pharmaceutical composition in the production of a therapeutic agent (for the prevention, diagnosis or treatment of a disease).

[0062] In one aspect, provided herein is a method for preventing or treating a disease (in a subject in need thereof), the method comprising administering to the subject a therapeutically effective amount of an antibody (prepared by the above method and having site-specific modification). BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The accompanying drawings are briefly described below and are intended to illustrate exemplary embodiments disclosed herein and not to limit the invention.

[0064] Figure 1 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 32 is shown. HIC-HPLC is an abbreviation for hydrophobic interaction chromatography-high performance liquid chromatography.

[0065] Figure 2 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 33 is shown.

[0066] Figure 3 AH shows the HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates prepared in Examples 34 to 41, wherein the reducing agent is TCEP-1, TCEP-2, TCEP-3, TCEP-4, TCEP-5, TCEP-6, TCEP-7 and TCEP-8.

[0067] Figure 4 AH shows the HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates prepared in Examples 42 to 49, wherein the reducing agent is TCEP-9, TCEP-10, TCEP-15, TCEP-18, TCEP-19, TCEP-20, TCEP-23 and TCEP-24.

[0068] Figure 5 AH shows the HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates prepared in Examples 50-57, wherein the reducing agent is TCEP-25, TCEP-26, TCEP-28, TCEP-A, TCEP-A, TCEP-30, TCEP-31 and TCEP-33.

[0069] Figure 6 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Comparative Example 5 is shown, wherein the reducing agent is TCEP.

[0070] Figure 7 AB shows the HIC-HPLC chromatograms of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Examples 58-59, wherein the molar ratio of the reducing agent to the antibody was 2.8:1 and 3.5:1.

[0071] Figure 8 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 60 is shown.

[0072] Fig. 9 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 61 is shown.

[0073] Fig.10 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 62 is shown.

[0074] Fig.11The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 63 is shown.

[0075] Fig.12 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 64 is shown.

[0076] Fig.13 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 65 is shown.

[0077] Fig.14 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 66 is shown.

[0078] Fig.15 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 67 is shown.

[0079] Fig.16 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 68 is shown.

[0080] Fig.17 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 69 is shown.

[0081] Fig.18 AC shows the HIC-HPLC chromatograms of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugates prepared in Examples 70-72, wherein the molar ratio of the reducing agent to the antibody is 5:1, 6:1, and 7:1.

[0082] Fig.19 AF show HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates prepared in Examples 73-78, wherein the molar ratio of reducing agent to antibody is 8:1, 9:1, 10:1, 11:1, 12:1, 13:1.

[0083] Fig. 20 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Comparative Example 1 is shown, wherein the molar ratio of the transition metal ion to the reducing agent is 0:1.

[0084] Fig.21AC shows the HIC-HPLC chromatograms of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Comparative Example 2-4, wherein the molar ratio of the transition metal ion to the reducing agent is 0:1.

[0085] Fig. 22 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 79 is shown.

[0086] Fig.23 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 80 is shown.

[0087] Fig.24 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 81 is shown.

[0088] Fig.25 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 82 is shown.

[0089] Fig.26 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 83 is shown.

[0090] Fig. 27 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 84 is shown.

[0091] Fig.28 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 85 is shown.

[0092] Fig.29 AE shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Examples 86-90, wherein the molar ratio of transition metal ion to reducing agent is 7.5:1, 15:1, 30:1, 0.22:1, 3.33:1.

[0093] Fig.30 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 91 is shown.

[0094] Fig.31The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 92 is shown.

[0095] Fig.32 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 93 is shown.

[0096] Fig.33 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 94 is shown.

[0097] Fig.34 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate prepared in Example 95 is shown.

[0098] Fig.35 AG shows the HIC-HPLC chromatograms of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugates prepared in Examples 96-102, wherein the buffer systems were different.

[0099] Fig.36 AE shows the HIC-HPLC chromatograms of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugates prepared in Examples 103-107, wherein the buffer systems were different.

[0100] Fig.37 AC shows the HIC-HPLC chromatograms of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugates prepared in Examples 108-110, wherein the concentration of the buffer system is different.

[0101] Fig.38 AF show HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]6 conjugates prepared in Examples 111-116, wherein the incubation time in step (1) was different.

[0102] Fig.39 AC shows the HIC-HPLC chromatograms of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugates prepared in Examples 117-119, wherein the incubation time and temperature in step (1) were different.

[0103] Fig.40 The HIC-HPLC chromatogram of the trastuzumab-[bismaleimide-DBCO]3 conjugate of Example 120 is shown.

[0104] Fig.41A shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6 conjugate of Example 121; Fig.41 B shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]6[MC-GGFG-DXd]2 conjugate of Example 121.

[0105] Fig.42 A shows the HIC-HPLC chromatogram of the trastuzumab-[MC-GGFG-DXd]6 conjugate of Example 122; Fig.42 B shows the HIC-HPLC chromatogram of the trastuzumab-[MC-GGFG-DXd]6[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate of Example 122.

[0106] Fig.43 A shows the HIC-HPLC of the trastuzumab-[maleimide]6 conjugate of Example 123; Fig.42 B shows the HIC-HPLC of the trastuzumab-[maleimide]6[MC-VC-PAB-MMAE]2 conjugate of Example 123.

[0107] Fig.44 HIC-HPLC of the trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate of Example 124 is shown.

[0108] Fig.45 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 125 is shown.

[0109] Fig.46 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 126 is shown.

[0110] Fig.47 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 127 is shown.

[0111] Fig.48 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 128 is shown.

[0112] Fig.49 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 129 is shown.

[0113] Fig.50 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 130 is shown.

[0114] Fig.51 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 131 is shown.

[0115] Fig.52 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 132 is shown.

[0116] Fig.53 The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Example 133 is shown.

[0117] Fig.54 AH show the HIC-HPLC chromatograms of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 134-141, wherein the molar ratio of DHAA to antibody, the molar ratio of reducing agent to antibody, and / or the incubation time in step (1) are different.

[0118] Fig.55 AF show HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 142-147, wherein the molar ratio of DHAA to antibody, and / or the molar ratio of reducing agent to antibody were different.

[0119] Fig.56 AC shows the HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 148-150, wherein the oxidation temperature and time were different.

[0120] Fig.57 AH show the HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 151-158, wherein the buffer systems were different.

[0121] Fig.58 AH show the HIC-HPLC chromatograms of trastuzumab-[MC-VC-PAB-MMAE]2 conjugates prepared in Examples 159-166, wherein the buffer systems were different.

[0122] Fig.59The HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate prepared in Comparative Example 6 is shown.

[0123] Fig.60 HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate in Example 167 is shown.

[0124] Fig.61 The HIC-HPLC chromatogram of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]6 conjugate of Example 168 is shown.

[0125] Fig.62 The HIC-HPLC chromatogram of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2 conjugate of Example 169 is shown.

[0126] Fig.63 A shows the HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 conjugate of Example 170; BC shows the HIC-HPLC of the trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 conjugates of Examples 170 to 171.

[0127] Fig.64 A shows the HIC-HPLC chromatogram of the trastuzumab-[MC-GGFG-DXd]2 conjugate of Example 172; B shows the HIC-HPLC chromatogram of the trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4 conjugate of Example 172.

[0128] Fig.65 A shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2 conjugate of Example 173; BC shows the HIC-HPLC chromatogram of the trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]4 conjugate of Examples 173-174. DETAILED DESCRIPTION

[0129] 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.

[0130] 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.

[0131] reducing agent

[0132] The present disclosure provides examples of reducing agents for use in the preparation of antibody drug conjugates (ADCs).

[0133] Provided herein is a reducing agent having the following formula (I):

[0134]

[0135] or a salt, solvate or stereoisomer thereof, characterized in that

[0136] R1 is H, -NH2, -C(O)(R3R4), optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl;

[0137] R3 is N, NH or O;

[0138] R4 is H, optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl;

[0139] R2 is H, optionally substituted C1-C5 alkyl, or optionally substituted C1-C5 hydroxyalkyl;

[0140] X is OH, optionally substituted C1-C5 alkoxy or -NR5R6,

[0141] 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),

[0142] R7 is C0-C5 hydroxyalkyl, -NHOH,

[0143] R8 is H, optionally substituted aralkyl,

[0144] n 1 and n 2 Each is independently digitized 0, 1, 2, 3, 4,

[0145] Y is the same as X, or Y is an ester or amide of X,

[0146] Z is the same as X or Y, or

[0147] Y and Z are each independently selected from:

[0148]

[0149] X, Y and Z are not the same

[0150] The term "C1-C5 alkyl" refers to an aliphatic hydrocarbon group having 1 to 3 carbon atoms in the chain or ring. Exemplary alkyl groups include methyl, ethyl, n-propyl and isopropyl.

[0151] 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.

[0152] The term "C2-C8 carboxyalkyl" refers to a C2-C8 alkyl group substituted with one, two, three, four, five, six, or seven carboxyl groups. Exemplary C2-C8 carboxyalkyl groups are -COOH, -CH2COOH, -CH2CH2COOH, -CH2(CH2)2COOH, -CH2(CH2)3COOH, -CH2(CH2)4COOH, -CH2(CH2)5COOH, -CH2(CH2)6COOH, or -CH(CH3)COOH.

[0153] The term "C1-C5 alkylsulfonyl" refers to a C1-C5 alkyl group in which one or more H atoms are replaced by one, two or three sulfonyl groups. Exemplary C1-C5 alkylsulfonyl groups are -CH2S(O)2OH, -CH2CH2S(O)2OH or -CH2(CH2)2S(O)2OH.

[0154] The term "aryl" refers to an aromatic or heteroaromatic group consisting of one or several rings, which contains 3 to 14 carbon atoms, preferably 6 to 10 carbon atoms. An exemplary aryl group is phenyl.

[0155] The term "aryl" also refers to aromatic groups in which one or more H atoms are independently replaced by groups such as F, Cl, Br, I, hydroxy, carboxyl, sulfonyl, amino, methoxy or ethoxy, N-hydroxyformamide, N-hydroxyacetamide, 4-pyridyl, 2-pyridyl, The invention may be substituted by other groups such as .

[0156] 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.

[0157] 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.

[0158] 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, -CH(CH3)CH2C6H5, -CH(CH3)CH2CH2C6H5.

[0159] 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

[0160] 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-.

[0161] The term "C1-C5 alkoxy" refers to an oxygen atom attached to a C1-C5 alkyl group. Exemplary C1-C5 alkoxy groups are -OCH3, -OCH2CH3, -OCH(CH3)2, -OCH2CH2CH3.

[0162] The term "halogen" refers to F, Cl, Br or I.

[0163] 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.

[0164] The term "cyano" refers to a substituent in which a carbon atom is triple bonded to a nitrogen atom, for example -CN.

[0165] In some embodiments, R1 is H and R2 is H.

[0166] In some embodiments, X is -OCH3, -OCH2CH3, or -OCH(CH3)2.

[0167] In some embodiments, X is -NR5R6, R5 is H, and

[0168] 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),

[0169] R7 is C0-C3 hydroxyalkyl or -NHOH,

[0170] R8 is H or an optionally substituted aralkyl group,

[0171] n 1 and n 2 Each independently is the number 0.

[0172] In some embodiments, 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),

[0173] R7 is OH or -NHOH,

[0174] R8 is H or benzyl optionally substituted by OH, halogen, cyano or nitro.

[0175] In some embodiments, X is -NR5R6, R5 is H, and

[0176] 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),

[0177] R7 is OH, C1 hydroxyalkyl, C2 hydroxyalkyl, C3 hydroxyalkyl or -NHOH,

[0178] R8 is H, or aralkyl optionally substituted by OH, halogen, cyano or nitro, wherein halogen is selected from F, Cl, Br or I,

[0179] n 1 and n 2 Each independently is the number 0.

[0180] In some embodiments, X is -NR5R6, R5 is H, and

[0181] 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,

[0182] In some embodiments, X is -NR5R6, R5 is H, and

[0183] R6 is H, OH, -CH2COOH, -CH2CONHOH, -OC(C6H5)3,

[0184]

[0185] In some embodiments, 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.

[0186] In some embodiments, X is -NR5R6, R5 is OH,

[0187] 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),

[0188] R7 is C0-C5 hydroxyalkyl,

[0189] R8 is H,

[0190] n 1 and n 2 Each independently is the number 0, 1, 2, 3, 4.

[0191] In some embodiments, R6 is C1-C3 alkyl, heteroarylalkyl containing a heteroatom N, optionally substituted benzyl, optionally substituted phenyl, or -CH(R8)CO(R7),

[0192] R7 is C0-C3 hydroxyalkyl,

[0193] R8 is H.

[0194] In some embodiments, R6 is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, heteroarylmethyl, heteroarylethyl, heteroarylpropyl, benzyl, arylethyl, arylpropyl, or -CH(R8)CO(R7),

[0195] R7 is hydroxy, C1 hydroxyalkyl, C2 hydroxyalkyl, C3 hydroxyalkyl, C4 hydroxyalkyl or C5 hydroxyalkyl,

[0196] R8 is H.

[0197] In some embodiments, R6 is -CH3, -CH2COCH3, -CH2COOH,

[0198] In some embodiments, R6 is -CH2COOH.

[0199] In some embodiments, X is -NR5R6,

[0200] R5 and R6 are independently C1-C5 alkyl, C0-C5 hydroxyalkyl, optionally substituted heteroarylalkyl or -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7),

[0201] R7 is C 0-5 Hydroxyalkyl or -NHOH,

[0202] R8 is H,

[0203] n 1 and n 2 Each independently is the number 0, 1, 2, 3, 4.

[0204] In some embodiments, X is -NR5R6,

[0205] R5 and R6 are 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),

[0206] R7 is hydroxy, C1 hydroxyalkyl, C2 hydroxyalkyl, C3 hydroxyalkyl, C4 hydroxyalkyl, C5 hydroxyalkyl or -NHOH,

[0207] R8 is H,

[0208] n 1 and n 2 Independently the numbers 0, 1, 2, 3, 4.

[0209] In some embodiments, X is -NR5R6, R5 and R6 are independently methyl, ethyl, -(CH2)2OH, -CH2COOH, -CH2CONHOH or

[0210] In some embodiments, Y is Z is In some embodiments, the reducing agent is selected from:

[0211]

[0212]

[0213] The present disclosure provides a composition comprising the above reducing agent and transition metal ions.

[0214] In some embodiments, the transition metal ion is Zn 2+ 、Cd2+ , Hg 2+ 、Ni 2+ 、Co 2+ or a combination thereof. In some embodiments, the transition metal ion is Zn 2+ .

[0215] In some embodiments, the molar ratio of the transition metal ion to the first reducing agent is 0.05:1 to 40:1, 0.25:1 to 30:1, 0.25:1 to 15:1, 0.1:1 to 10:1, 0.25:1 to 9:1, 0.2:1 to 7.5:1, 0.2:1 to 6:1, 0.2:1 to 5:1, 0.25:1 to 4:1, 0.5:1 to 7.5:1, 1:1 to 7.5:1, or 2:1 to 4:1.

[0216] The reducing agent having the above formula (I) can be prepared according to the following steps:

[0217] By introducing a condensation reagent under an inert atmosphere, at least one X' is linked to a compound of formula II,

[0218]

[0219] R1 is H, -NH2, -C(O)(R3R4), optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl;

[0220] R3 is N, NH or O;

[0221] R4 is H, optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl;

[0222] R2 is H, optionally substituted C1-C5 alkyl, or optionally substituted C1-C5 hydroxyalkyl;

[0223] X' is an optionally substituted C1-C5 alkyl alcohol or NR5R6,

[0224] 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),

[0225] R7 is C0-C5 hydroxyalkyl, -NHOH,

[0226] R8 is H, optionally substituted aralkyl,

[0227] n 1 and n 2 Each is independently digitized 0, 1, 2, 3, 4,

[0228] Y is the same as X, or Y is an ester or amide of X,

[0229] Z is the same as X or Y, or

[0230] Y and Z are each independently selected from:

[0231]

[0232] The term "condensation reagent" refers to a condensation reaction reagent that helps two molecules (functional groups) to covalently combine to form a single molecule. Condensation reagents include, but are not limited to, 1-hydroxybenzotriazole (HOBT), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), o-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate and o-benzo(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU).

[0233] The term "inert atmosphere" refers to a chemically inactive atmosphere, such as nitrogen, carbon dioxide, helium.

[0234] In some embodiments, the compound of Formula II is

[0235]

[0236] In some embodiments, it is characterized in that X' is 2-phenoxy-ethylamine, aniline, benzylamine, 4-aminobenzene-1,2-diol, 5-amino-2-hydroxybenzoic acid, bis(pyridin-2-ylmethyl)amine, 5-amino-8-hydroxyquinoline, bis(pyridin-2-yl)methylamine, 4-aminophthalic acid, L-tyrosine tert-butyl ester, DL-3-(4-fluorophenyl)alanine, DL-4-cyanophenylalanine, DL-4-nitrophenylalanine, N-benzylhydroxylamine hydrochloride, N-phenylhydroxylamine,

[0237]

[0238] Uses of reducing agents

[0239] The reducing agent of formula (I) provided above has reducing properties and can reduce the disulfide bonds of antibodies, and thus the reducing agent can be used to modify proteins or antibodies.

[0240] The term "disulfide bond" herein refers to a covalent bond having a RSS-R' structure. The amino acid cysteine ​​includes 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 located on two polypeptide chains, respectively, thereby forming an interchain bridge or interchain bond.

[0241] In some embodiments, the reducing agent having formula (I) can reduce the interchain disulfide bonds of the antibody.

[0242] In some embodiments, the reducing agent having formula (I) can selectively reduce three disulfide bonds among the interchain disulfide bonds of the antibody.

[0243] In some embodiments, reducing agents having formula (I) can selectively reduce interchain disulfide bonds, thereby selectively modifying antibodies.

[0244] In some embodiments, provided herein is the use of a reducing agent having the above formula (I) and the above composition in reducing antibody interchain bonds; optionally, provided herein is the use of a reducing agent having the above formula (I) and the above composition in reducing three interchain bonds among antibody interchain bonds.

[0245] In some embodiments, the reducing agent of formula (I) is used to prepare an antibody with a site-specific modification; optionally, the antibody with a site-specific modification is an antibody drug conjugate (ADC).

[0246] A mixture of antibody drug conjugates will be generated by a conventional coupling process or a bioconjugation process of the present invention. Typically, an antibody molecule belonging to an IgG1 or IgG4 subclass has four interchain disulfide bonds, each bond being formed by two -SH groups. Partial reduction or complete reduction of one or more interchain disulfide bonds may occur in the antibody molecule, thereby forming 2n (n is an integer selected from 1, 2, 3 or 4) reactive -SH groups, and therefore, the number of drugs (or payload toxins) 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, D2, D4, D6, D8, D3, D1, D6+D1, D6+D2, D3+D1, D3+D2, D0+D1, D0+D2, D1+D6, D1+D2, D1+D4 or D2+D4. Therefore, the concept of "homogeneity" of antibody drug conjugates refers to the dominant position of a specific type of antibody drug conjugate (i.e., a type selected from D0, D2, D4, D6, D8, D3, D1, D6+D1, D6+D2, D3+D1, D3+D2, D0+D1, D0+D2, D1+D6, D1+D2, D1+D4 or D2+D4 conjugate) in a given antibody drug conjugate mixture.

[0247] The drug-to-antibody ratio (DAR) of an ADC is the average number of drugs attached to each antibody. DAR is a key attribute to measure the quality of an ADC because it can significantly affect ADC efficacy. The DAR distribution (D0, D2, D4, D6, D8) can reflect the uniformity of the ADC.

[0248] 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.

[0249] As used herein, the term "D0" or "ADC having D0" refers to an ADC in which the average number of drugs conjugated to a single antibody molecule is about zero.

[0250] 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, 2.5 molecules) are 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 between the heavy chain and the heavy chain via a linker.

[0251] As used herein, the term "D4" or "ADC having D4" refers to an ADC having an average of about four drug molecules (e.g., 3.5, 4.0, 4.5 molecules) coupled to a single antibody molecule, wherein the drug molecule may be coupled to four -SH groups (generated by reducing two interchain disulfide bonds or intrachain disulfide bonds).

[0252] As used herein, the term "D6" or "ADC with D6" refers to an ADC having an average of about six drug molecules (e.g., 5.5, 6.0, 6.5 molecules) coupled to a single antibody molecule, wherein the drug molecules may be coupled to six -SH groups (generated by reducing three disulfide bonds).

[0253] As used herein, the term "D8" or "ADC having D8" refers to an ADC having an average of about eight drug molecules (e.g., 7.5, 8.0, 8.5 molecules) coupled to a single antibody molecule, wherein the drug molecules may be coupled to eight -SH groups (generated by reducing four disulfide bonds).

[0254] As used herein, the term "D1" or "ADC with D1" refers to an ADC in which one of the first thio-bridging groups with the first linker-payload toxin rebridges two thiol groups of a single antibody molecule.

[0255] As used herein, the term "D3" or "ADC with D3" refers to an ADC in which three of the first thio-bridging groups with the first linker-payload toxin rebridge the six thiol groups of a single antibody molecule.

[0256] As used herein, the term "D6+D1" or "dual payload toxin ADC with D6+D1" refers to an ADC in which six of the first linker-payload toxins and one of the second thio-bridging groups with the second linker-payload toxin are conjugated to a single antibody molecule.

[0257] The term "D6+D2" or "D6+D2-type dual-payload ADC" herein refers to an ADC in which six of the first linker-payloads and two of the second linker-payloads are coupled to a single antibody molecule.

[0258] As used herein, the term "D3+D1" or "dual payload toxin ADC with D3+D1" refers to: in this ADC, three of the first thio-bridging groups with the first linker-payload toxin and one of the second thio-bridging groups with the second linker-payload toxin re-bridge the eight thiol groups of a single antibody molecule.

[0259] As used herein, the term "D3+D2" or "dual payload toxin ADC with D3+D2" refers to an ADC in which three of the first thio-bridging groups with the first linker-payload toxin are rebridged to six thiol groups and two of the second linker-payload toxins are conjugated to a single antibody molecule.

[0260] As used herein, the term "D0+D2" or "ADC with D0+D2" refers to: in this ADC, one, two or three of the first thio-bridging groups are re-bridged to six thiol groups, and two of the second linker-payload toxins are coupled to a single antibody molecule; or refers to: in this ADC, two, four or six of the capping reagents and two of the second linker-payload toxins are coupled to a single antibody molecule.

[0261] As used herein, the term "D0+D1" or "ADC with D0+D1" refers to: in the ADC, three of the first thio-bridging groups rebridge six thiol groups and one of the second thio-bridging groups with a linker-payload toxin rebridges two thiol groups of a single antibody molecule; or refers to: in the ADC, six of the capping agents react with six thiol groups and one of the second thio-bridging groups with a linker-payload toxin rebridges two thiol groups of a single antibody molecule.

[0262] As used herein, the term "D1+D6" or "ADC with dual payload toxins having D1+D6" refers to an ADC in which one of the first thio-bridging groups with a first linker-payload toxin rebridges two thiol groups and six of the second linker-payload toxins are conjugated to a single antibody molecule, wherein the first linker-payload toxin and the second linker-payload toxin may be the same or different.

[0263] As used herein, the term "D1+D2" or "dual payload toxin ADC with D1+D2" refers to an ADC in which one of the first thio-bridging groups with a first linker-payload toxin rebridges two thiol groups and two of the second linker-payload toxins are conjugated to a single antibody molecule, wherein the first linker-payload toxin and the second linker-payload toxin may be the same or different.

[0264] As used herein, the term "D1+D4" or "dual payload toxin ADC with D1+D4" refers to an ADC in which one of the first thio-bridging groups with a first linker-payload toxin rebridges two thiol groups and four of the second linker-payload toxins are conjugated to a single antibody molecule, wherein the first linker-payload toxin and the second linker-payload toxin may be the same or different.

[0265] The term "D2+D4" or "D2+D4-type dual-payload ADC" herein refers to an ADC in which two of the first linker-payloads and four of the second linker-payloads are coupled to a single antibody molecule.

[0266] 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.

[0267] 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.

[0268] In some embodiments, the reducing agent having the above formula (I) or the composition provided above can be used to prepare ADC with better homogeneity.

[0269] In some embodiments, the present disclosure provides use of a reducing agent of formula (I) or a composition provided above in the preparation of an ADC; optionally, it can be used to prepare an ADC having D2, an ADC having D4, an ADC having D1, an ADC having D6, an ADC having D3, an ADC having D1+D6, an ADC having D1+D2, an ADC having D1+D4, an ADC having D2+D4, an ADC having D6+D2, an ADC having D6+D1, an ADC having D3+D1, an ADC having D3+D2, an ADC having D0+D6, or an ADC having D0+D2.

[0270] In some embodiments, the ADC includes D6, and the content of D6 is at least up to 50% of the total weight of DO, D2, D4, D6 and D8. In some embodiments, the ADC includes D6, and the content of D6 is up to 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96% of the total weight of DO, D2, D4, D6 and D8.

[0271] In some embodiments, the ADC comprises D2, and the content of D2 is at least up to 70% of the total weight of DO, D2, D4, D6 and D8. In some embodiments, the ADC comprises D6, and the content of D6 is up to 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% of the total weight of DO, D2, D4, D6 and D8.

[0272] In some embodiments, the uniformity of the ADC with D3 is as high as 82%, 83%, or 85%.

[0273] In some embodiments, the uniformity of the ADC having D6+D2 is up to 80%, 81%, 82%, 83%, 84%, 85% or 86%.

[0274] In some embodiments, the uniformity of the ADC having D6+D1 is up to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% or 92%.

[0275] In some embodiments, the uniformity of the ADC with D0+D2 is up to 65%, 67%, 69%, 70%, 71% or 73%.

[0276] In some embodiments, the uniformity of the ADC with D0+D1 is up to 70%, 71%, 73%, 75%, 77%, 79%, 80% or 83%.

[0277] In some embodiments, the uniformity of the ADC with D1 is up to 77%, 79%, 80%, 83% or 85%.

[0278] In some embodiments, the uniformity of the ADC with D1+D6 is up to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%.

[0279] In some embodiments, the uniformity of the ADC with D1+D2 is up to 65%, 67%, 69%, 70%, 71%, 73%, 75% or 77%.

[0280] In some embodiments, the uniformity of the ADC with D1+D4 is up to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%.

[0281] In some embodiments, the homogeneity of the ADC with D2+D4 is up to 60%, 77%, 79%, 80%, 83% or 85%.

[0282] Antibodies with site-specific modifications and preparation thereof

[0283] The present disclosure also provides a method for preparing an antibody with site-specific modification using a reducing agent having formula (I) and a modified antibody thereof. Optionally, the present invention provides a method for preparing an ADC with better homogeneity using a reducing agent having formula (I) or a salt, solvate, or stereoisomer thereof.

[0284] In some embodiments, a method of preparing an antibody having a site-specific modification comprises the following steps:

[0285] (A1) In a buffer system, a reducing agent having formula (I) or a salt, solvate, or stereoisomer thereof (as a first reducing agent) and a transition metal ion are co-incubated with an antibody to selectively reduce the interchain disulfide bonds within the antibody, thereby obtaining an antibody with reduced thiol groups.

[0286] In some embodiments, two interchain disulfide bonds in the Fab region of the antibody and one interchain disulfide bond in the hinge region of the antibody are reduced in step (A1).

[0287] In some embodiments, the method of preparing an antibody having a site-specific modification further comprises the following steps:

[0288] (A2) Introducing an oxidizing agent to selectively reoxidize the reduced thiol groups produced in step (A1).

[0289] In some embodiments, the oxidizing agent in step (A2) reoxidizes the reduced thiol groups in the antibody Fab region. Optionally, four of the reduced thiol groups are reoxidized to form two disulfide bonds.

[0290] In some embodiments, the product obtained in step (A2) is purified after oxidation. Purification methods include, but are not limited to, desalting columns, ultrafiltration (UF) and diafiltration (DF).

[0291] In some embodiments, the method for preparing an antibody having a site-specific modification further comprises the following steps,

[0292] (A3) Incubating a second reducing agent in a buffer system to reduce the interchain disulfide bonds in step (A2).

[0293] In some embodiments, the second reducing agent reduces the interchain disulfide bonds of the antibody in step (A3), optionally, one interchain disulfide bond in the hinge region is reduced.

[0294] In some embodiments, the method for preparing an antibody having a site-specific modification further comprises the following steps,

[0295] (B1) introducing a metal chelator and a first payload toxin unit to react with the reduced thiol group produced in step (A1), step (A2) or step (A3); wherein the first payload toxin unit is a capping agent, a first linker payload toxin or a first thio-bridging agent; and, as the case may be, the first thio-bridging agent may carry a first linker payload toxin or a reactive group.

[0296] In some embodiments, when the first payload toxin unit is a first thio-bridging reagent carrying a reactive group, step (B1) further includes the following steps: incubating the metal chelator and the first linker-payload toxin in a buffer system to react with the reactive group carried by the first thio-bridging reagent.

[0297] In some embodiments, the method of preparing an antibody having a site-specific modification further comprises the following steps:

[0298] (B2) incubating a second reducing agent in a buffer system to reduce the interchain disulfide bonds in step (B1), and optionally introducing transition metal ions; and

[0299] (B3) introducing a second payload toxin unit to react with the reduced thiol group produced in step (B2), and optionally introducing a metal chelator; wherein the second payload toxin unit is a second linker-payload toxin or a second thio-bridging reagent; optionally, the second thio-bridging reagent may carry a second linker-payload toxin or a reactive group.

[0300] In some embodiments, in the absence of transition metal ions, the second reducing agent reduces all interchain disulfide bonds of the antibody in step (B2). In some embodiments, one interchain disulfide bond in the hinge region of the antibody is reduced using steps (A1), (B1), and (B2). In some embodiments, three interchain disulfide bonds are reduced using steps (A1), (A2), (B1), and (B2). In some embodiments, two interchain disulfide bonds are reduced using steps (A1), (A2), (A3), (B1), and (B2).

[0301] In some embodiments, in the presence of transition metal ions, the second reducing agent selectively reduces the interchain disulfide bonds of the antibody, and optionally reduces one or two interchain disulfide bonds. In some embodiments, using steps (A1), (A2), (B1) and (B2), the second reducing agent and transition metal ions selectively reduce one or two interchain disulfide bonds of the antibody.

[0302] In some embodiments, when the second payload toxin unit is a second thio-bridging reagent carrying a reactive group, step (B3) further includes the following steps: incubating the second linker-payload toxin in a buffer system to react with the reactive group carried by the second thio-bridging reagent, and optionally introducing a metal chelator.

[0303] In some embodiments, if transition metal ions are introduced in step (B2), a metal chelator is introduced in step (B3) to capture excess transition metal ions.

[0304] In some embodiments, the molar ratio of the reducing agent having formula (I) or the second reducing agent to the antibody in steps (A1), (A3), and (B2) is independently 1:1 to 20:1, 1:1 to 10:1, 1:1 to 8:1, 1:1 to 5:1, 2:1 to 5:1, 3:1 to 5:1, 1:1 to 3:1, 1:1 to 2:1, 2:1 to 4:1, or 3:1 to 4:1.

[0305] In some embodiments, the molar ratio of the first reducing agent to the antibody in step (A1) is 2.8: 1 to 13: 1; optionally, 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, the molar ratio of the first reducing agent to the antibody in step (A1) 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.

[0306] In some embodiments, the incubation temperatures in steps (A1), (A3), and (B2) are independently 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.

[0307] In some embodiments, the incubation temperature in step (A1) is 37°C, 35°C, 33°C, 30°C, 28°C, 24°C, 20°C, 18°C, 15°C, 13°C, 10°C, 8°C, 4°C or 0°C.

[0308] In some embodiments, the incubation time in step (A1) is 2 hours to 24 hours, 14 hours to 24 hours, 16 hours to 20 hours, or 16 hours to 18 hours. In some embodiments, the incubation time in step (A1) 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.

[0309] In some embodiments, in step (A1), the molar ratio of the first reducing agent to the antibody is 2.8: 1 to 3: 1, and the incubation time is 10 to 24 hours. In some embodiments, in step (A1), 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.

[0310] In some embodiments, the incubation time in step (A1) is shortened as the molar ratio of the first reducing agent to the antibody increases. In some embodiments, in step (A1), the molar ratio of the first reducing agent to the antibody is 4: 1 to 20: 1, and the incubation time is 1 hour to 24 hours. In some embodiments, in step (A1), the molar ratio of the first reducing agent to the antibody is 4: 1 to 10: 1, and the incubation time is 2 hours to 16 hours. In some embodiments, in step (A1), the molar ratio of the first reducing agent to the antibody is 4: 1, 4.5: 1, 5: 1, 5: 1, 7: 1, 8: 1, 9: 1 or 10: 1, and the incubation time is 1 hour, 2 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.

[0311] In some embodiments, in step (A1), the molar ratio of the first reducing agent to the antibody is 5: 1 to 20: 1, and the incubation time is 3 hours to 24 hours. In some embodiments, in step (A1), the molar ratio of the first reducing agent to the antibody is 6: 1 to 13: 1, and the incubation time is 4 hours to 16 hours. In some embodiments, in step (A1), 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 or 13: 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.

[0312] In some embodiments, the molar ratio of the transition metal ion to the first reducing agent in step (A1) is 0.05:1 to 40:1, 0.08:1 to 30:1, 0.1:1 to 20:1, 0.2:1 to 8:1, or 0.25:1 to 7.5:1.

[0313] In some embodiments, the molar ratio of the transition metal ion to the first reducing agent in step (A1) is 0.1:1 to 20:1, 0.2:1 to 20:1, 0.1:1 to 10:1, 0.1:1 to 8:1, 0.2:1 to 8:1, 0.25:1 to 7.5:1, or 0.4:1 to 1:1.

[0314] In some embodiments, the molar ratio of the transition metal ion to the first reducing agent in step (A1) is 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, 25:1, 27:1, 29:1, 32:1, 34:1, 36:1, 38:1 or 40:1.

[0315] In some embodiments, the molar ratio of the transition metal ion to the antibody in step (A1) 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.

[0316] 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 transition metals.

[0317] In some embodiments, the transition metal ion is selected from Zn 2+ 、Cd 2+ , Hg 2+ 、Ni 2+ 、Co 2+ , or a combination thereof.

[0318] In some embodiments, the transition metal ion is Zn 2+ .

[0319] In some embodiments, there is no specific limitation on the salt of the transition metal ion, as long as the transition metal ion is soluble in the reaction solution so that free transition metal ions can be released in the reaction solution. In some embodiments of the present invention, the salt of the transition metal ion is chloride, nitrate, sulfate, acetate, iodide, bromide, formates or tetrafluoroborate.

[0320] In some embodiments, Zn 2+ The salt is ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, zinc formate, or zinc tetrafluoroborate.

[0321] In some embodiments, Zn 2+ The salt is ZnCl2.

[0322] In some embodiments, 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 0.01mM to 0.2mM. In some embodiments, the concentration of the first reducing agent is 0.02mM to 0.15mM. In some embodiments, the concentration of the first reducing agent is 0.05mM to 0.1mM. In some embodiments, the concentration of the first reducing agent is 0.01mM, 0.02mM, 0.03mM, 0.04mM, 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.10mM, 0.11mM, 0.12mM, 0.13mM, 0.14mM, 0.15mM, 0.16mM, 0.17mM, 0.18mM, 0.19mM or 0.20mM.

[0323] In some embodiments, there is no particular limitation on the concentration of the transition metal ion in step (A1), as long as the concentrations of the first reducing agent and the antibody are increased or decreased in equal proportions.

[0324] In some embodiments, there is no particular limitation on the concentration of the antibody in step (A1), as long as the concentrations of the first reducing agent and the transition metal ion are increased or decreased in equal proportions.

[0325] In some embodiments, there is no particular limitation on the oxidant of step (A2), as long as the oxidant can reoxidize the reduced thiol group. In some embodiments, the oxidant is dehydroascorbic acid (DHAA).

[0326] In some embodiments, in step (A2), the molar ratio of the oxidant to the antibody is 2: 1 to 25: 1. In some embodiments, in step (A2), the molar ratio of the oxidant to the antibody is 4: 1 to 22: 1 or 3: 1 to 15: 1. In some embodiments, in step (A2), the molar ratio of the oxidant to the antibody is 2: 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.

[0327] In some embodiments, in step (A2), the oxidation temperature is 0°C to 37°C, and / or the oxidation time is 1 hour to 48 hours; depending on the circumstances, in step (A2), the oxidation temperature is 0°C to 30°C, and / or the oxidation time is 1 hour to 5 hours.

[0328] In some embodiments, in step (A2), 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. In some embodiments, in step (A2), the oxidation temperature is 0°C, 3°C, 6°C, 8°C, 10°C, 12°C, 15°C, 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, 32°C, 35°C, or 37°C. In some embodiments, in step (A2), the oxidation temperature is room temperature.

[0329] In some embodiments, in step (A2), the oxidation time is 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. In some embodiments, in step (A2), the oxidation time is 1 hour, 3 hours, 5 hours, 7 hours, 9 hours, 11 hours, 13 hours, 15 hours, 18 hours, 20 hours, 23 hours, 25 hours, 27 hours, 30 hours, 33 hours, 35 hours, 37 hours, 40 hours, 43 hours, 45 hours or 48 hours.

[0330] In some embodiments, in step (A2), the oxidation reaction is carried out in the dark.

[0331] In some embodiments, in step (A2), to increase the content of D2-type ADC, D4-type ADC, and D1-type ADC, it is important to remove excess oxidant to purify the oxidation product.

[0332] In some embodiments, there is no specific limitation on the second reducing agent, as long as the second reducing agent can reduce the interchain disulfide bonds in the antibody. In some embodiments, the second reducing agent in step (A3) is the same as the first reducing agent in step (A1). In some embodiments, the second reducing agent in step (A3) is different from the first reducing agent in step (A1). In some embodiments, the second reducing agent in step (A3) is independently tris(2-carboxyethyl)phosphine (TCEP).

[0333] In some embodiments, in step (A3), a metal chelator and a second reducing agent are introduced.

[0334] In some embodiments, in step (A3), 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.

[0335] In some embodiments, the molar ratio of the second reducing agent to the antibody in step (A3) 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, 1:1 to 2:1.

[0336] In some embodiments, in step (A3), 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. In some embodiments, the incubation temperature in step (A3) is room temperature.

[0337] As used herein, the term "room temperature" refers to 23°C±2°C, 25°C±5°C or 20°C±5°C.

[0338] In some embodiments, the incubation time in step (A3) is 0.5 hours to 12 hours, 1 hour to 10 hours, 1 hour to 8 hours, 1 hour to 5 hours, 1 hour to 3 hours, 2 hours to 4 hours, 1 hour to 4 hours, or 2 hours to 5 hours.

[0339] In some embodiments, the reaction temperature in step (B1) and / or (B3) is 0° C. to 40° C. In some embodiments, the reaction time in step (B1) and / or (B3) is 0.5 hour to 10 hours.

[0340] In some embodiments, the reaction temperature in step (B1) and / or (B3) 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. In some embodiments, the reaction time in step (B1) and / or (B3) is 0.5 hour to 5 hours, 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.

[0341] In some embodiments, the reaction in steps (B1) and / or (B3) is carried out at room temperature.

[0342] In some embodiments, the reaction temperature in step (B1) and / or (B3) is 15°C to 25°C, and the reaction time in step (B1) and / or (B3) is 1 hour to 3 hours.

[0343] In some embodiments, in step (B1) and / or in step (B3), the temperature for reacting with the reduced thiol group is 4°C to 37°C, and the time for reacting with the reduced thiol group is 0.5 hour to 6 hours.

[0344] In some embodiments, in step (B1) and / or in step (B3), the temperature for reacting with the reduced thiol group is 20°C to 30°C or 20°C to 25°C. In some embodiments, in step (B1) and / or in step (B3), the temperature for reacting with the reduced thiol group is room temperature. In some embodiments, in step (B1) and / or in step (B3), the temperature for reacting with the reduced thiol group is 4°C, 6°C, 8°C, 10°C, 13°C, 17°C, 20°C, 23°C, 27°C, 30°C, 34°C or 37°C.

[0345] In some embodiments, in step (B1) and / or in step (B3), the time for reacting with the reduced thiol group is 0.5 hour to 6 hours, 0.5 hour to 4 hours, 0.5 hour to 2 hours, 1 hour to 2 hours or 0.5 hour to 1 hour. In some modifications, in step (B1) and / or in step (B3), the time for reacting with the reduced thiol group is 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours.

[0346] In some embodiments, in step (B1) and / or in step (B3), the temperature and time of the reaction with the reduced thiol groups are independent.

[0347] In some embodiments, in step (B1) and / or in step (B3), the temperature for reacting with the reactive group is 10°C to 37°C, and the time for reacting with the reduced thiol group is 2 hours to 12 hours.

[0348] In some embodiments, in step (B1) and / or in step (B3), the temperature for reacting with the reactive group is 10° C. to 30° C., 15° C. to 30° C., or 25° C. to 30° C. In some embodiments, in step (B1) and / or in step (B3), the temperature for reacting 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.

[0349] In some embodiments, in step (B1) and / or in step (B3), the time of reaction with the reactive group is 2 hours to 10 hours, 4 hours to 10 hours, 8 hours to 10 hours. In some embodiments, in step (B1) and / or in step (B3), the time of reaction 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.

[0350] In some embodiments, in step (B1) and / or in step (B3), the temperature and time of reacting with the reactive group are independent.

[0351] In some embodiments, in step (B1), the first payload toxin unit is in excess based on the amount of antibody.

[0352] In some embodiments, the molar ratio of the first payload toxin unit to the antibody in step (B1) 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.

[0353] In some embodiments, in step (B1), the molar ratio of the first thio-bridging agent to the antibody is 1: 1 to 10: 1. In some embodiments, in step (B1), the molar ratio of the first thio-bridging agent to the antibody is 1: 1, 1.05: 1, 2: 1, 3: 1, 3.3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1.

[0354] In some embodiments, in step (B1), when the first linker-payload toxin reacts with the reactive group in the first thio-bridging reagent, the molar ratio of the first linker-payload toxin to the antibody is 1: 1 to 10: 1. In some embodiments, in step (B1), when the first linker-payload toxin reacts with the reactive group in the first thio-bridging reagent, the molar ratio of the first linker-payload toxin to the antibody is 1: 1, 1.05: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1.

[0355] In some embodiments, in step (B1), when the first linker-payload toxin reacts with the reduced thiol group, the molar ratio of the first linker-payload toxin to the antibody is 2: 1 to 20: 1. In some embodiments, in step (B1), when the first linker-payload toxin reacts with the reduced thiol group, the molar ratio of the first linker-payload toxin to the antibody is 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 20: 3, 7: 1, 8: 1, 9: 1, 10: 1, 15: 1 or 20: 1.

[0356] In some embodiments, in step (B3), the second payload toxin unit is in excess based on the amount of antibody.

[0357] In some embodiments, the molar ratio of the second payload toxin unit to the antibody in step (B3) 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.

[0358] In some embodiments, in step (B3), the molar ratio of the second thio-bridging agent to the antibody is 1: 1 to 10: 1. In some embodiments, in step (b), the molar ratio of the second thio-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.

[0359] In some embodiments, in step (B3), when the second linker-payload toxin reacts with the reactive group in the second thio-bridging reagent, the molar ratio of the second linker-payload toxin to the antibody is 1: 1 to 10: 1. In some embodiments, in step (B3), when the second linker-payload toxin reacts with the reactive group in the second thio-bridging reagent, the molar ratio of the second linker-payload toxin to the antibody is 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1.

[0360] In some embodiments, in step (B3), when the second linker-payload toxin reacts with the reduced thiol group, the molar ratio of the second linker-payload toxin to the antibody is 2: 1 to 20: 1. In some embodiments, in step (B3), when the second linker-payload toxin reacts with the reduced thiol group, the molar ratio of the second linker-payload toxin 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.

[0361] In the present disclosure, metal chelators are used to capture excess transition metal ions and perform site-specific modification on antibodies.

[0362] In the present disclosure, there is no particular limitation on the metal chelator, as long as the metal chelator can capture excess transition metal ions and does not affect the reduction of disulfide bonds in the antibody. In some embodiments of the present invention, the metal chelator is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), citric acid (CA), tartaric acid (TA), gluconic acid (GA) or N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA).

[0363] In some embodiments, the metal chelator is selected from EDTA, NTA and DTPA and sodium salts thereof. In some embodiments, the metal chelator in steps (B1) and (B3) is disodium ethylenediaminetetraacetic acid (EDTA-2Na).

[0364] In some embodiments, the molar ratio of the metal chelator to the antibody in step (B1) 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.

[0365] In some embodiments, the molar ratio of the metal chelator to the antibody in step (B3) 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.

[0366] In some embodiments, the second reducing agent in step (B2) is the same as the first reducing agent in step (A1). In some embodiments, the second reducing agent in step (B2) is different from the first reducing agent in step (A1). In some embodiments, the second reducing agent in step (B2) is independently tris(2-carboxyethyl)phosphine (TCEP).

[0367] In some embodiments, in step (B2), one of the interchain disulfide bonds in the product prepared by steps (A1) and (B1) is completely reduced in the absence of transition metal ions. In some embodiments, in step (B2), three of the interchain disulfide bonds in the product prepared by steps (A1), (A2) and (B1) are completely reduced in the absence of transition metal ions. In some embodiments, one or two of the interchain disulfide bonds in the product prepared by steps (A1), (A2) and (B1) are reduced in the presence of transition metal ions.

[0368] In some embodiments, the molar ratio of the second reducing agent to the antibody in step (B2) is independently 0.05:1 to 20:1, 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, 1:1 to 5:1, 1:1 to 1:4:1, 1:1 to 3:1, 1:1 to 2:1, 1.5:1 to 3:1.

[0369] In some embodiments, step (A1), step (B1) and step (B2) are employed in the absence of transition metal ions, and the molar ratio of the second reducing agent to the antibody in step (B2) is independently 1:1 to 3:1, 2:1 to 1:1, 1.5:1 to 1:1 or 1.2:1 to 1:1.

[0370] In some embodiments, step (A1), step (A2), step (B1), and step (B2) are used in the absence of transition metal ions, and the molar ratio of the second reducing agent to the antibody in step (B2) is independently 3: 1 to 20: 1, 6: 1 to 20: 1, 4: 1 to 10: 1. In some embodiments, step (A1), step (A2), step (B1), and step (B2) are used in the absence of transition metal ions, and the molar ratio of the second reducing agent to the antibody in step (B2) is independently 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.

[0371] In some embodiments, in step (B2), the incubation temperature is 0°C to 37°C, and the incubation time is 0.2 hours to 24 hours.

[0372] In some embodiments, in step (B2), the incubation temperature is 0°C, 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C or 36°C.

[0373] In some embodiments, step (A1), step (B1) and step (B2) are used in the absence of transition metal ions, and the incubation temperature of the second reducing agent in step (B2) is 5° C. to 37° C., 10° C. to 37° C., 15° C. to 37° C., 20° C. to 37° C. or 25° C. to 37° C. In some embodiments, step (A1), step (B1) and step (B2) are used in the absence of transition metal ions, and the incubation temperature in step (B2) is room temperature or 37° C.

[0374] As used herein, the term "room temperature" refers to 23°C±2°C, 25°C±5°C or 20°C±5°C.

[0375] In some embodiments, the incubation temperature of the second reducing agent in step (B2) is 0° C. to 30° C., 0° C. to 25° C., 0° C. to 20° C., 0° C. to 15° C., or 0° C. to 10° C. In some embodiments, the incubation temperature in step (A1), step (A2), step (B1), and step (B2) is 4° C., 6° C., 8° C., 10° C., 12° C., or 14° C.

[0376] In certain embodiments, the incubation time in step (B2) is independently 0.5 hours to 24 hours, 1 hour to 10 hours, 1 hour to 8 hours, 1 hour to 5 hours, 1 hour to 3 hours, 2 hours to 4 hours, 1 hour to 4 hours, or 2 hours to 5 hours. In certain embodiments, in step (B2), the incubation time is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours.

[0377] In some embodiments, step (A1), step (B1) and step (B2) are used in the absence of transition metal ions, and the incubation time of the second reducing agent in step (B2) is 0.5 hours to 18 hours, 0.5 hours to 12 hours, 1 hour to 10 hours, 1 hour to 8 hours, 1 hour to 5 hours, 1 hour to 3 hours, 2 hours to 4 hours, 1 hour to 4 hours, or 2 hours to 5 hours. In some embodiments, step (A1), step (B1) and step (B2) are used in the absence of transition metal ions, and the incubation time in step (B2) is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours.

[0378] In some embodiments, steps (A1), (A2), (B1) and (B2) are performed in the absence of transition metal ions, and the incubation time in step (B2) is 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours.

[0379] In some embodiments, the transition metal ion is introduced to selectively reduce two interchain disulfide bonds in the interchain disulfide bonds. In some embodiments, steps (A1), (A2), (B1) and (B2) are used, and in step (B2), 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, steps (A1), (A2), (B1) and (B2) are used, and in step (B2), 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, step (A1), step (A2), step (B1) and step (B2) are used, and in step (B2), 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, step (A1), step (A2), step (B1) and step (B2) are used, and in step (B2), 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, using step (A1), step (A2), step (B1) and step (B2), in step (B2), 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, using step (A1), step (A2), step (B1) and step (B2), in step (B2), 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.

[0380] In some embodiments, the transition metal ion is introduced to selectively reduce one of the interchain disulfide bonds. In some embodiments, steps (A1), step (A2), step (B1) and step (B2) are used, and in step (B2), 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, steps (A1), step (A2), step (B1) and step (B2) are used, and in step (B2), 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, step (A1), step (A2), step (B1) and step (B2) are used, and in step (B2), the molar ratio of the second reducing agent to the antibody is 0.8: 1, 1: 1, 1.2: 1, 1.4: 1, 1.6: 1, 1.8: 1, 2: 1, 2.2: 1, 2.4: 1, 2.5: 1. In some embodiments, step (A1), step (A2), step (B1) and step (B2) are used, and in step (B2), the incubation time is 0.5 hours, 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, using step (A1), step (A2), step (B1) and step (B2), in step (B2), 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 2:1, and the incubation time is 0.5 hours to 24 hours. In some embodiments, using step (A1), step (A2), step (B1) and step (B2), in step (B2), the molar ratio of the second reducing agent to the transition metal ion is 1:0.54 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.

[0381] Without being bound by theory, the reducing agent selectively reduces disulfide bonds in the buffer system. The buffer systems in steps (A1), (A3) and (B2) 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, acetate buffer, ADA buffer, BTP buffer, HEPPSO buffer, POPSO buffer, EPPS buffer or Tris buffer.

[0382] As used herein, the term "MES buffer" refers to 2-(N-morpholino)ethanesulfonic acid buffer.

[0383] As used herein, the term "Bis-Tris buffer" refers to a bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane buffer.

[0384] The term "PIPES buffer" herein refers to piperazine-1,4-bisethanesulfonic acid buffer.

[0385] The term "MOPS buffer" herein refers to 3-morpholinepropanesulfonic acid buffer.

[0386] The term "BES buffer" herein refers to N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer.

[0387] As used herein, the term "HEPES buffer" refers to 4-hydroxyethylpiperazineethanesulfonic acid buffer.

[0388] As used herein, the term "DIPSO buffer" refers to 3-[bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid buffer.

[0389] The term "MOBS buffer" herein refers to 3-morpholinepropanesulfonic acid buffer.

[0390] The term "MOPSO buffer" herein refers to 3-(N-morpholino)-2-hydroxy-1-propanesulfonic acid buffer.

[0391] The term "TES buffer" herein refers to 2-[tris(hydroxymethyl)methylamino]-1-ethanesulfonic acid buffer.

[0392] The term "ACES buffer" herein refers to N-(carbamoylmethyl)taurine buffer.

[0393] As used herein, the term "TAPSO buffer" refers to 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid buffer.

[0394] The term "PBS" herein refers to phosphate buffered saline.

[0395] The term "ADA buffer" herein refers to N-(carbamoylmethyl)iminodiacetic acid buffer.

[0396] The term "PB buffer" herein refers to phosphate buffered saline.

[0397] The term "BTP buffer" herein refers to Bis-tris propane buffer.

[0398] The term "Heppso buffer" herein refers to N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid buffer.

[0399] The term "POPSO buffer" herein refers to piperazine-N,N'-bis(2-hydroxy-propanesulfonic acid) buffer.

[0400] The term "EPPS buffer" herein refers to 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid buffer.

[0401] The term "Tris buffer" herein refers to tris(hydroxymethyl)aminomethane buffer.

[0402] In some embodiments, the buffer systems in steps (A1), (A3) and (B2) are independently MES buffer, Bis-Tris buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer or TAPSO buffer.

[0403] In some embodiments, the buffer systems in steps (A1), (A3) and (B2) are independently Bis-Tris buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer or TAPSO buffer.

[0404] In some embodiments, the buffer system is BES buffer.

[0405] 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 6.7 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.

[0406] In some embodiments, the buffer systems in steps (A1) and (A3) are the same. In some embodiments, the buffer systems in steps (A1) and (A3) are different. In some embodiments, the buffer systems in steps (A1) and (B2) are the same. In some embodiments, the buffer systems in steps (A1) and (B2) are different.

[0407] The concentration of the buffer system in steps (A1), (A3) and (B2) of the method is independently in the range of 10mM to 100mM (mmol / L). Optionally, the concentration of the buffer system in steps (A1), (A3) and (B2) of the method 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.

[0408] In some embodiments, the concentration of the buffer system in steps (A1) and (A3) is the same. In some embodiments, the concentration of the buffer system in steps (A1) and (A3) is different. In some embodiments, the concentration of the buffer system in steps (A1) and (B2) is the same. In some embodiments, the concentration of the buffer system in steps (A1) and (B2) is different.

[0409] In some embodiments, the method further comprises the step of introducing a compound containing at least one thiol group to consume excess first linker-payload and excess second linker-payload. In some embodiments, the compound is cysteine.

[0410] In some embodiments, the method further comprises the steps of purifying and recovering the product from (B1) and / or (B3).

[0411] In some embodiments, the resulting antibody drug conjugate is recovered by any suitable purification method, such as using a desalting column, size exclusion chromatography, ultrafiltration, dialysis, ultrafiltration (UF)-diafiltration (DF), etc. If desired, in some cases, further ADC enrichment (e.g., D2) can be applied using hydrophobic interaction chromatography (HIC).

[0412] In some embodiments, in step (B1) and / or (B3), the obtained ADC is purified by desalting column, size exclusion chromatography, ultrafiltration, dialysis, etc. In some embodiments of the present application, in step (B1) and / or (B3), the obtained ADC is purified by desalting column.

[0413] In some embodiments, the linkers of the first linker-payload toxin and the second linker-payload toxin are selected from any linker that meets the following requirements: one end of which can be connected to a reduced thiol group of an antibody or a reactive group of a thio-bridging reagent and the other end of which can be connected to a payload toxin.

[0414] As used herein, the term "linker" refers to a substituted molecule containing at least two substituent groups, one of which can covalently bond to a drug molecule and the other of which can covalently couple to a reactive group of an antibody or a thio-bridging agent.

[0415] In some embodiments, when the first linker-payload and the second linker-payload react with the 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 can be a chemically unstable linker and an enzyme-cleavable linker. Due to having higher plasma stability and good intracellular cleavage selectivity and effectiveness, the enzyme-cleavable linker is widely selected as a cleavable linker candidate in ADC. In some embodiments, the enzyme-unstable linker can include a peptide unit (-AAs-), -maleimidocaproyl-(-MC-), p-aminobenzyl alcohol-(-PAB-) or -MC-peptide unit-PAB-. In some embodiments, the peptide unit is a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.

[0416] In some embodiments, the dipeptide can be, but is not limited to, valine-alanine (VA), valine-citrulline (VC), alanine-asparagine (AD), alanine-phenylalanine (AF), phenylalanine-lysine (FK), alanine-lysine (AK), alanine-valine (AV), valine-lysine (VK), lysine-lysine (KK), phenylalanine-citrulline (FC), leucine-citrulline (LC), isoleucine-citrulline (IC), tryptophan-citrulline (WC) or phenylalanine-alanine (FA). In some embodiments, the dipeptide can 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.

[0417] In some embodiments, the tripeptide can be, but is not limited to, alanine-alanine-asparagine (AAD), glycine-valine-citrulline (GVC), glycine-glycine-glycine (GGG), phenylalanine-phenylalanine-lysine (FFK), glutamic acid-valine-citrulline (EVC), or glycine-phenylalanine-lysine (GFK).

[0418] In some embodiments, the tetrapeptide can be, but is not limited to, glycine-glycine-phenylalanine-glycine (GGFG).

[0419] 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.

[0420] 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.

[0421] 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.

[0422] In some embodiments, when the first linker-payload toxin and / or the second linker-payload toxin reacts with a reactive group in a thio-bridging reagent, the linker of the first linker-payload toxin and / or the second linker-payload toxin further comprises an azide group and / or dibenzocyclooctyne (DBCO). In some embodiments, when the linker of the first linker-payload toxin and / or the second linker-payload toxin contains an azide group, the reactive group of the thio-bridging group contains DBCO. In some embodiments, when the linker of the first linker-payload toxin and / or the second linker-payload toxin contains DBCO, the reactive group of the thio-bridging group contains an azide group.

[0423] In some embodiments, when the linker of the first linker-payload toxin and / or the second linker-payload toxin reacts with the reactive group in the thio-bridging reagent, the linker of the first linker-payload toxin and the second linker-payload toxin are independently selected from:

[0424]

[0425]

[0426] Wherein n of the linker is an integer of 0-20, 0-18, 0-15, 0-13, 0-10, 0-7, 0-5 or 0-3, and m is an integer of 0-20, 0-18, 0-15, 0-13, 0-10, 0-7, 0-5 or 0-3; depending on the circumstances, 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.

[0427] 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.

[0428] In some embodiments, the payload is a cytotoxic drug, a cytokine, a nucleic acid, a radionuclide, a kinase, or a derivative thereof. In some embodiments, the payload includes, but is not limited to, a topoisomerase inhibitor and a tubulin inhibitor.

[0429] 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.

[0430] In some embodiments, the payload is deludecanoic acid (DXd), cyanine 3 (Cy3), MMAE, MMAD or MMAF. In some embodiments of the invention, the payload is MMAE, DXd or Cy3.

[0431] 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 a reactive group for coupling to the antibody by a covalent bond. In the present invention, a drug-maleimide complex (i.e., maleimide-connected drug) is used as an example of a payload carrying a reactive group. In ADC preparation, the most common reactive group that can be combined with a thiol group is maleimide. In addition, organic chlorides, bromides, and iodides are also often used.

[0432] The linker-payload toxin can be any physically active compound, or any compound used to diagnose, prevent or treat a disease, such as MC-GGFG-DXd, MC-VC-PAB-MMAE, MC-VC-PAB-MMAD, and MC-VC-PAB-MMAF.

[0433] 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.

[0434] The first thio-bridging reagent and the second thio-bridging reagent each independently contain at least two substituent groups to re-bridge the thiol group.

[0435] In some embodiments, without limitation, the first thio-bridging reagent and the second thio-bridging reagent are each independently selected from:

[0436]

[0437] In some embodiments, the reactive group contains an azide and / or dibenzocyclooctyne (DBCO).

[0438] In some embodiments, the thio-bridging reagent and the reactive group are linked via an alkyl group or polyethylene glycol (PEG).

[0439] In some embodiments, without limitation, the first thio-bridging reagent having a reactive group and the second thio-bridging reagent having a reactive group are each independently selected from:

[0440]

[0441] Wherein, n of the first thio-bridging reagent with a reactive group and the second thio-bridging reagent with a reactive group is an integer of 0-20, 0-18, 0-15, 0-13, 0-10, 0-7, 0-5 or 0-3.

[0442] In some embodiments, the first thio-bridging reagent with a reactive group may be different from the second thio-bridging reagent with a reactive group. In some embodiments, the first thio-bridging reagent with a reactive group may be the same as the second thio-bridging reagent with a reactive group.

[0443] In some embodiments, the first thio-bridging reagent having a reactive group and the second thio-bridging reagent having a reactive group are dibromomaleimide-PEG4-N3 having the formula:

[0444]

[0445] Under appropriate conditions, the reactive group may react with the linker-payload toxin, and the linker-payload toxin is linked to the thio-bridging reagent by a covalent bond. As the reactive group or linker-payload toxin changes, the reaction product may change. In the present disclosure, products with different reactive groups and linker-payload toxins are collectively referred to as "thio-bridging reagents with linker-payload toxins".

[0446] In some embodiments, the first thio-bridging reagent with the first linker-payload toxin and the second thio-bridging reagent with the second linker-payload toxin have the following formula:

[0447] QST

[0448] Wherein Q is selected from the group consisting of:

[0449]

[0450] S is selected from, but not limited to, a cleavable linker or a non-cleavable linker, S is selected from the group consisting of:

[0451]

[0452] 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.

[0453] T is the effective load.

[0454] In some embodiments, the first thio-bridging reagent with a first linker-payload toxin and the second thio-bridging reagent with a second linker-payload toxin are selected from the group consisting of:

[0455]

[0456]

[0457] Thus, the first payload toxin unit includes a first linker-payload toxin, a first thio-bridging reagent with a reactive group, and a first thio-bridging reagent with a first linker-payload toxin.

[0458] The second payload toxin unit includes a second linker-payload toxin, a second thio-bridging reagent with a reactive group, and a second thio-bridging reagent with a second linker-payload toxin.

[0459] In some embodiments, the first payload toxin unit is a first linker-payload toxin, a first thio-bridging reagent with a reactive group, or a first thio-bridging reagent with a first linker-payload toxin.

[0460] In some embodiments, the second payload toxin unit is a second linker-payload toxin, a second thio-bridging reagent with a reactive group, or a second thio-bridging reagent with a second linker-payload toxin.

[0461] In some embodiments, the payload toxin of the first thio-bridging reagent with the first linker-payload toxin and the payload toxin of the second thio-bridging reagent with the second linker-payload toxin are different. In some embodiments, the linker of the first thio-bridging reagent with the first linker-payload toxin and the linker of the second thio-bridging reagent with the second linker-payload toxin may be different. In some embodiments, the linker of the first thio-bridging reagent with the first linker-payload toxin and the linker of the second thio-bridging reagent with the second linker-payload toxin may be the same. In some embodiments, the thio-bridging reagent of the first thio-bridging reagent with the first linker-payload toxin and the thio-bridging reagent of the second thio-bridging reagent with the second linker-payload toxin may be different. In some embodiments, the thio-bridging reagent of the first thio-bridging reagent with the first linker-payload toxin and the thio-bridging reagent of the second thio-bridging reagent with the second linker-payload toxin may be the same.

[0462] As used herein, the term "capping agent" refers to a compound that does not carry a drug but contains at least one substituent that can be covalently coupled to an antibody.

[0463] 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.

[0464] In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a monospecific antibody, or a multispecific antibody.

[0465] As used herein, the term "antibody" refers to any immunoglobulin 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 and the first, second and third constant regions, while each light chain consists of a variable region and a constant region. The heavy chains from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG and IgM.

[0466] 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 includes approximately 25 amino acid residues and is flexible so that the two N-terminal antigen binding regions can move independently.

[0467] 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. Fab fragments can be obtained by protease digestion of full-length antibodies. Under the action of papain, human immunoglobulin G can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into F(ab')2 fragments and pFc' fragments. The F(ab')2 fragment can be further reduced to form two Fab' fragments.

[0468] The term "Fc region" herein refers to a monomeric protein, a dimeric protein or a heterodimeric protein having at least an immunoglobulin CH2 and CH3 domains. The CH2 and CH3 domains may form at least a portion of a dimer region of a protein / molecule (eg, an antibody).

[0469] In some embodiments, the antibody is a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding portion thereof.

[0470] 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.

[0471] 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.

[0472] 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.

[0473] There is no particular limitation on the antibody. Depending on the antigen associated with the disease, a person skilled in the art can select a suitable antibody that can be used in the bioconjugation process of the present invention.

[0474] In some embodiments of the invention, 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.

[0475] In some embodiments of the invention, the class of the antibody is IgG1, IgG2, IgG3 or IgG4. In some embodiments, the antibody is IgG1 or IgG4.

[0476] In some embodiments of the present application, the antibody is wild-type. The term "wild-type" herein refers to naturally occurring and without mutation.

[0477] In some embodiments of the present application, the antibody comprises at least one mutation in the Fc region. In some embodiments, the at least one mutation modulates effector function, or reduces or eliminates Fc-g receptor binding.

[0478] 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.

[0479] 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.

[0480] 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.

[0481] 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.

[0482] 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.

[0483] In some embodiments, the Fc region includes a mutation at residue positions 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 positions 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 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.

[0484] 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.

[0485] 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.

[0486] 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 / ).

[0487] 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.

[0488] 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.

[0489] 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.

[0490] In some embodiments of the present application, the antibody is selected from any one of the following: a cytotoxic antibody, a cell proliferation inhibitor, a cell activation and interaction regulator, a human immune system regulator, an antigen neutralizer, an antibody with immune specificity for a viral antigen, or an antibody with immune specificity for a microbial antigen.

[0491] There is no particular limitation on the antibody. Depending on the antigen associated with the disease, a person skilled in the art can select a suitable antibody that can be used in the bioconjugation process of the present invention.

[0492] In some embodiments, the antibody has targeting specificity, and it targets HER2 (human epidermal growth factor receptor 2), TROP2 (TACSTD2, tumor-associated calcium signal transducer 2), BCMA (TNFRSF17, TNF receptor superfamily member 17).

[0493] In some embodiments, the antibody is trastuzumab, gosartuzumab, or mabelantuzumab.

[0494] In some embodiments, the antibodies can be purchased commercially or produced by any method known to those skilled in the art.

[0495] The present application provides a modification prepared by the above method.

[0496] The resulting modified antibodies include ADCs. In some embodiments, the ADCs include ADCs with D2, ADCs with D4, ADCs with D1, ADCs with D6, ADCs with D3, ADCs with D1+D6, ADCs with D1+D2, ADCs with D1+D4, ADCs with D2+D4, ADCs with D6+D2, ADCs with D6+D1, ADCs with D3+D1, ADCs with D3+D2, ADCs with D0+D6, or ADCs with D0+D2.

[0497] In some embodiments, the ADC is 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-GGFG-DXd]6[maleimide-PEG4-N3-DBCO-Cy3]1, trastuzumab-[maleimide]6[MC-VC-PAB-MMAE]2, trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1 , trastuzumab-[MC-VC-PAB-MMAE]2, trastuzumab-[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 and trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4.

[0498] The present disclosure also provides antibodies with site-specific modifications, wherein two interchain disulfide bonds in the Fab region and one interchain disulfide bond in the hinge region of the antibody are reduced, coupled or modified.

[0499] In some embodiments, an antibody with site-specific modification (ADC with D6) is prepared by a method comprising steps (A1) and (B1), wherein the first payload toxin unit is a first linker-payload toxin.

[0500] In some embodiments, an antibody with site-specific modification (ADC with D3) is prepared by a method comprising steps (A1) and (B1), wherein the first payload toxin unit is a first thio-bridging reagent with a reactive group (reacting with a first linker-payload toxin).

[0501] In some embodiments, an antibody with site-specific modification (ADC with D3) is prepared by a method comprising steps (A1) and (B1), wherein the first payload toxin unit is a first thio-bridging reagent with a first linker-payload toxin.

[0502] The present disclosure also provides antibodies with site-specific modifications, wherein one interchain disulfide bond in the hinge region of the antibody is reduced, coupled or modified.

[0503] In some embodiments, provided herein is an antibody with site-specific modification (ADC with D2) prepared by a method comprising steps (A1), (A2) and (B1), wherein the first payload toxin unit is a first linker-payload toxin.

[0504] In some embodiments, an antibody with site-specific modification (ADC with D1) is prepared by a method comprising steps (A1), (A2) and (B1), wherein the first payload toxin unit is a first thio-bridging reagent with a reactive group (reacting with a first linker-payload toxin).

[0505] In some embodiments, an antibody with site-specific modification (ADC with D1) is prepared by a method comprising steps (A1), (A2) and (B1), wherein the first payload toxin unit is a first thio-bridging reagent with a first linker-payload toxin.

[0506] The present disclosure also provides antibodies with site-specific modifications, wherein two interchain disulfide bonds in the hinge region of the antibody are reduced, coupled or modified.

[0507] In some embodiments, an antibody with site-specific modification (ADC with D4) is prepared by a method comprising steps (A1), (A2), (A3) and (B1), wherein the first payload toxin unit is a first linker-payload toxin.

[0508] In some embodiments, provided herein is an antibody with site-specific modification (ADC with D1+D2) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging agent with a first linker-payload toxin, and the second payload toxin unit is a second linker-payload toxin. At the same time, a transition metal ion is introduced in step (B2).

[0509] In some embodiments, provided herein is an antibody with site-specific modification (ADC with D1+D2) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging reagent with a reactive group (reacting with the first linker-payload toxin), and the second payload toxin unit is a second linker-payload toxin. At the same time, a transition metal ion is introduced in step (B2).

[0510] In some embodiments, provided herein is an antibody with site-specific modification (ADC with D2+D2) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first linker-payload toxin, and the second payload toxin unit is a second linker-payload toxin. At the same time, a transition metal ion is introduced in step (B2).

[0511] In some embodiments, provided herein is an antibody with site-specific modification (ADC with D2+D1) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first linker-payload toxin, and the second payload toxin unit is a second thio-bridging agent with a second linker-payload toxin. At the same time, a transition metal ion is introduced in step (B2).

[0512] In some embodiments, provided herein is an antibody with site-specific modification (ADC with D2+D1) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first linker-payload toxin, and the second payload toxin unit is a second thio-bridging agent with a reactive group (reacting with the second linker-payload toxin). At the same time, a transition metal ion is introduced in step (B2).

[0513] In some embodiments, provided herein is an antibody with site-specific modification (ADC with D1+D4) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging agent with a first linker-payload toxin, and the second payload toxin unit is a second linker-payload toxin. At the same time, a transition metal ion is introduced in step (B2).

[0514] In some embodiments, provided herein is an antibody with site-specific modification (ADC with D1+D4) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging reagent with a reactive group (reacting with the first linker-payload toxin), and the second payload toxin unit is a second linker-payload toxin. At the same time, a transition metal ion is introduced in step (B2).

[0515] In some embodiments, provided herein is an antibody with site-specific modification (ADC with D2+D4) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first linker-payload toxin, and the second payload toxin unit is a second linker-payload toxin. At the same time, a transition metal ion is introduced in step (B2).

[0516] The present disclosure also provides antibodies with site-specific modifications, wherein two interchain disulfide bonds in the Fab region and two interchain disulfide bonds in the hinge region of the antibody are reduced, coupled or modified.

[0517] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D6+D2) prepared by a method comprising steps (A1), (B1), (B2), and (B3), wherein the first payload toxin unit is a first linker-payload toxin and the second payload toxin unit is a second linker-payload toxin.

[0518] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D6+D1) prepared by a method comprising steps (A1), (B1), (B2), and (B3), wherein the first payload toxin unit is a first linker-payload toxin, and the second payload toxin unit is a second thio-bridging reagent (reacting with a second linker-payload toxin) with a reactive group.

[0519] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D6+D1) prepared by a method comprising steps (A1), (B1), (B2), and (B3), wherein the first payload toxin unit is a first linker-payload toxin and the second payload toxin unit is a second thio-bridging agent with a second linker-payload toxin.

[0520] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D3+D2) prepared by a method comprising steps (A1), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging reagent with a first linker-payload toxin, and the second payload toxin unit is a second linker-payload toxin.

[0521] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D3+D2) prepared by a method comprising steps (A1), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging reagent with a reactive group (reacting with a first linker-payload toxin), and the second payload toxin unit is a second linker-payload toxin.

[0522] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D3+D1) prepared by a method comprising steps (A1), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging reagent with a first linker-payload toxin, and the second payload toxin unit is a second thio-bridging reagent with a second linker-payload toxin.

[0523] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D3+D1) prepared by a method comprising steps (A1), (B1), (B2) and (B3), wherein the first payload toxin unit is a first thio-bridging reagent with a reactive group (reacting with a first linker-payload toxin), and the second payload toxin unit is a second thio-bridging reagent with a reactive group (reacting with a second linker-payload toxin).

[0524] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D0+D2) prepared by a method comprising steps (A1), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging agent and the second payload toxin unit is a second linker-payload toxin.

[0525] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D0+D1) prepared by a method comprising steps (A1), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging agent and the second payload toxin unit is a second thio-bridging agent with a second linker-payload toxin.

[0526] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D0+D1) prepared by a method comprising steps (A1), (B1), (B2), and (B3), wherein the first payload toxin unit is a thio-bridging reagent and the second payload toxin unit is a second thio-bridging reagent with a reactive group (reacting with a second linker-payload toxin).

[0527] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D2+D6) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first linker-payload toxin and the second payload toxin unit is a second linker-payload toxin.

[0528] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D1+D6) prepared by a method comprising steps (A1), (A2), (B1), (B2) and (B3), wherein the first payload toxin unit is a first thio-bridging reagent with a reactive group (reacting with a first linker-payload toxin), and the second payload toxin unit is a second linker-payload toxin.

[0529] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D1+D6) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging reagent with a first linker-payload toxin, and the second payload toxin unit is a second linker-payload toxin.

[0530] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D2+D3) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first linker-payload toxin and the second payload toxin unit is a second thio-bridging agent with a second linker-payload toxin.

[0531] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D2+D3) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first linker-payload toxin and the second payload toxin unit is a second thio-bridging reagent (reacting with a second linker-payload toxin) with a reactive group.

[0532] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D1+D3) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging reagent with a first linker-payload toxin, and the second payload toxin unit is a second thio-bridging reagent with a second linker-payload toxin.

[0533] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D1+D3) prepared by a method comprising steps (A1), (A2), (B1), (B2) and (B3), wherein the first payload toxin unit is a first thio-bridging reagent with a reactive group (reacting with a first linker-payload toxin), and the second payload toxin unit is a second thio-bridging reagent with a reactive group (reacting with a second linker-payload toxin).

[0534] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D0+D6) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging agent and the second payload toxin unit is a second linker-payload toxin.

[0535] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D0+D3) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a first thio-bridging agent and the second payload toxin unit is a second thio-bridging agent with a second linker-payload toxin.

[0536] In some embodiments, provided herein is an antibody with site-specific modifications (ADC with D0+D3) prepared by a method comprising steps (A1), (A2), (B1), (B2), and (B3), wherein the first payload toxin unit is a thio-bridging reagent and the second payload toxin unit is a second thio-bridging reagent with a reactive group (reacting with a second linker-payload toxin).

[0537] 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. HIC-HPLC can separate antibodies loaded with different numbers of linker-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.

[0538] The method for generating antibodies with site-specific modifications does not require protein engineering or enzyme catalysis, but is based on natural interchain disulfide bonds, and only requires the use of novel 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.

[0539] Pharmaceutical composition

[0540] The present application also provides a pharmaceutical composition, which comprises an antibody (prepared by the above method and having site-specific modification) and at least one pharmaceutically acceptable ingredient.

[0541] 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.

[0542] A pharmaceutical composition is formulated to be compatible with its intended route of administration (eg, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal administration).

[0543] Pharmaceutically acceptable carriers 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.

[0544] 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.

[0545] 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.

[0546] In some embodiments of the present application, 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.

[0547] In some embodiments of the present application, the anticancer agent may include, but is not limited to, erlotinib, bortezomib, fulvestrant, sunitinib imatinib, mesylate, letrozole, finasute, platinums such as oxaliplatin, carboplatin and cisplatin, finasute, fluorouracil, rapamycin, folinic acid, lapatinib, lonafarnib, sorafenib, gefitinib, cartosicin, topotecan, bryostatin, adelaxin, anthracyclines, kazeresin, biszepam, dolastatin, auristatin, duocarmycin, elucerobin, taxanes such as paclitaxel or docetaxel, cyclosporine ... Phosphamide, 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 such as vinblastine, vinblastine, vinorelbine and vindesine as well as taxanes), epipodophyllotoxins (etoposide, teniposide, etoposide phosphate and epipodophyllotoxin), topoisomerase inhibitors, other cytotoxins such as actinomycin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin, mitomycin, etc.

[0548] The present disclosure provides use of the antibodies having site-specific modifications described herein in the preparation of therapeutic agents for preventing, diagnosing or treating diseases.

[0549] 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.

[0550] 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.

[0551] In some embodiments, the disease is a tumor or cancer. In some embodiments, the disease is an autoimmune disease, etc.

[0552] In some embodiments of the present application, cancer may include, but is not limited to, epithelial cancer, lymphoma, blastoma, sarcoma, 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 cell 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.

[0553] The present disclosure provides a method for preventing, diagnosing or treating a disease (in a subject in need thereof), the method comprising administering to the subject a therapeutically effective amount of an antibody (prepared by the above method and having site-specific modification).

[0554] 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.

[0555] 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.

[0556] 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.

[0557] Example

[0558] The invention is further described in the following examples, which are not intended to limit the scope of the invention described in the claims.

[0559] Reagents and manufacturers

[0560] Trastuzumab is commercially available from Roche.

[0561] Gosartuzumab and mabelantuzumab are commercially available from MedChemExpress.

[0562] EDTA is commercially available from Aladdin.

[0563] DMA (dimethylacetamide) can be purchased from Aldrich Sigma.

[0564] MC-VC-PAB-MMAE can be purchased from Levena biopharma.

[0565] MC-GGFG-DXd can be purchased from Levena biopharma.

[0566] Desalting columns (type: 40K, 0.5 mL, reference number: 87766, batch number: SJ251704) can be purchased from ThermoScientific.

[0567] DBCO-Cy3 can be purchased from Confluore.

[0568] TCEP is commercially available from Bidepharm.

[0569] Dibromomaleimide is commercially available from Aladdin.

[0570] The reagents used in the examples are all commercially available reagents, including but not limited to 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).

[0571] Synthetic Procedure A of Compounds According to the Invention

[0572] 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.

[0573] Synthesis Procedure A-1 of Compounds According to the Invention

[0574] 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.

[0575] Synthetic Procedure B for Compounds According to the Invention

[0576] 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.

[0577] Synthesis Procedure B-1 of Compounds According to the Invention

[0578] 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.

[0579] Homogeneity determination

[0580] Drug / antibody ratio (DAR) and product distribution were analyzed at 25°C with a flow rate of 0.5 mL / min using HIC-HPLC (Agilent 1200) with a TSK gel butyl-NPR column (2.5 μm, 4.6 mM*3.5 cm) (available from Tosoh Biosciences). Solvent A was 1.5 M (NH4)2SO4 and 50 mM K2HPO4·3H2O. Solvent B was 75% v / v of 21.3 mM KH2PO4, 28.6 mM K2HPO4·3H2O and 25% v / v isopropanol. The flushing procedure was as follows:

[0581] 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

[0582] Example 1: Synthesis of TCEP-NO-Trtyl and TCEP-NO

[0583]

[0584] 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. 1H 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).

[0585] 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).

[0586] Example 2: Synthesis of TCEPA

[0587]

[0588] 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. 1H NMR (400 MHz, deuterium oxide) δ 2.85-2.70 (m, 4H), 2.61-2.43 (m, 6H), 2.15-2.06 (m, 2H).

[0589] Example 3: Synthesis of TCEP-1

[0590]

[0591] 1.TCEP-1-int2

[0592] 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.

[0593] 2.TCEP-1-int3

[0594] 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).

[0595] 3.TCEP-1

[0596] 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. 1H-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).

[0597] Example 4: Synthesis of TCEP-2

[0598]

[0599] 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).

[0600] Example 5: Synthesis of TCEP-3

[0601]

[0602] TCEP-3 was synthesized in Procedure B, where compound 6 (DL-phenylalanine, Adamas) was the amine reagent to give 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).

[0603] Example 6: Synthesis of TCEP-4

[0604]

[0605] 1.TCEP-4-int1

[0606] 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.

[0607] 2.TCEP-4

[0608] 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).

[0609] Example 7: Synthesis of TCEP-5

[0610]

[0611] TCEP-5 was synthesized in Procedure B, where (2-phenoxy-ethylamine, Bidepharm) was used as the amine reagent to obtain 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).

[0612] Example 8: Synthesis of TCEP-6

[0613]

[0614] 1.TCEP-6-int1

[0615] 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.

[0616] 2.TCEP-6

[0617] TCEP-6 was synthesized in Procedure A-1, wherein 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).

[0618] Example 9: Synthesis of TCEP-7

[0619]

[0620] 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).

[0621] Example 10: Synthesis of TCEP-8

[0622]

[0623] TCEP-8 was synthesized in the synthetic procedure B, wherein (benzylamine, Adamas) was used as the amine reagent to obtain TCEP-8 (85.6 mg, yield: 50.5%) as 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).

[0624] Example 11: Synthesis of TCEP-9

[0625]

[0626] 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).

[0627] Example 12: Synthesis of TCEP-10

[0628]

[0629] 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. 1H 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).

[0630] Example 13: Synthesis of TCEP-11

[0631]

[0632] 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).

[0633] Example 14: Synthesis of TCEP-12

[0634]

[0635] TCEP-12 was synthesized in Synthesis Procedure A, where (5-amino-8-hydroxyquinoline, Bidepharm) was used as an 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. 1H 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).

[0636] Example 15: Synthesis of TCEP-15

[0637]

[0638] 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).

[0639] Example 16: Synthesis of TCEP-18

[0640]

[0641] 1.TCEP-18-int1

[0642] 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) and 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%) in the form of a transparent liquid.

[0643] 2.TCEP-18

[0644] 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 15 The 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).

[0645] Example 17: Synthesis of TCEP-19

[0646]

[0647] 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).

[0648] Example 18: Synthesis of TCEP-20

[0649]

[0650] 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).

[0651] Example 19: Synthesis of TCEP-21

[0652]

[0653] 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).

[0654] Example 20: Synthesis of TCEP-23

[0655]

[0656] 1.TCEP-23-int1

[0657] 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.

[0658] 2.TCEP-23

[0659] 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 H 21 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).

[0660] Example 21: Synthesis of TCEP-24

[0661]

[0662] 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).

[0663] Example 22: Synthesis of TCEP-25

[0664]

[0665] 1.TCEP-25-int1

[0666] 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.

[0667] 2.TCEP-25 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): δ 8.60 (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).

[0668] Example 23: Synthesis of TCEP-26

[0669]

[0670] 1.TCEP-26-int1

[0671] 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 reactant was 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.

[0672] 2.TCEP-26-int2

[0673] 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%).

[0674] 3.TCEP-26

[0675] The compound was synthesized according to the synthetic procedure A-1, wherein TCEP-26-int2 was an amine reagent, to obtain TCEP-26 (31.5 mg, yield 21.3%) in the form of 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).

[0676] Example 24: Synthesis of TCEP-28

[0677]

[0678] 1.TCEP-28-int1

[0679] To a solution of o-tritylhydroxylamine (1.4 g, 5.0 mmol, 1.0 equiv) in DMF (15 mL) 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.

[0680] 2.TCEP-28-int2

[0681] 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.

[0682] 3.TCEP-28

[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 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).

[0684] Example 25: Synthesis of TCEP-30

[0685]

[0686] 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. 1 H 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).

[0687] Example 26: Synthesis of TCEP-31

[0688]

[0689] 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).

[0690] Example 27: Synthesis of TCEP-32

[0691]

[0692] 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 23The 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).

[0693] Example 28: Synthesis of TCEP-33

[0694]

[0695] 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).

[0696] Example 29: Synthesis of TCEP-34

[0697]

[0698] 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. 1H 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).

[0699] Example 30: Synthesis of TCEP-35

[0700]

[0701] 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).

[0702] Example 31: Synthesis of TCEP-37

[0703]

[0704] 1.TCEP-37-int1

[0705] 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).

[0706] 2.TCEP-37

[0707] 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. 1 H 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).

[0708] Example 32: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate (ADC with D6) ADC was prepared by a one-pot reaction:

[0709] (1) ZnCl2 (0.024 mM) and the reducing agent TCEP-NO-Trtyl (0.048 mM) prepared by Example 1 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;

[0710] (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;

[0711] (3) Purify the reaction mixture using a desalting column. Example 33: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugate (ADC with D6) The method of Example 33 is similar to that of Example 32, except that the reducing agent is TCEP-NO prepared in Example 1.

[0712] Examples 34-57 and Comparative Example 5: Preparation of D6-type ADC

[0713] The methods of Examples 34-57 are similar to those of Example 32, 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 is shown in the following table. Meanwhile, in Examples 55-57, the incubation time in step (1) is 16 hours.

[0714]

[0715] “E” is the abbreviation of Example, “C” is the abbreviation of Comparative Example, and mAb is the abbreviation of monoclonal antibody.

[0716] The uniformity measurement results of Examples 32-57 and Comparative Example 5 are shown below:

[0717] Table 1

[0718]

[0719]

[0720] As shown in the table above, the linker-payload toxin (MC-VC-PAB-MMAE) was successfully linked to the antibody, indicating that the ADC prepared using the reducing agent in the present application in Examples 32-57 was successfully synthesized.

[0721] According to Examples 32-57 and Comparative Example 5, compared with the use of a Zn-free 2+ Compared with the traditional method of TCEP, the reducing agent in the present application can improve the uniformity of ADC with D6, which successfully proves that the combination of transition metal ions and novel reducing agents can increase the level of D6 in the resulting ADC. In addition, TCEP-3 has the best selective reducing ability, with a D6 content of up to 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 reducing abilities, with a D6 content of up to 90%.

[0722] According to the results of Examples 53 and 54, the reducing agent in the present application is suitable for preparing ADCs with different antibodies.

[0723] Examples 58 to 78: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugate

[0724] Examples 58-78 were prepared in a manner similar to the preparation of the ADC with D6 in Example 33, except that the reducing agent, the molar ratio of the reducing agent to the monoclonal antibody, the molar ratio of ZnCl2 to the monoclonal antibody, and / or the reduction time were adjusted in step (a), as shown below:

[0725]

[0726]

[0727] The results of the homogeneity test are as follows:

[0728] Table 2

[0729]

[0730] The results of Examples 58-78 are shown in Table 2. As shown in Table 2, the linker-payload toxin (MC-VC-PAB-MMAE) was successfully linked to trastuzumab, indicating that the ADC prepared using TCEPA or TCEP-NO in Examples 58-78 was successfully synthesized. TCEPA and TCEP-NO can be used as reducing agents for antibody modification and preparation of ADCs with D6.

[0731] According to the results in Table 2, 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 D6 level of the obtained ADC is high, which indicates that when the molar ratio of the reducing agent to the antibody is in the range of 2.5 to 13, it is beneficial to improve the uniformity of the ADC.

[0732] According to Examples 70-78, 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%.

[0733] Examples 79 to 90: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugates with Different Molar Ratios of ZnCl2 to Reducing Agent

[0734] The preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate was similar to the preparation of ADC in Example 32, but the reducing agent was TCEPA or TCEP-NO.

[0735] The reducing agent, the molar ratio of ZnCl2 to the reducing agent and the reducing agent time in step (1) are shown in the following table.

[0736] Comparative Example 1-4: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugate (Zn 2+ The concentration is 0)

[0737] The preparation of Comparative Example 1 is similar to the preparation of ADC in Example 32, but the reducing agent is TCEPA. The preparation of Comparative Example 2 is similar to the preparation in Example 73, the preparation of Comparative Example 3 is similar to the preparation in Example 75, and the preparation of Comparative Example 4 is similar to the preparation in Example 77, except that the concentration of ZnCl2 in step (1) is 0.

[0738]

[0739] The uniformity measurement results of Examples 79-90 and Comparative Examples 1-4 are shown below:

[0740] Table 3

[0741]

[0742] As shown in Table 3, the linker-payload toxin (MC-VC-PAB-MMAE) was successfully linked to trastuzumab, indicating that the use of TCEPA / TCEP-NO and Zn in Examples 79-90 2+ The prepared ADC has been successfully synthesized.

[0743] According to the results in Table 3, the proportion of D6 in Comparative Examples 1-4 is only 11%, 17%, 20% or 31%, but the proportion of D6 in Examples 79-90 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 in the range of 0.05 to 30, it is beneficial to improve the uniformity of the ADC. Examples 91 to 102: Preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate using different buffers

[0744] The preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate is similar to the preparation of ADC in Example 82, and the molar ratio of TCEPA to antibody and the buffer system are as follows:

[0745]

[0746] mAb is the abbreviation of monoclonal antibody. All buffers can be purchased from Macklin.

[0747] The results of the homogeneity test are as follows:

[0748] Table 4

[0749] serial number Attached photos D0 (wt%) D2 (wt%) D4 (wt%) D6 (wt%) D8 (wt%) E91 30 0 2 5 85 8 E92 31 0 0 4 87 9 E93 32 0 0 4 88 8 E94 33 0 3 5 85 7 E95 34 0 0 7 86 7 E96 35A 0.96 3.89 4.05 84.65 6.46 E97 35B 1.09 11.27 14.10 71.45 2.09 E98 35C 0 1.22 4.83 86.55 7.40 E99 35D 1.04 4.67 5.54 83.54 5.21 E100 35E 0 3.70 3.90 88.11 4.28 E101 35F 1.41 8.83 20.36 66.55 2.85 E102 35G 0 7.59 17.42 52.10 22.89

[0750] The results of Examples 91-102 are shown in Table 4. As shown in Table 4, the linker-payload toxin (MC-VC-PAB-MMAE) was successfully linked to trastuzumab, indicating that ADCs in different buffers were successfully synthesized.

[0751] According to the results in Table 4, in Examples 91-102, the proportion of D6 is greater than 50%. In addition, in Examples 91-100, the proportion of D6 is greater than 70%, and the highest proportion of D6 is 88%. The results show that the buffer system in Examples 91-102 is beneficial to improve the uniformity of the ADC with D6 and the proportion of D6.

[0752] Examples 103 to 107: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugates with Different pH Values

[0753] The preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate is similar to the preparation of ADC in Example 82, but the pH value of the buffer and the molar ratio of TCEPA to antibody are adjusted as follows:

[0754]

[0755] The results of the homogeneity test are as follows:

[0756] Table 5

[0757] serial number Attached photos D0 (wt%) D2 (wt%) D4 (wt%) D6 (wt%) D8 (wt%) E103 36A 0 6.21 8.05 80.67 5.07 E104 36B 0 5.34 13.90 77.02 3.75 E105 36C 0 2.73 14.61 79.28 3.38 E106 36D 1.43 14.67 25.41 58.49 0 E107 36E 0 13.45 24.28 60.53 1.74

[0758] The results of Examples 103-107 are shown in Table 5. As shown in the results in Table 5, the linker-payload toxin (MC-VC-PAB-MMAE) was successfully linked to trastuzumab, indicating that ADC was successfully synthesized when the buffer pH was in the range of 5.8 to 6.7.

[0759] According to the results of Table 5, in Examples 103 to 107, the proportion of D6 is greater than 58%. In addition, in Examples 103 to 105, the proportion of D6 is greater than 75%, and the highest proportion of D6 is 80%.

[0760] Examples 108 to 110: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugates Using Buffer Systems of Different Concentrations

[0761] The preparation of trastuzumab-[MC-VC-PAB-MMAE]6 conjugate is similar to the preparation of ADC in Example 82, but the buffer concentration of the buffer system and the molar ratio of TCEPA to antibody are adjusted as shown below:

[0762]

[0763] The results of the homogeneity test are as follows:

[0764] Table 6

[0765] serial number Attached photos D0 (wt%) D2 (wt%) D4 (wt%) D6 (wt%) D8 (wt%) E108 37A 0 5.83 6.32 84.06 3.79 E109 37B 1.70 5.14 3.72 85.50 3.95 E110 37C 0 7.10 6.95 83.64 2.31

[0766] As shown in Table 6, the results show that the concentration of the buffer system of Examples 108-110 helps to increase the content of D6.

[0767] Examples 111-119: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6 Conjugate (reduction time and / or reducing agent temperature in step (1) are different)

[0768] The methods of Examples 111-119 are substantially the same as those of Example 32, except that 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) are as follows:

[0769]

[0770]

[0771] The results of the homogeneity test are as follows:

[0772] Table 7

[0773]

[0774] As shown in Examples 111-116 in Table 7, 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%, or even as high as 80%, 85% or 88%, which also shows that when using the present method, the reduction time can be shortened by increasing the molar ratio of the reducing agent to the antibody.

[0775] As shown in Examples 116-119 in Table 7, 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 as high as 87%.

[0776] Example 120: Preparation of trastuzumab-[bismaleimide-DBCO]3 conjugate

[0777] (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;

[0778] (2) EDTA-2Na (0.6 mM) and bismaleimide-DBCO (0.045 mM) in DMA were introduced and the reaction was continued at room temperature for 1 hour; then trastuzumab-[bismaleimide-DBCO]3 was recovered using a desalting column.

[0779] Wherein, the preparation of bismaleimide-DBCO is as follows:

[0780]

[0781]

[0782] 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.

[0783] 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.

[0784] 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.

[0785] Intermediate 5: HATU (0.92 g, 2.4 mmol, 2.4 eq) and DIPEA (0.65 g, 5.0 mmol, 5.0 eq) were added to a mixture of intermediate 4 (50 mg, 1.00 mmol, 1.0 eq) in DCM (5 mL). 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% to 70%), and the eluent was lyophilized to give the product 5 (0.51 g, 80.3%) as a white solid. Intermediate 6: TFA (100 uL) was added to a mixture of intermediate 5 (100 mg, 0.16 mmol, 1.0 eq) in DCM (2 mL). The mixture was stirred at room temperature for 1 hour. LCMS showed complete consumption of intermediate 5. 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.

[0786] 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. 1 H NMR (400MHz, DMSO-d6): δ8.17(t,J=6.0Hz,2H),7.85(d,J=6.4Hz,1H),7.64(dd,J=18.4,7.3Hz,2H),7.47(d,J=10.3Hz,3H),7.38–7.24(m,3H),6.96 (s,4H),5.02(d,J=14.0Hz,1H),4.22(s,4H),3.68(t,J=6.6Hz,2H),3.65- 3.58(m,4H),3.43(t,J=6.2Hz,3H),3.24-3.20(m,3H),3.16-3.10(m,5H).

[0787] The results of the homogeneity test are as follows:

[0788] serial number Attached photos ADC Type D3(％) D4(％) E120 40 D3 82.93 17.07

[0789] As shown in the table, the results confirmed that the content of trastuzumab-[bismaleimide-DBCO]3 was generally as high as 82%, which indicates that the process of this method is conducive to site-specific modification of D3-type antibodies and can improve uniformity.

[0790] Example 121: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]6[MC-GGFG-DXd]2 (ADC with D6+D2)

[0791] (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;

[0792] (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, and then the obtained product was recovered using a desalting column;

[0793] (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 toxin MC-GGFG-DXd (0.05 mM) was added and the reaction was continued at room temperature for 2 hours, and then the product was recovered using a desalting column.

[0794] Among them, an average of about six drug molecules MC-VC-PAB-MMAE were coupled to trastuzumab, and an average of about two drug molecules MC-GGFG-DXd were coupled to trastuzumab.

[0795] The results of the homogeneity test are as follows:

[0796] serial number Attached photos D2(％) D4(％) D6(％) D8(％) E121-Step (2) 41A 4.48 4.97 86.49 4.06 serial number Attached photos D2(％) D4(％) D6+D2(%) D8(％) E121-Step (3) 41B 3.67 3.88 84.81 7.65

[0797] As shown in the table above, the results indicate that the content of ADC with D6+D2 is generally as high as 84.81%, which indicates that this method is beneficial for site-specific modification of antibodies with D6+D2 and improves uniformity.

[0798] Example 122: Preparation of trastuzumab-[MC-GGFG-DXd]6[maleimide-PEG4-N3-DBCO-Cy3]1 (ADC with D6+D1)

[0799] 1. Synthesis of dibromomaleimide-PEG4-N3

[0800]

[0801] 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%).

[0802] A solution of azido-PEG4-amine (105 mg, 0.4 mmol, 1 eq., Xi'an Kangfuno Biotechnology Co., Ltd.) in dichloromethane (2 mL) was added to a stirred solution of product 1 (139 mg, 0.4 mmol, 1 eq.) in dichloromethane (2 mL).

[0803] 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 MgSO4. After vacuum concentration, column chromatography purification (100% EtOAc as mobile phase) was performed to give dibromomaleimide-PEG4-N3 (title product 2) as a light yellow oil (150 mg, 0.3 mmol, 75%).

[0804] 2. Preparation of Trastuzumab-[MC-GGFG-DXd]6[Maleimide-PEG4-N3-DBCO-Cy3]1

[0805] (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;

[0806] (2) EDTA-2Na (0.6 mM) and MC-GGFG-DXd (0.096 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;

[0807] (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.

[0808] (4) Purify the obtained product using a desalting column.

[0809] Among them, an average of about 6 drug molecules MC-GGFG-DXd were coupled to trastuzumab, and an average of about 1 drug molecule maleimide-PEG4-N3-DBCO-Cy3 was coupled to trastuzumab.

[0810] The results of the homogeneity test are as follows:

[0811] serial number Attached photos D4(％) D6(％) D8(％) E122-Step (2) 42A 4.58 92.20 3.23 serial number Attached photos D4+D1(%) D6+D1(%) D8(％) E122-Step (4) 42B 4.26 92.25 3.50

[0812] As shown in the table above, the results indicate that the content of ADC with D6+D1 is generally as high as 90%, which indicates that this method is beneficial for site-specific modification of antibodies with D6+D1 and improves uniformity.

[0813] Example 123: Preparation of trastuzumab-[maleimide]6[MC-VC-PAB-MMAE]2 (D0+D2 type ADC)

[0814] (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;

[0815] (2) introducing EDTA (0.6 mM) and (2-aminoethyl)maleimide (0.1 mM) to react with the reduced thiol group produced in step (1) at room temperature for 1 hour, and then recovering the product using a desalting column to obtain trastuzumab-[maleimide] 6;

[0816] (3) Incubate trastuzumab-[maleimide]6 and the second reducing agent TCEP (0.02 mM) at 37°C for 18 hours; add the second linker-payload toxin MC-VC-PAB-MMAE (0.05 mM) and continue the reaction at room temperature for 2 hours.

[0817] (4) Purify the obtained product using a desalting column.

[0818] The results of the homogeneity test are as follows:

[0819] serial number Attached photos D0(％) / / E123-Step (2) 43A 100 / / serial number Attached photos D0(％) D0+D1(%) D0+D2(%) E123-Step (4) 43B 24.30 5.18 70.53

[0820] As shown in the table above, the results confirmed that the D0+D2 type ADC content was generally as high as 70.53%, which indicates that the process of this method is conducive to site-specific modification of D0+D2 type antibodies and can improve uniformity.

[0821] Example 124: Preparation of trastuzumab-[maleimide]6[maleimide-PEG4-N3-DBCO-Cy3]1 (D0+D1 type ADC)

[0822] (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;

[0823] (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;

[0824] (3) Incubate trastuzumab-[maleimide]6 and the second reducing agent TCEP (0.02 mM) at 37°C for 18 hours. The second thio-bridging reagent with a reactive group dibromomaleimide-PEG4-N3 (0.013 mM) reacts with the reduced thiol group at 24°C for 3 hours;

[0825] (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.

[0826] The results of the homogeneity test are as follows:

[0827] serial number Attached photos D0(％) D0+D1(%) D0+D2(%) E124 44 17.00 79.40 3.60

[0828] As shown in the table above, the results confirmed that the D0+D1 type ADC content was generally as high as 79.40%, which indicates that the process of this method is conducive to site-specific modification of D0+D1 type antibodies and can improve uniformity.

[0829] Example 125: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2 conjugate (ADC with D2)

[0830] (1) ZnCl2 (0.012 mM) and the prepared 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;

[0831] (2) DHAA (0.072 mM) was added to the mixture of step (1) and vortexed to mix. The mixture was then incubated at room temperature in the dark for 2 hours;

[0832] (3) Purify using a desalting column to remove excess DHAA, if necessary;

[0833] (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.

[0834] (5) Purify the reaction mixture using a desalting column.

[0835] Examples 126 to 147: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugate (ADC with D2) The methods of Examples 126-147 are similar to those of Example 125, except for the parameters in step (1) and step (2), which are as follows:

[0836]

[0837] RT is the abbreviation for room temperature.

[0838]

[0839]

[0840] The results of the homogeneity test are as follows:

[0841] Table 8

[0842] serial number Attached photos D0 (wt%) D2 (wt%) D4 (wt%) D6 (wt%) D8 (wt%) E125 45 4 93 3 0 0 E126 46 7 93 0 0 0 E127 47 8 92 0 0 0 E128 48 10 90 0 0 0 E129 49 12 88 0 0 0 E130 50 5 95 0 0 0 E131 51 6 86 5 3 0 E132 52 6 94 0 0 0 E133 53 7 88 5 0 0 E134 54A 7.24 81.27 11.49 0 0 E135 54B 22.43 71.72 5.85 0 0 E136 54C 26.93 73.07 0 0 0 E137 54D 12.42 85.32 2.26 0 0 E138 54E 32.55 67.45 0 0 0 E139 54F 33.57 66.43 0 0 0 E140 54G 2.34 60.64 33.30 3.72 0 E141 54H 4.50 87.68 7.82 0 0 E142 55A 6.19 92.05 1.77 0 0 E143 55B 8.49 91.51 0 0 0 E144 55C 2.23 71.71 26.06 0 0 E145 55D 4.48 91.94 3.58 0 0 E146 55E 7.36 92.64 0 0 0 E147 55F 8.06 91.94 0 0 0

[0843] The results of Examples 125-147 are shown in Table 8. As shown in the results in Table 8, the linker-payload toxin (MC-VC-PAB-MMAE) was successfully linked to trastuzumab, indicating that the ADC with D2 was successfully synthesized.

[0844] According to the results in Table 8, in Examples 125-133, the proportion of D2 is greater than 80%. The results show that the oxidation reaction is beneficial to improving the uniformity of ADCs with D2, and also show that the oxidation reaction is beneficial to site-specific modification of antibodies, especially to ADCs with site-specific conjugation.

[0845] 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.

[0846] As shown in Examples 134-147, when the molar ratio of the reducing agent to the antibody is 4:1 to 10:1, the content of D2 is as high as 60%, even as high as 70%, 80% and 90%, and the reduction time in step (1) is shortened to 2 hours or 4 hours, i.e., the reduction time cost is saved. As shown in Examples 125-147, when the reducing agent time in step (1) is 2 hours to 18 hours, the content of D2 is as high as 60%, even as high as 70%, 80% and 90%.

[0847] As shown in Examples 125-147, when the molar ratio of DHAA to antibody was 4:1 to 22:1, the content of D2 was as high as 60%, 70%, 80%, 85%, or even as high as 90% or 95%.

[0848] Examples 148 to 150: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugate (reduction time and / or reducing agent temperature in step (2) are different)

[0849] The methods of Examples 148-150 are similar to Example 126, except for the oxidation temperature and / or time in step (2) and the molar ratio of the reducing agent to the antibody, as shown in Table 9.

[0850] Table 9

[0851]

[0852] As shown in Table 9 and Example 126, the results show that when the oxidation temperature in step (2) is 4°C to 37°C, the content of D6 is as high as 80%, even as high as 95%, and the oxidation time in step (2) is 1 hour to 48 hours. Examples 151 to 166 and Comparative Example 6: Preparation of Trastuzumab-[MC-VC-PAB-MMAE]2 Conjugates Using Different Buffer Systems

[0853] The methods of Examples 151-166 and Comparative Example 6 are similar to Example 126, except for the buffer system shown in Table 9. Meanwhile, the molar ratio of the reducing agent to the antibody in Examples 151-166 and Comparative Example 6 is 3.5:1.

[0854] The results of the homogeneity test are as follows:

[0855] Table 10

[0856] serial number Attached photos Buffer system D0(％) D2(％) D4(％) D6(％) D8(％) E151 57A Bis-Tris, pH 6.7 5.51 87.96 6.53 0 0 E152 57B PIPES, pH 6.7 4.46 79.81 15.73 0 0 E153 57C PB, pH 6.7 38.34 61.67 0 0 0 E154 57D HEPES, pH 7.0 6.06 91.51 2.43 0 0 E155 57E MOPS, pH 7.0 6.36 87.38 6.26 0 0 E156 57F DIPSO, pH 7.4 7.19 92.81 0 0 0 E157 57G MOBS, pH 7.4 6.18 93.83 0 0 0 E158 57H MOPSO, pH 7.4 5.70 92.18 2.12 0 0 E159 58A TES, pH 7.4 5.66 91.75 2.59 0 0 E160 58B ACES, pH 7.4 7.71 92.29 0 0 0 E161 58C TAPSO, pH 7.4 4.77 92.26 2.98 0 0 E162 58D MES pH 5.8 3.48 60.48 32.66 3.39 0 E163 58E MES pH 6.1 4.12 73.51 22.37 0 0 E164 58F BES, pH 6.4 3.57 76.45 19.98 0 0 E165 58G BES, pH 6.7 1.79 82.25 14.77 1.20 0 E166 58H BES, pH 7.4 3.05 94.48 2.48 0 0 C6 59 ADA, pH 6.7 86.47 13.53 0 0 0

[0857] As shown in Table 10, the results show that the type and pH value of the buffer system will affect the content of D2 by affecting the reduction kinetics and selectivity. The buffer systems of Examples 151 to 166 can be used to increase the content of D2, and the pH value of the buffer system is 5.8 to 7.4.

[0858] Example 167: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 (D1-type ADC)

[0859] (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 18 h;

[0860] (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;

[0861] (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 hour, and then the product is recovered using a desalting column to obtain trastuzumab-[maleimide-PEG4-N3]1;

[0862] (4) Trastuzumab-[maleimide-PEG4-N3]1 (0.013 mM) and DBCO-Cy3 (0.02 mM) were incubated in BES buffer (20 mM, pH 7.0) at a reaction temperature of 25° C. and a reaction time of 8 hours.

[0863] (5) Purify the reaction mixture using a desalting column.

[0864] The results of the homogeneity test are as follows:

[0865] serial number Attached photos D0(％) D1(％) D2(％) E167 60 13.07 83.81 3.13

[0866] As shown in the table above, the results confirmed that the D1-type ADC content was generally as high as 83.81%, which indicates that the process of this method is conducive to site-specific modification of D1-type antibodies and can improve uniformity.

[0867] Example 168: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]6 (D1+D6 type ADC)

[0868] (1) introducing trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 (0.008 mM) prepared in Example 167 and TCEPA (0.08 mM) in BES buffer (20 mM, pH 7.0), the reaction temperature was 24° C., and the reaction time was 24 hours;

[0869] (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.

[0870] The results of the homogeneity test are as follows:

[0871] serial number Attached photos D6(％) D1+D6(%) D2+D6(%) E168 61 7.79 88.02 4.20

[0872] As shown in the table above, the results confirmed that the D1+D6 ADC content was generally as high as 88.02%, which indicates that the process of this method is conducive to site-specific modification of D1+D6 antibodies and can improve uniformity.

[0873] Example 169: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]2 (D1+D2 type ADC)

[0874] (1) incubating ZnCl2 (0.8 mM), a second reducing agent TCEP-6 (0.0144 mM) and trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1 (0.008 mM) prepared in Example 167 in BES buffer (pH 7.0, 20 mM), and allowing the reaction mixture to stand at 4°C for 4 hours;

[0875] (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;

[0876] (3) Purify the reaction mixture using a desalting column.

[0877] The results of the homogeneity test are as follows:

[0878] serial number Attached photos D1(％) D2(％) D1+D2(%) D4％ D1+D4(%) E169 62 3.72 7.92 73.80 4.06 10.51

[0879] Using step (1), one of the interchain disulfide bonds in the ADC with D1 is reduced. As shown in the table above, the results confirm that the content of D1+D2 type ADC is generally as high as 73.8%, which indicates that the process of this method is conducive to site-specific modification of D1+D2 type antibodies and can improve uniformity.

[0880] Examples 170 to 171: Preparation of trastuzumab-[maleimide-PEG4-N3-DBCO-Cy3]1[MC-VC-PAB-MMAE]4 (D1+D4 type ADC)

[0881] (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;

[0882] (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;

[0883] (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;

[0884] (4) incubating trastuzumab-[maleimide-PEG4-N3]1 from step (3) and DBCO-Cy3 (0.02 mM) in BES buffer (20 mM, pH 7.0) at a reaction temperature of 25° C. and a reaction time of 8 hours;

[0885] (5) purifying the reaction mixture using a desalting column;

[0886] (6) incubating ZnCl2 (0.8 mM), a second reducing agent TCEP-3 (0.0384 mM) or TCEP-6 (0.0336 mM) and the product from step (5) in BES buffer (pH 7.0, 20 mM), and allowing the reaction mixture to stand at 4°C for 16 hours;

[0887] (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;

[0888] (8) Purify the reaction mixture using a desalting column.

[0889] The results of the homogeneity test are as follows:

[0890]

[0891] For step (6), two of the interchain disulfide bonds in the D1-type ADC are reduced. As shown in the table above, the results confirm that the D1+D4-type ADC content is generally as high as 84%, which indicates that the process of this method is conducive to site-specific modification of D1+D4-type antibodies and can improve uniformity.

[0892] Example 172: Preparation of trastuzumab-[MC-GGFG-DXd]2[MC-VC-PAB-MMAE]4 conjugate (ADC with D2+D4)

[0893] (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;

[0894] (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;

[0895] (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;

[0896] (4) purifying the reaction mixture using a desalting column;

[0897] (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;

[0898] (6) Introducing EDTA (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;

[0899] (7) Purify the reaction mixture using a desalting column.

[0900] Examples 173 to 174: Preparation of trastuzumab-[MC-VC-PAB-MMAE]2[MC-GGFG-DXd]4 conjugate (D2+D4 type ADC)

[0901] (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;

[0902] (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;

[0903] (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 1.5 h;

[0904] (4) purifying the reaction mixture using a desalting column;

[0905] (5) Incubate ZnCl2 (Example 173: 0.36 mM; Example 174: 0.024 mM), a second reducing agent TCEP-6 (Example 173: 0.042 mM; Example 174: 0.054 mM) and the product from step (4) in BES buffer (pH 7.0, 20 mM), and allow the reaction mixture to stand at 3°C ​​for 1 hour;

[0906] (6) Introducing EDTA (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;

[0907] (7) Purify the reaction mixture using a desalting column.

[0908] The results of the homogeneity test are as follows:

[0909]

[0910] As shown in the table above, the results confirmed that the D2+D4 ADC content was generally as high as 60%, indicating that the process of this method is conducive to site-specific modification of D2+D4 antibodies and can improve uniformity.

[0911] As shown in Examples 173-174, the results indicate that when the molar ratio of the transition metal ion to the reducing agent in step (5) increases, the selective reducing ability in step (5) increases.

[0912] Although specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are presently unforeseeable or possibly unforeseeable may occur to applicants or other persons skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. A reducing agent having the following formula (I): or a salt, solvate or stereoisomer thereof, characterized in that R1 is H, -NH2, -C(O)(R3R4), optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl; R3 is N, NH or O; R4 is H, optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl; R2 is H, optionally substituted C1-C5 alkyl, or optionally substituted C1-C5 hydroxyalkyl; X is OH, optionally substituted C1-C5 alkoxy or -NR5R6, R5 and R6 are each independently H, C0-C5 hydroxyalkyl, optionally substituted C1-C5 alkyl, optionally substituted C2-C8 carboxyalkyl, optionally substituted C1-C5 alkoxy, optionally substituted heteroarylalkyl, optionally substituted arylalkoxy, optionally substituted arylalkyl, optionally substituted aryl, C1-C5 alkylsulfonyl, or -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7), R7 is C0-C5 hydroxyalkyl, -NHOH, R8 is H, optionally substituted aralkyl, n 1 and n 2 Each of them is independently digitized 0, 1, 2, 3, 4, Y is the same as X, or Y is an ester or amide of X, Z is the same as X or Y, or Y and Z are each independently selected from: X, Y and Z are not the same 2. The reducing agent according to claim 1, characterized in that R1 is H, and R2 is H.

3. The reducing agent according to claim 1, characterized in that X is -OCH3, -OCH2CH3, or -OCH(CH3)2.

4. The reducing agent according to claim 1 or 2, characterized in that X is -NR5R6, R5 is H, and 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), R7 is C0-C3 hydroxyalkyl or -NHOH, R8 is H or an optionally substituted aralkyl group, n 1 and n 2 Each independently is the number 0.

5. The reducing agent according to claim 4, characterized in that R6 is H, C0-C2 hydroxyalkyl, C1-C3 alkoxy, C1-C3 alkylsulfonyl, bipyridyl, benzyl, arylalkoxy, phenyl optionally substituted by OH, carboxyl or pyridyl, or -CH(R8)CO(R7), R7 is OH or -NHOH, R8 is H or benzyl optionally substituted by -OH, halogen, cyano or nitro.

6. The reducing agent according to claim 1 or 2, characterized in that X is -NR5R6, R5 is H, and <h2 style=";text-align:left;direction:ltr">R6H、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、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 7. The reducing agent according to claim 1 or 2, characterized in that X is -NR5R6, R5 is OH, 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), R7 is C0-C5 hydroxyalkyl, R8 is H, n 1 and n 2 Each independently is the number 0, 1, 2, 3, 4.

8. The reducing agent according to claim 7, characterized in that R6 is C1-C3 alkyl, heteroarylalkyl containing heteroatom N, optionally substituted benzyl, optionally substituted phenyl, or -CH(R8)CO(R7), R7 is C0-C3 hydroxyalkyl, R8 is H.

9. The reducing agent according to claim 7, characterized in that R6 is -CH3, -CH2COOH, 10. The reducing agent according to claim 1 or 2, characterized in that X is -NR5R6, R5 and R6 are independently C1-C5 alkyl, C0-C5 hydroxyalkyl, optionally substituted heteroarylalkyl or -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7), R7 is C0-C5 hydroxyalkyl or -NHOH, R8 is H, n 1 and n 2 Each independently is the number 0, 1, 2, 3, 4.

11. The reducing agent according to claim 10, characterized in that R5 and R6 are independently methyl, ethyl, -(CH2)2OH, -CH2COOH, -CH2CONHOH or 12. The reducing agent according to claim 1 or 2, characterized in that Y is Z is 13. The reducing agent according to any one of claims 1 to 12, characterized in that The reducing agent is selected from:

14. A composition comprising the reducing agent of any one of claims 1 to 13 and a transition metal ion.

15. The composition according to claim 14, 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+ .

16. The composition according to claim 14 or 15, characterized in that The molar ratio of the transition metal ion to the first reducing agent is 0.05:1 to 40:1; optionally, the molar ratio of the transition metal ion to the first reducing agent may be 0.25:1 to 30:1; optionally; the molar ratio of the transition metal ion to the first reducing agent may further be 0.25:1 to 15:

1.

17. A method for preparing the reducing agent according to any one of claims 1 to 13, characterized in that: By introducing a condensation reagent under an inert atmosphere, at least one X' is linked to a compound of formula II, R1 is H, -NH2, -C(O)(R3R4), optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl; R3 is N, NH or O; R4 is H, optionally substituted C1-C5 alkyl, optionally substituted C1-C5 hydroxyalkyl, or optionally substituted aryl; R2 is H, optionally substituted C1-C5 alkyl, or optionally substituted C1-C5 hydroxyalkyl; X' is an optionally substituted C1-C5 alkyl alcohol or NR5R6, R5 and R6 are independently H, C0-C5 hydroxyalkyl, optionally substituted C1-C5 alkyl, optionally substituted C2-C8 carboxyalkyl, optionally substituted C1-C5 alkoxy, optionally substituted heteroarylalkyl, optionally substituted arylalkoxy, optionally substituted arylalkyl, optionally substituted aryl, C1-C5 alkylsulfonyl, or -(CH2)n 1 (OCH2CH2O)n 2 CH(R8)CO(R7), R7 is C0-C5 hydroxyalkyl, -NHOH, R8 is H, optionally substituted aralkyl, n 1 and n 2 Each is independently digitized 0, 1, 2, 3, 4, Y is the same as X, or Y is an ester or amide of X, Z is the same as X or Y, or Y and Z are independently selected from:

18. The method according to claim 17, characterized in that The compound of formula II is 19. The method according to claim 17, characterized in that The X' is 2-phenoxy-ethylamine, aniline, benzylamine, 4-aminobenzene-1,2-diol, 5-amino-2-hydroxybenzoic acid, bis(pyridin-2-ylmethyl)amine, 5-amino-8-hydroxyquinoline, bis(pyridin-2-yl)methylamine, 4-aminophthalic acid, L-tyrosine tert-butyl ester, DL-3-(4-fluorophenyl)alanine, DL-4-cyanophenylalanine, DL-4-nitrophenylalanine, N-benzylhydroxylamine hydrochloride, N-phenylhydroxylamine, 20. Use of the reducing agent of any one of claims 1 to 13 or the composition of any one of claims 14 to 16 for reducing interchain disulfide bonds of an antibody.

21. The use according to claim 20, characterized in that In preparing an antibody with a site-specific modification, the antibody with a site-specific modification may be an antibody drug conjugate (ADC) as appropriate; the ADC may further be an ADC with D2, an ADC with D4, an ADC with D1, an ADC with D6, an ADC with D3, an ADC with D1+D6, an ADC with D1+D2, an ADC with D1+D4, an ADC with D2+D4, an ADC with D6+D2, an ADC with D6+D1, an ADC with D3+D1, an ADC with D3+D2, an ADC with D0+D6, or an ADC with D0+D2.

22. A method for preparing an antibody having a site-specific modification, comprising the following steps: (A1) In a buffer system, a reducing agent or a salt, solvate, or stereoisomer thereof as described in any one of claims 1 to 13 (as a first reducing agent) and a transition metal ion are co-incubated with an antibody to selectively reduce the interchain disulfide bonds in the antibody, thereby obtaining an antibody with reduced thiol groups.

23. The method of claim 22, wherein: Two interchain disulfide bonds in the Fab region of the antibody and one interchain disulfide bond in the hinge region of the antibody are reduced.

24. The method of claim 22, wherein: The method further comprises the following steps: (A2) Introducing an oxidizing agent to selectively reoxidize the reduced thiol groups produced in step (A1), and optionally reoxidize the reduced thiol groups in the Fab region of the antibody.

25. The method of claim 24, wherein: The method further comprises the following steps: (A3) Incubating a second reducing agent in a buffer system to selectively reduce the interchain disulfide bonds generated in step (A2), and optionally, reducing the interchain disulfide bonds in the hinge region of the antibody.

26. The method according to any one of claims 22 to 24, characterized in that The method further comprises the following steps: (B1) introducing a metal chelator and a first payload toxin unit to react with the reduced thiol group produced in step (A1), step (A2) or step (A3); wherein the first payload toxin unit is a capping agent, a first linker payload toxin or a first thio-bridging agent; and, as the case may be, the first thio-bridging agent may carry a first linker payload toxin or a reactive group.

27. The method of claim 26, wherein: The method further comprises the following steps: (B2) incubating a second reducing agent in a buffer system to reduce the interchain disulfide bonds generated in step (B1), and optionally introducing transition metal ions; and (B3) introducing a second payload toxin unit to react with the reduced thiol group produced in step (B2), and optionally introducing a metal chelator; wherein the second payload toxin unit is a second linker-payload toxin or a second thio-bridging reagent; optionally, the second thio-bridging reagent may carry a second linker-payload toxin or a reactive group.

28. The method according to claim 26 or 27, characterized in that The first thio-bridging reagent and the second thio-bridging reagent independently contain at least two substituent groups to re-bridge the thiol group.

29. The method of claim 28, wherein: The first thio-bridging reagent and the second thio-bridging reagent are each independently selected from:

30. The method according to any one of claims 22, 25 and 27, characterized in that The molar ratio of the reducing agent to the antibody in steps (A1), (A3) and (B2) is each independently 1:1 to 20, 1:1 to 5:1, 1:1 to 3:1, 1:1 to 2:1 or 3:1 to 5:

1.

31. The method of claim 30, wherein: The molar ratio of the first reducing agent to the antibody in step (A1) is 2.8:1 to 13:1, and optionally, 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.

32. The method according to any one of claims 22, 25 and 27, characterized in that The incubation temperatures in steps (A1), (A3) and (B2) are each independently 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.

33. The method according to any one of claims 22, 25 and 27, characterized in that The incubation time in step (A1) is 2 hours to 24 hours, 14 hours to 24 hours, 16 hours to 20 hours, or 16 hours to 18 hours; The incubation time in steps (A3) and (B2) is each independently 0.5 hour to 24 hours, 0.5 hour to 12 hours, 1 hour to 10 hours, 1 hour to 8 hours, or 1 hour to 5 hours.

34. The method of claim 33, wherein: In step (A1), the molar ratio of the first reducing agent to the antibody is 4:1 to 10:1, and the incubation time is 1 hour to 16 hours.

35. The method of claim 33, wherein: In step (A1), the molar ratio of the first reducing agent to the antibody is 6:1 to 13:1, and the incubation time is 4 to 16 hours.

36. The method of claim 33, wherein: In step (A1), the molar ratio of the first reducing agent to the antibody is 2.8:1 to 3:1, and the incubation time is 10-24 hours.

37. The method of claim 22, wherein: The molar ratio of the transition metal ion to the first reducing agent in step (A1) is 0.05:1 to 40:1, 0.08:1 to 30:1, 0.1:1 to 20:1, 0.2:1 to 8:1, or 0.25:1 to 7.5:

1.

38. The method of claim 27, wherein: In step (B2), 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.

39. The method of claim 27, wherein: In step (B2), 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.

40. The method according to any one of claims 22 or 27, characterized in that The transition metal ion is selected from Zn 2+ 、Cd 2+ , Hg 2+ 、Ni 2+ 、Co 2+ or a combination thereof, optionally, the transition metal ion is Zn 2+ .

41. The method of claim 25 or 27, wherein: The second reducing agent in steps (A3) and (B2) is the same as the first reducing agent in step (A1).

42. The method of claim 25 or 27, wherein: The second reducing agent in step (A3) and the second reducing agent in step (B2) are independently tris(2-carboxyethyl)phosphine (TCEP).

43. The method of claim 24, wherein: The molar ratio of the oxidant to the antibody in step (A2) is 2:1 to 25:

1. Depending on the situation, the molar ratio of the oxidant to the antibody in step (A2) is 4:1 to 22:1 or 3:1 to 15:

1.

44. The method of claim 24, wherein: The oxidant is dehydroacetic acid (DHAA).

45. The method of claim 24, wherein: In step (A2), the oxidation temperature is 0°C to 37°C, and / or the oxidation time is 1 hour to 48 hours; depending on the situation, the oxidation temperature is 0°C to 30°C, and / or the oxidation time is 1 hour to 5 hours.

46. ​​The method of any one of claims 22, 25 and 27, wherein: The buffer systems described in steps (A1), (A3) and (B2) are independently selected from: 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, acetate buffer, ADA buffer, BTP buffer, HEPPSO buffer, POPSO buffer, EPPS buffer or Tris buffer; Optionally, the buffer systems described in steps (A1), (A3) and (B2) are independently Bis-Tris buffer, MOPS buffer, BES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, MOPSO buffer, TES buffer, ACES buffer or TAPSO buffer.

47. The method of claim 45, wherein: 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.

48. The method of claim 45, wherein: The pH value of the buffer system is 5.5 to 8; preferably, the pH value of the buffer system is 6.7 to 7.

4.

49. The method according to any one of claims 26 or 27, characterized in that The metal chelating agent described in steps (B1) and (B3) is disodium ethylenediaminetetraacetic acid (EDTA-2Na).

50. The method of claim 26, wherein: When the first payload toxin unit is a first thio-bridging agent with a reactive group, the step (B1) further comprises the following steps: The metal chelator and the first linker payload toxin are incubated in the buffer system to react with the reactive group carried by the first thio-bridging reagent.

51. The method of claim 27, wherein: When the second payload toxin unit is a second thio-bridging agent with a reactive group, the step (B3) further comprises the following steps: The second linker-payload toxin is incubated in the buffer system to react with the reactive group carried by the second thio-bridging reagent, and the metal chelator may be introduced as appropriate.

52. The method of any one of claims 22, wherein: The antibody is a monoclonal antibody, a polyclonal antibody, a monospecific antibody or a multispecific antibody; optionally, the antibody is IgG1 or IgG4.

53. The method according to any one of claims 26 or 27, characterized in that The linkers of the first linker-payload toxin and the second linker-payload toxin are selected from any linker that meets the following requirements: one end of which can be connected to the reduced thiol group of the antibody or the reactive group of the thio-bridging reagent and the other end can be connected to the payload toxin.

54. The method according to any one of claims 26 or 27, characterized in that The payload toxin is selected from any payload toxin that contains at least one substituent group to attach the payload toxin to a linker.

55. A modified antibody prepared by the method of any one of claims 22-54.

56. The modified antibody according to claim 55, characterized in that The modified antibody is an antibody with site-specific modification; optionally, the modified antibody includes an ADC with D2, an ADC with D4, an ADC with D1, an ADC with D6, an ADC with D3, an ADC with D1+D6, an ADC with D1+D2, an ADC with D1+D4, an ADC with D2+D4, an ADC with D6+D2, an ADC with D6+D1, an ADC with D3+D1, an ADC with D3+D2, an ADC with D0+D6, or an ADC with D0+D2.

57. A pharmaceutical composition comprising an antibody with site-specific modification prepared by the method of any one of claims 22-54 and at least one pharmaceutically acceptable ingredient.

58. Use of an antibody with site-specific modification prepared by the method of any one of claims 22 to 54 or a pharmaceutical composition of claim 57 in preparing a therapeutic agent for preventing, diagnosing or treating a disease.

59. A method for preventing or treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody having a site-specific modification prepared by the method of any one of claims 22-54.

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