Antibodies, antibody-drug conjugates, and methods of making the same

Abstract

This invention provides a hinge area comprising X1X2X3X4X5CPPX6 or X 11 X 12 THX 15 An antibody containing the CPPC amino acid sequence, and an antibody-drug conjugate prepared using the antibody. The antibody helps improve the homogeneity of the antibody-drug conjugate.

Description

Technical Field

[0001] This disclosure relates to antibodies, antibody-drug conjugates, and methods for preparing said antibody-drug conjugates. Background Technology

[0002] The statements in this section are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Antibody-drug conjugates (ADCs) typically consist of an antibody and a small molecule drug conjugated to the antibody via a chemical linker. After decades of preclinical and clinical studies, a range of ADCs have been approved for the treatment of specific tumor types.

[0004] A key step in ADC preparation is the covalent coupling of the payload to the antibody. WO2022084560A1 discloses enzymatic coupling methods (such as microbial transglutaminase (MTG)-mediated coupling and engineered antibodies), achieving coupling through sequence insertion—these reactions are typically fast, highly site-specific, and can be performed under physiological conditions. However, sequence insertion may increase the immunogenicity of the antibody and reduce its overall stability.

[0005] Most ADCs currently in clinical development are conjugated to lysine or cysteine ​​residues on the antibody itself, requiring strict control of the average degree of modification. The number of drug loads linked to each antibody molecule may vary, leading to heterogeneity in ADC formulations, increasing product complexity, and potentially negatively impacting the stability, pharmacokinetics, aggregation, and ultimately, safety of the ADC.

[0006] Given the above, there is still a need in the art to develop improved methods for generating ADCs with higher uniformity. Summary of the Invention

[0007] To achieve the above objectives, this disclosure provides a novel antibody having one or more amino acid substitutions in the hinge region, which helps to improve the homogeneity of the antibody-drug conjugate.

[0008] In one aspect, this disclosure provides an antibody comprising a hinge region, wherein the hinge region comprises the amino acid sequence X1X2X3X4X5CPPX6, wherein X1 is D or H; X2 is E, D, K, or S; X3 is E, T, H, or D; X4 is T, H, D, S, or E; X5 is E, H, T, or D; and X6 is G, A, H, D, E, Y, C, S, V, L, I, or F; or, the hinge region comprises the amino acid sequence X 11 X 12 THX 15 CPPC (SEQ ID NO: 30), where X 11 For H, D, or S; X12 For K or S; X 15 For T or R; X 11 X 12 THX 15 The amino acid sequence of CPPC or X1X2X3X4X5CPPX6 is not DKTHTCPPC (SEQ ID NO: 26).

[0009] In some implementations, X1X2X3X4X5CPPX6 is selected from any of the following groups: (1) X1 is D or H; X2 is D, K or S; X3 is T, H or D; X4 is T, H, D, S or E; X5 is H, T or D; X6 is C, S, V, L, I or F.

[0010] (2) X1 is H; X2 is D, K or S; X3 is T, H or D; X4 is T, H, D, S or E; X5 is H, T or D; X6 is C, S, V, L, I or F; (3) X1 is H; X2 is D, K or S; X3 is T, H or D; X4 is D; X5 is H, T or D; X6 is G, A, H, D, E, Y, C, S, V, L, I or F; (4) X1 is H; X2 is D, K or S; X3 is T, H or D; X4 is D; X5 is H, T or D; X6 is C, S, V, L, I or F; (5) X1 is H; each X2, X3 and X5 is independently E; (6) X1 is H; X4 is D; X6 is G, A, H, D, E, Y, C, S, V, L, I or F; (7) X1 is D; X2 is K; X3 is T or H; X4 is T or H; X5 is H, T or D; X6 is C.

[0011] In some implementation schemes, X 11 X 12 THX 15 CPPC is selected from any of the following groups: (1) X 11 For H or S; X 15 For T; (2) X 11 For D; X 15 Let R be the value.

[0012] In another aspect, this disclosure provides an antibody-drug conjugate comprising the antibody described above and at least one linker-load, optionally having a drug-antibody ratio (DAR) of about 2 to 8, or 2 to 6.

[0013] In some embodiments, the linker-load includes a first linker-load and / or a second linker-load, each of which independently includes at least one thiol reactive group or a thiobridging agent.

[0014] In some implementations, the DAR of the antibody-drug conjugate is approximately 4, and the proportion of antibody-drug conjugates with a DAR of approximately 4 can reach up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or higher; or In some implementations, the DAR of the antibody-drug conjugate is approximately 2, meaning that the proportion of antibody-drug conjugates carrying the linker-load via a sulfur-bridged reagent is approximately 2, up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 90%, or higher; or The DAR of antibody-drug conjugates is approximately 6, and the proportion of antibody-drug conjugates with a DAR of approximately 6 can reach up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90% or higher; or The DAR of antibody-drug conjugates is approximately 3, meaning that the proportion of antibody-drug conjugates with a DAR of approximately 3 via sulfur-bridged reagents can reach up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, or higher; or The antibody-drug conjugate is a dual-load type with a DAR of approximately 2+4, meaning it carries a first linker-load with a DAR of approximately 2 and a second linker-load with a DAR of approximately 4. The proportion of antibody-drug conjugates with a DAR of 2+4 can be up to 50%, optionally up to 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher; or The antibody-drug conjugate is a dual-load type with a DAR of approximately 4+2, meaning it carries a first linker-load with a DAR of approximately 4 and a second linker-load with a DAR of approximately 2. The proportion of antibody-drug conjugates with a DAR of 4+2 can reach up to 60%, optionally up to 65%, 66%, 67%, 68%, 69%, 70%, 75%, or higher; or The antibody-drug conjugate is a dual-load type with a DAR of approximately 4+4, meaning it carries a first linker-load with a DAR of approximately 4 and a second linker-load with a DAR of approximately 4. The proportion of antibody-drug conjugates with a DAR of 4+4 can reach up to 60%, optionally up to 65%, 70%, 75%, 80%, 85%, 90%, or higher; or The antibody-drug conjugate is a dual-load type with a DAR of approximately 6+2, that is, carrying a first linker-load with a DAR of approximately 6 and a second linker-load with a DAR of approximately 2. The proportion of antibody-drug conjugates with a DAR of 6+2 can be up to 60%, and optionally up to 65%, 70%, 75%, 80%, 85%, 90% or higher.

[0015] In another aspect, this disclosure provides a method for producing antibody-drug conjugates (ADCs), comprising the following steps: (a) In a first reaction buffer, in the presence of a first transition metal ion, the antibody described above is incubated with a first reducing agent to reduce at least one interchain disulfide bond of the antibody. (b) Introducing a first metal chelating agent and a first linker-loading agent to react with the reduced thiol group generated in step (a). (c) The mixture from step (b) is purified to obtain the product ADC.

[0016] In some implementations, the method further includes: (d) In a second reaction buffer, in the presence of a second transition metal ion, the ADC from step (c) is incubated with a second reducing agent to reduce at least one remaining interchain disulfide bond of the ADC. (e) Introduce a second linker-load to react with the reduced thiol group generated in step (d). (f) The mixture from step (e) is purified to obtain the dual-loaded ADC product.

[0017] In another aspect, this disclosure provides a polynucleotide encoding the antibody described above. In another aspect, this disclosure provides a vector containing polynucleotides.

[0018] In another aspect, this disclosure provides a host cell comprising a polynucleotide or a vector.

[0019] In another aspect, this disclosure provides a kit comprising the antibodies, antibody-drug conjugates, polynucleotides, vectors, or host cells described above.

[0020] In another aspect, this disclosure provides a pharmaceutical composition comprising an antibody, an antibody-drug conjugate, an antibody-drug conjugate prepared by the above method, and at least one pharmaceutically acceptable carrier.

[0021] In another aspect, this disclosure provides the use of antibodies, antibody-drug conjugates, polynucleotides, vectors, host cells, kits, or pharmaceutical compositions in the preparation of therapeutic agents for the diagnosis, prevention, and treatment of diseases.

[0022] In another aspect, this disclosure provides a method for preventing, diagnosing, or treating a disease in a subject in need, comprising administering to the subject a therapeutically effective amount of an antibody, antibody-drug conjugate, polynucleotide, vector, host cell, kit, or pharmaceutical composition. Attached Figure Description

[0023] The accompanying drawings are briefly described below. These drawings are used to illustrate exemplary embodiments disclosed herein and are not intended to limit the invention.

[0024] Figures 1-10 The hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) chromatograms of the ADCs prepared in Examples 1-1 to 1-8 and Comparative Examples 1-1 to 1-2 are shown respectively. "D" in the figure represents "DAR".

[0025] Figure 11 The HIC-HPLC chromatograms of the ADCs prepared in Examples 2-6 are shown.

[0026] Figure 12 The HIC-HPLC chromatograms of the ADCs prepared in Comparative Examples 1-3 are shown.

[0027] Figures 13-39 The HIC-HPLC chromatograms of the ADCs prepared in Examples 2-1 to 2-5 and Examples 2-7 to 29 are shown respectively.

[0028] Figures 40-63 The HIC-HPLC chromatograms of the ADCs prepared in Examples 3-1 to 3-24 are shown respectively.

[0029] Figures 64-66 The HIC-HPLC chromatograms of the ADCs prepared in Examples 4-1 to 4-3 are shown respectively.

[0030] Figures 67-68 The HIC-HPLC chromatograms of the ADCs prepared in Examples 5-1 to 5-2 are shown respectively.

[0031] Figures 69-70 The HIC-HPLC chromatograms of the ADCs prepared in steps (c) and (f) of Example 6 are shown respectively.

[0032] Figures 71-72 The HIC-HPLC chromatograms of the ADCs prepared in steps (c) and (f) of Example 6-2 are shown respectively.

[0033] Figures 73-83 The HIC-HPLC chromatograms of the ADCs prepared in Examples 8-1 to 8-11 are shown respectively.

[0034] Figures 84-87 The HIC-HPLC chromatograms of the ADCs prepared in Examples 4-4 to 4-7 are shown respectively.

[0035] Figures 88-93 The HIC-HPLC chromatograms of the ADCs prepared in Examples 1-9 to 1-14 are shown respectively.

[0036] Figure 94 The HIC-HPLC chromatogram of the ADC prepared in Example 7 is shown.

[0037] Figure 95 The HIC-HPLC chromatogram of the ADC prepared in Comparative Example 8-1 is shown.

[0038] Figure 96 The HIC-HPLC chromatogram of the ADC prepared in Comparative Example 3-1 is shown.

[0039] Figures 97-99 The HIC-HPLC chromatograms of the ADCs prepared in Examples 8-15 to 8-17 are shown respectively.

[0040] Figures 100-101 The HIC-HPLC chromatograms of the ADCs prepared in Examples 1-15 to 1-16 are shown respectively.

[0041] Figures 102-106 The HIC-HPLC chromatograms of the ADCs prepared in Examples 1-17 to 1-21 are shown respectively.

[0042] Figures 107-108 The HIC-HPLC chromatograms of the ADCs prepared in Examples 8-12 to 8-13 are shown respectively.

[0043] Figure 109 The HIC-HPLC chromatograms of the ADCs prepared in Examples 1-22 are shown. Detailed Implementation

[0044] The present invention will now be explained in more detail. This description is not a detailed list of all different ways in which the invention can be practiced, nor is it a detailed list of all features that can be included in the invention. For example, a feature shown with respect to one embodiment may be incorporated into other embodiments, and a feature shown with respect to a particular embodiment may be removed from that embodiment. Furthermore, many variations and additions to the various embodiments presented herein will be apparent to those skilled in the art without departing from this invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention, and not to exhaustively describe all permutations, combinations, and variations thereof.

[0045] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described and used herein may be used in the testing practices of this invention, preferred materials and methods are described herein. The following terms will be used in describing and asserting this invention.

[0046] Antibody This disclosure provides examples of antibodies containing one or more amino acid substitutions in the hinge region.

[0047] Generally, the term "antibody" refers to any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen. A naturally occurring complete antibody consists of two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR"), a hinge region, and first, second, and third constant regions (CH1, CH2, and CH3), while each light chain consists of a variable region ("LCVR") and a constant region (CL). Mammalian heavy chains are classified into α, δ, ε, γ, and μ types, while mammalian light chains are classified into λ or κ types. Antibodies are Y-shaped, with the backbone of the Y consisting of the second and third constant regions of two heavy chains linked together via disulfide bonds. Each arm of the Y includes a variable region and a first constant region of a single heavy chain that bind to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in two chains generally contain three highly variable rings, called complementary determinant regions (CDRs) (the CDRs for light (L) chains include LCDR1, LCDR2, and LCDR3, and the CDRs for heavy (H) chains include HCDR1, HCDR2, and HCDR3). The boundaries of antibody CDRs can be defined or identified by the rules of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, Journal of Molecular Biology, 273(4), 927 (1997); Chothia, C. et al., Journal of Molecular Biology, Dec 5; 186(3):651-63 (1985); Chothia, C. and Lesk, AM, Journal of Molecular Biology, 196, 901 (1987); Chothia, C. et al., Nature, Dec 21-28; 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, MD. (1991)). The three CDRs are located between flanking segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support the hypervariable loop. Each HCVR and LCVR contains four FRs, and the CDRs and FRs are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are subdivided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0048] As used in this article, the term "hinge region" refers to the hinge region connecting the constant domains CH1 and CH2 of an antibody. In the structure of an antibody, the two heavy chains are interconnected by disulfide bonds in the hinge region. The hinge region and its variants used in this article have meanings known in the art, for example, see Janeway et al., Immunobiology: The Immune System in Health and Disease (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al., Protein Science (1997), 6:407-415; Humphreys et al., Journal of Immunological Methods (1997), 209:193-202.

[0049] The antibody provided in this article contains a hinge region comprising the amino acid sequence X1X2X3X4X5CPPX6, where X1 is D or H; X2 is E, D, K, or S; X3 is E, T, H, or D; X4 is T, H, D, S, or E; X5 is E, H, T, or D; and X6 is G, A, H, D, E, Y, C, S, V, L, I, or F; or The hinge region contains the amino acid sequence X. 11 X 12 THX 15 CPPC (SEQ ID NO: 30), where X 11 It is H, D, or S; X 12 Is it K or S, X 15 Is it T or R? X 11 X 12 THX 15 The amino acid sequence of CPPC or X1X2X3X4X5CPPX6 is not DKTHTCPPC (SEQ ID NO: 26).

[0050] The amino acids in this disclosure are represented by standard single-letter codes according to the IUPAC (International Union of Pure and Applied Chemistry) amino acid abbreviations. For example: alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y).

[0051] X1X2X3X4X5CPPX6 or X 11 X 12 THX 15The amino acids in CPPC can be substituted with conserved amino acids; therefore, the substitution of conserved amino acids is also within the scope of this disclosure.

[0052] The term "conservative amino acid" in this article generally refers to amino acids that belong to the same class or have similar properties (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity).

[0053] For example, the following six groups of amino acids are considered to be examples of conserved substitution for each other: 1) A, S and T; 2) D and E; 3) N and Q; 4) R, K and H; 5) I, L, M and V; and 6) F, Y and W.

[0054] In some implementations, X1 is D or H; X2 is D, K or S; X3 is T, H or D; X4 is T, H, D, S or E; X5 is H, T or D; and X6 is C, S, V, L, I or F.

[0055] In some implementations, X1 is H. In some implementations, X1 is H; X2 is D, K, or S; X3 is T, H, or D; X4 is T, H, D, S, or E; X5 is H, T, or D; X6 is C, S, V, L, I, or F.

[0056] In some implementations, X1 is H and X2 is S or D.

[0057] In some implementations, X1 is H and X3 is D.

[0058] In some embodiments, X1 is H, and X4 is D, S, or E. In some embodiments, X1 is H, and X4 is D or E. In some embodiments, X1 is H, and X4 is D. In some embodiments, X1 is H; X4 is D; and X6 is G, A, H, D, E, Y, C, S, V, L, I, or F. In some embodiments, X1 is H; X2 is D, K, or S; X3 is T, H, or D; X4 is D; X5 is H, T, or D; and X6 is G, A, H, D, E, Y, C, S, V, L, I, or F.

[0059] In some implementations, X1 is H; X4 is D; and X6 is A, Y, C, S, V, L, I, or F. In some implementations, X1 is H; X2 is D, K, or S; X3 is T, H, or D; X4 is D; X5 is H, T, or D; and X6 is C, S, V, L, I, or F.

[0060] In some embodiments, X1 is H; X3 is T or D; X4 is D, S, or E; and X5 is T or D. In some embodiments, X1 is H; X2 is K; X3 is T or D; X4 is D, S, or E; and X5 is T or D. In some embodiments, X1 is H; X2 is K; X3 is T; X4 is D, S, or E; and X5 is T or D. In some embodiments, X1 is H; X2 is K; X3 is T; X4 is D or E; and X5 is T. In some embodiments, X1 is H; X2 is D; and X3 is S.

[0061] In some implementations, X1 is H; X2 is K; X3 is T; X4 is D or E; X5 is T; and X6 is S, V, L, I, or F.

[0062] In some embodiments, X1X2X3X4X5CPPX6 has the amino acid sequence of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 29, 31 or its conservative substitutions.

[0063] In some implementations, X1 is H; each X2, X3, and X5 is independently E.

[0064] In some implementations, X1 is D; X2 is K; X6 is C. In some implementations, X1 is D; X2 is K; X3 is T or H; X4 is T or H; X5 is H, T, or D; X6 is C.

[0065] In some embodiments, X1X2X3X4X5CPPX6 has the amino acid sequence of SEQ ID NO: 23, 24, 25 or its conservative substitution form.

[0066] In some embodiments, X1X2X3X4X5CPPX6 has the amino acid sequence of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 31 or its conservative substitutions.

[0067] In some embodiments, X1X2X3X4X5CPPX6 has the amino acid sequence of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 29, 31 or its conservative substitutions.

[0068] In some embodiments, X1X2X3X4X5CPPX6 has the amino acid sequence of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 29, 31 or its conserved substitutions. In some embodiments, X 11 It is H or S; X 15 It is T. In some implementations, X 11 It is D; X 15 It is R.

[0069] In some implementation schemes, X 11 X 12 THX 15 CPPC has the amino acid sequence of SEQ ID NO: 11, 13, 27, 28 or its conserved substitutions.

[0070] In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody has a heavy chain constant region comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:1, and a light chain constant region comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:2.

[0071] In some embodiments, the antibody comprises a heavy chain constant region containing one or more amino acid substitutions selected from the group consisting of: K218H, D221H, K222S, K222D, T223H, T223D, H224S, H224D, H224E, H224T, T225H, T225R, T225D, C229S, C229V, C229L, C229I, C229E, or combinations thereof, wherein the first amino acid at the N-terminus of SEQ ID NO: 1 is defined as position 118 compared to SEQ ID NO: 1. This position is defined by an EU number.

[0072] In this disclosure, the amino acid substitution D221H indicates that amino acid D at position 221 of the heavy chain, as defined by the EU number, is mutated to amino acid H. Other mutations in the heavy chain have similar meanings.

[0073] Some amino acid substitutions (e.g., K218H) are located in the Fab region of the antibody. The Fab fragment consists of the complete L chain along with the variable region domain (VH) of the H chain and the first constant domain (CH1) of a heavy chain. Each Fab fragment is monovalent in terms of antigen binding, meaning it has a single antigen-binding site. Treatment of IgG antibodies with pepsin yields a large F(ab′)2 fragment, which roughly corresponds to two Fab fragments linked by disulfide bonds, exhibiting bivalent antigen-binding activity and still capable of cross-linking antigens. The Fab′ fragment differs from the Fab fragment in that it has several additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab′-SH is the nomenclature of Fab′ used herein, where the cysteine ​​residue in the constant domain carries a free thiol group. The F(ab′)2 antibody fragment is initially produced as a pair of Fab′ fragments with a hinge cysteine ​​residue between them. Other chemical coupling mechanisms of antibody fragments are also known.

[0074] Mutation sites in antibodies play a crucial role in determining reductive selectivity, thereby improving the homogeneity of antibody-drug conjugates.

[0075] In some implementations, the antibody constant region contains amino acid substitutions selected from the group consisting of: (1) D221H; (2) D221H and T225D; (3) D221H and K222S; (4) D221S, H224S and T225D; (5) D221H, T223D and H224S; (6) D221H and H224E; (7) D221H and H224D; (8) D221H, H224D and C229S; (9) D221H, H224D and C229V; (10) D221H, H224D and C229L; (11) D221H, H224D and C229I; (12) D221H, H224D and C229E; (13) K218H, D221H and H224S; (14) T225R; (15) D221H and K222D; (16) D221H and H224S; or (17) D221H, K222D and H224S.

[0076] In some embodiments, the antibody comprises an antigen-binding fragment capable of binding to one or more target antigens, optionally the target antigens being expressed on tumor cells or immune checkpoints.

[0077] In some embodiments, the antigen-binding fragment binds to at least one antigen expressed on tumor cells. In some embodiments, the modified antibody binds to epidermal growth factor receptor (EGFR), erb-b2 receptor tyrosine kinase 2 (HER2), CD3, calcium channels, programmed cell death 1 ligand 1 (PDL1), CD19, kinase insertion domain receptor (VEGFR), glucagon-like peptide 1 receptor (GLP1R), dopamine receptor D2 (DRD2), glutamate ionotropic receptor NMDA (NMDAR), bacterial DNA gyrase, programmed cell death 1 (PD1), sodium channel protein (VGSC), cytochrome c oxidase subunit II (COX2), DNA topoisomerase 2 (TOP2), V-set, and immunoglobulin domain-containing protein 4 (VSI). G4), CD276 molecule (B7-H3), V-set domain containing T cell activation inhibitory factor 1 (B7-H4), receptor tyrosine kinase-like orphan receptor 1 (ROR1), receptor tyrosine kinase-like orphan receptor 2 (ROR2), erb-b2 receptor tyrosine kinase 3 (HER3), MET proto-oncogene, receptor tyrosine kinase (c-MET), CEA cell adhesion molecule 5 (CEACAM5), interleukin 2 receptor subunit α (CD25), transferrin receptor (CD71), CD70, CD74, CD38, TNF receptor superfamily member 17 (BCMA), tumor-associated calcium signaling transducer 2 (TROP2), CD79b, claudin 6 (CLDN6), and claudin 18.2. Mesothelin, trophoblast glycoprotein (5T4), solute carrier family 39 member 6 (LIV-1), archaea proteasome endopeptide complex subunit α (PSMA), coagulation factor III (CD142), mucin 1 (MUC-1), mucin 16 (MUC-16), delta-like typical Notch ligand 3 (DLL3), cadherin 6 (CDH6), cadherin 3 (CDH3), fibroblast growth factor receptor 2 (FGFR2), solute carrier family 34 member 2 (NaPi2b), fibroblast growth factor receptor 3 (FGFR3), G protein-coupled receptor class C, group 5. Member D (GPRC5D), Leucine-rich repeat-containing protein 15 (LRRC15), Protein tyrosine kinase 7 (PTK7), Eukaryotic translation elongation factor 1α2 (STn), KIT proto-oncogene (CD117), Protein tyrosine phosphatase receptor type C (CD45), Lymphocyte antigen 6 family member E (Ly6E), Prolactin subfamily 3 member A1 (BTN3A1), G protein-coupled receptor 65 (GPR65), GABA receptor (GABA), Discoid domain receptor tyrosine kinase 1 (DDR1), ADP-ribosyltransferase 1 (ART1), Ariadne RBR E3 ubiquitin protein ligase 1 (ARIH1), lysine demethylase 5B (KDM5B), PVR cell adhesion molecule (CD155), CD200 receptor 1 (CD200R1), LIF interleukin-6 family cytokines (LIF), leukocyte immunoglobulin-like receptor B (LILRB), cytotoxic lectin-like receptor B1 (CD161), SET domain bifurcated histone lysine methyltransferase 1 (SETB1), COP1 E3 ubiquitin ligase (COP1), CAP-Gly domain-containing ligin 1 (CLIP1), CAP-Gly domain-containing ligin 1 (LTK), lysine demethylase 1A (LSD1), methyltransferase 3, N6-adenosylmethyltransferase complex catalytic subunit (METTL3), myelodysplastic syndrome, transient transient abnormality (TAM), basal cell adhesion molecule (BCAM), nuclear receptor-binding SET domain protein 3 (NSD3), selectin P ligand (PSGL-1), interleukin 8 (IL8), protein tyrosine phosphatase non-receptor type 2 (PTPN2), CC motif chemokine receptor 8 (CCR8), SRY-box transcription factor 4 (SOX4), elastase, neutrophil expression (ELANE), folate receptor (FOLR), cadherin 17 (CDH17), Nectin 4, tyrosine kinase non-receptor 1 (TNK1), or combinations thereof.

[0078] In some implementations, the antigen-binding fragment targets HER2, FOLR, CDH17, DLL3, or combinations thereof.

[0079] In some implementations, the antibody is derived from a commercially available anti-HER2 antibody, the parental anti-HER2 antibody (also known as wild-type, WT) having the heavy chain of SEQ ID NO: 3 and the light chain of SEQ ID NO: 4, namely trastuzumab.

[0080] In some implementations, the antibody is derived from a commercially available anti-DLL3 antibody, the parental anti-DLL3 antibody (also known as wild-type, WT) having the heavy chain of SEQ ID NO: 5 and the light chain of SEQ ID NO: 6, namely Rovalpituzumab.

[0081] In some implementations, the antibody is derived from a commercially available anti-FOLR antibody, the parental anti-FOLR antibody (also known as wild-type, WT) having the heavy chain of SEQ ID NO: 7 and the light chain of SEQ ID NO: 8, namely Farletuzumab.

[0082] In some implementations, the antibody is derived from a commercially available anti-CDH17 antibody, the parental anti-CDH17 antibody (also known as wild-type, WT) having the heavy chain of SEQ ID NO: 9 and the light chain of SEQ ID NO: 10.

[0083] In some embodiments, this disclosure provides examples of antibodies having one or more mutations in the hinge region, with the parent antibody and mutation (underlined) shown below:

[0084] The mutations listed in the table above help enhance the selectivity of thiol reduction under reducing conditions, thereby improving the homogeneity of antibody-drug conjugates.

[0085] In some implementations, antibodies containing the amino acid sequence X1X2X3X4X5CPPX6 can improve the homogeneity of antibody-drug conjugates with a drug-antibody ratio (DAR) of 4.

[0086] In some implementations, X is included 11 X 12 THX 15 Antibodies with CPPC amino acid sequences can improve the homogeneity of antibody-drug conjugates with a drug-antibody ratio (DAR) of 6.

[0087] In some implementations, X is included 11 X 12 THX 15Antibodies with the amino acid sequence CPPC or X1X2X3X4X5CPPX6 are suitable for preparing dual-loaded antibody-drug conjugates.

[0088] Antibody-drug conjugates This disclosure provides an antibody-drug conjugate comprising the antibody described above.

[0089] In some implementations, antibody-drug conjugates contain a single type of payload, i.e., only one type of linker-load is chemically covalently conjugated to the antibody.

[0090] In some implementations, the antibody-drug conjugate is dual-loaded, meaning that two different linker-loads are chemically covalently conjugated to the same antibody.

[0091] Typically, linker-loading contains at least one (e.g., one or two) reactive groups for covalently coupling with an antibody, which are capable of reacting with thiol groups. In some embodiments, the reactive groups include thiol reactive groups and / or thiol bridging agents.

[0092] The term "thiol reactive group" refers to a chemical group that reacts with a thiol group on an antibody. The most common thiol reactive group that can bond with a thiol group is maleimide, or its organochloride, bromide, or iodide.

[0093] The term "thiol bridging agent" refers to a chemical group that reacts with two thiol groups on an antibody, allowing for the re-bridging of thiol groups within the antibody. Thiol bridging agents can have two reaction sites within a single chemical group, or in two chemical groups. Thiol bridging agents are well known to those skilled in the art; an exemplary thiol bridging agent is dibromomaleimide. In some embodiments, thiol bridging agents and click chemistry reagents, such as azides and dibenzocyclooctylene, can be used in combination to produce ADCs.

[0094] As used herein, a linker-load is a chemical component synthesized by attaching a linker to a load. One end of the linker is covalently attached to the load, and the other end is attached to the antibody via a thiol reactive group or a thiol bridging reagent.

[0095] There are no specific restrictions on the linker-load; it can be cleavable or non-cleavable, chemically unstable or enzyme-unstable.

[0096] In some embodiments, the ADC includes a first linker-loader and / or a second linker-loader, each of which independently contains at least one thiol reactive group or thiol bridging agent.

[0097] In some embodiments, the first linker-loader and the second linker-loader each independently contain one, two, or three thiol reactive groups to react with the reduced thiol groups of the antibody. In some embodiments, the first linker-loader and the second linker-loader each independently contain one thiol reactive group.

[0098] In some embodiments, the first linker-load and the second linker-load each independently comprise one or two thiol bridging agents. In some embodiments, the first linker-load and the second linker-load each independently comprise one thiol bridging agent.

[0099] In some embodiments, the first linker-load and the second linker-load each independently comprise a load, such as a physiologically active substance. In some embodiments, the load includes cytotoxic agents, markers, nucleic acids, radionuclides, hormones, immunomodulators, prodrug-converting enzymes, ribonucleases, agonist antibodies, antagonist antibodies and fragments thereof, fusion proteins or derivatives thereof, or combinations thereof.

[0100] In some implementations, the first connector-load and the second connector-load each independently contain a cytotoxic agent.

[0101] In some implementations, the first connector-load and the second connector-load are each independently including, but not limited to, MC-VC-PAB-MMAE, MC-VA-PAB-MMAE, MC-GGFG-DXd, MC-VC-PAB-MMAD, MC-VC-PAB-Eribulin, MC-MMAF, or MC-VC-PAB-MMAF.

[0102] In some embodiments, the first connector-load and the second connector-load are each independently including, but not limited to, Dibromomaleimide-PEG4-VC-PAB-MMAE, Dibromomaleimide-PEG4-VC-PAB-MMAF, Bis-Maleimide-PEG2-VC-MMAE, Bis-Maleimide-PEG4-VC-MMAE, Bis-Maleimide-PEG4-VC-DX8951, Bis-Maleimide-PEG4-VC-Eribulin, Bis-Maleimide-PEG2-VC-Eribulin, Bis-Maleimide-PEG2-MMAF, or Bis-Maleimide-PEG4-MMAF.

[0103] In some implementations, the first connector-load and the second connector-load are each independently MC-VC-PAB-MMAE, MC-GGFG-DXd, or Dibromomaleimide-PEG4-VC-PAB-MMAE.

[0104] ADCs are heterogeneous, and their homogeneity can be assessed using the drug-to-antibody ratio (DAR), which is the average number of drugs attached to a single antibody molecule. Typically, DAR4 indicates that approximately four payloads (drugs) are attached to one antibody molecule. DAR2, DAR3, or DAR6 have similar meanings.

[0105] As used herein, the term “about” refers to a number that varies by as much as 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference figure. In a particular embodiment, the term “about” precedes a numerical value to indicate a range of that value plus or minus 15%, 10%, 5%, or 1%.

[0106] The DAR of an ADC is approximately 2 to 8, 2 to 6, or 4 to 6. In some implementations, the DAR of an ADC is approximately 2, 3, 4, 5, 6, 7, or 8.

[0107] In some embodiments, the DAR of the antibody-drug conjugate is approximately 4. In some embodiments, the DAR of the antibody-drug conjugate is approximately 2, which is achieved by attaching a loaded sulfur-bridged reagent to the antibody.

[0108] In some embodiments, the DAR of the antibody-drug conjugate is approximately 6. In some embodiments, the DAR of the antibody-drug conjugate is approximately 3, which is achieved by attaching a loaded sulfur-bridged reagent to the antibody.

[0109] In some implementations, the DAR of the dual-loaded antibody-drug conjugate is approximately 2+4, 4+4, or 4+2. The DAR value before the "+" sign refers to the average number of the first linker-loads on each antibody molecule, and the DAR value after the "+" sign refers to the average number of the second linker-loads on each antibody molecule.

[0110] In some embodiments, antibodies containing the amino acid sequence SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 are suitable for producing antibody-drug conjugates with a DAR of approximately 4. Furthermore, in this case, the ADC can also be DAR2 by linking a sulfur-bridged reagent containing a physiologically active substance.

[0111] In some embodiments, antibodies containing the amino acid sequence SEQ ID NO: 11, 13, 27, or 28 are suitable for producing antibody-drug conjugates with a DAR of approximately 6. It is understood that antibody-drug conjugates with a DAR of approximately 3 can be produced by using a sulfur-bridged reagent containing a physiologically active substance.

[0112] In some embodiments, the DAR of the antibody-drug conjugate is approximately 4, and the proportion of antibody-drug conjugates with a DAR of approximately 4 can reach up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or higher. In some embodiments, the proportion of antibody-drug conjugates with a DAR of approximately 4 can reach up to 60%, 65%, 70%, 72%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or higher.

[0113] In some embodiments, the DAR of the antibody-drug conjugate carrying the linker-load via the sulfur bridging reagent is approximately 2, and the proportion of antibody-drug conjugates with a DAR of approximately 2 is up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 90%, or higher. In some embodiments, the proportion of antibody-drug conjugates with a DAR of approximately 2 is up to 60%, 65%, 70%, 72%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or higher.

[0114] In some embodiments, the DAR of the antibody-drug conjugate is approximately 6, and the proportion of antibody-drug conjugates with a DAR of approximately 6 can reach up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, or higher. In some embodiments, the proportion of antibody-drug conjugates with a DAR of approximately 6 can reach up to 60%, 65%, 70%, 72%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or higher.

[0115] In some embodiments, the DAR of the antibody-drug conjugate is approximately 3, meaning that the proportion of antibody-drug conjugates with a DAR of approximately 3 carried by a sulfur bridging reagent can reach up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, or higher. In some embodiments, the proportion of antibody-drug conjugates with a DAR of approximately 3 can reach up to 60%, 65%, 70%, 72%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or higher.

[0116] In some implementations, the antibody-drug conjugate is a dual-load type with a DAR of approximately 2+4, i.e., carrying a first linker-load with a DAR of approximately 2 and a second linker-load with a DAR of approximately 4. The proportion of antibody-drug conjugates with a DAR of approximately 2+4 can be up to 50%, optionally up to 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or higher.

[0117] In some implementations, the proportion of antibody-drug conjugates with a DAR of approximately 2+4 can reach up to 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 82%, 85%, 87%, 90%, 92%, 94% or higher.

[0118] In some implementations, the antibody-drug conjugate is a dual-load type with a DAR of approximately 4+2, that is, carrying a first linker-load with a DAR of approximately 4 and a second linker-load with a DAR of approximately 2. The proportion of antibody-drug conjugates with a DAR of approximately 4+2 can be up to 60%, optionally up to 65%, 66%, 67%, 68%, 69%, 70%, 75% or higher.

[0119] In some implementations, the proportion of antibody-drug conjugates with a DAR of approximately 4+2 can reach as high as 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 77%, 80%, 82%, 85% or higher.

[0120] In some implementations, the antibody-drug conjugate is a dual-load type with a DAR of approximately 4+4, that is, carrying a first linker-loader with a DAR of approximately 4 and a second linker-loader with a DAR of approximately 4. The proportion of conjugates with a DAR of approximately 4+4 can be up to 60%, optionally up to 65%, 70%, 75%, 80%, 85%, 90% or higher.

[0121] In some implementations, the proportion of antibody-drug conjugates with a DAR of approximately 4+4 can be as high as 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or higher.

[0122] In some embodiments, the antibody-drug conjugate is a dual-load type with a DAR of approximately 6+2, i.e., carrying a first linker-load with a DAR of approximately 6 and a second linker-load with a DAR of approximately 2. The proportion of antibody-drug conjugates with a DAR of approximately 6+2 can be up to 60%, optionally up to 65%, 70%, 75%, 80%, 85%, 90%, or higher. In some embodiments, the proportion of antibody-drug conjugates with a DAR of approximately 6+2 can be up to 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or higher.

[0123] The antibody-drug conjugates presented in this article exhibit improved homogeneity and excellent selectivity in the hinge region of the antibody, particularly the Fab region.

[0124] Methods for producing antibody-drug conjugates This disclosure provides a method for producing antibody-drug conjugates, the method comprising the following steps: (a) In a first reaction buffer, in the presence of a first transition metal ion, the antibody is incubated with a first reducing agent to reduce at least one interchain disulfide bond of the antibody. (b) Introduce a first metal chelating agent and a first linker-loader to react with the reduced thiol group generated in step (a). (c) Purify the mixture obtained in step (b) to obtain the product ADC.

[0125] The resulting ADC is a single-load type. In some implementations, the DAR of the antibody-drug conjugate is approximately 2, 3, 4, or 6. In some embodiments, the method further includes the step of producing a dual-load antibody-drug conjugate: (d) In a second reaction buffer, in the presence of a second transition metal ion, the ADC obtained in step (c) is incubated with a second reducing agent to reduce at least one remaining interchain disulfide bond in the ADC. (e) Introduce a second linker-loader to react with the reduced thiol group generated in step (d). (f) The mixture obtained in step (e) is purified to obtain the dual-load ADC product. In some implementations, the DAR of dual-load antibody-drug conjugates is approximately 4+2, 4+4, 2+4, or 6+2.

[0126] In this method, the first transition metal ion and the second transition metal ion are each independently Zn. 2+ or Mn 2+ The first transition metal ion and the second transition metal ion can be the same or different.

[0127] In some implementation schemes, Zn 2+ and / or Mn 2+ Salts or complexes derived from transition metal ions. There are no particular restrictions on the salts or complexes of transition metal ions, as long as the transition metal ions are soluble in the reaction solution and serve as a source of free transition metal ions, and perform any further function or form complexes in the reaction solution.

[0128] In some embodiments, the salts of transition metal ions include lactates, chlorides, nitrates, sulfates, acetates, iodides, bromides, formates, and / or tetrafluoroborates.

[0129] In some implementation schemes, Zn 2+ Derived from divalent zinc salts. In some embodiments, the divalent zinc salt is selected from ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2, zinc formate, and zinc tetrafluoroborate, or combinations thereof. Exemplary divalent zinc salts are ZnCl2, Zn(NO3)2, or ZnSO4.

[0130] In some implementation schemes, Mn 2+ Derived from divalent manganese salts. In some embodiments, the divalent manganese salt is selected from MnSO4, MnCl2, MnBr2, and Mn(NO3)2, or combinations thereof. Exemplary divalent manganese salts are MnSO4 or MnCl2.

[0131] In some embodiments, the first transition metal salt is ZnCl2, Zn(NO3)2, or ZnSO4. In some embodiments, the first transition metal salt is MnSO4 or MnCl2. In some embodiments, the second transition metal salt is ZnCl2, Zn(NO3)2, or ZnSO4. In some embodiments, the second transition metal salt is MnSO4 or MnCl2.

[0132] In some embodiments, the concentrations of the first transition metal ion and the second transition metal ion in the reaction buffer are the same. In other embodiments, the concentrations of the first transition metal ion and the second transition metal ion in the reaction buffer are different.

[0133] In some embodiments, the concentrations of the first and second transition metal ions in the reaction buffer are each independently 0.01 mM - 2 mM, 0.1 mM - 2 mM, 0.2 mM - 2 mM, 0.3 mM - 2 mM, 0.05 mM - 2 mM, 0.1 mM - 1.5 mM, 0.01 mM - 1 mM, or 0.05 mM - 1.5 mM. In some embodiments, the concentrations of the transition metal ions in steps (a) and (d) in the reaction buffer are each independently 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.8 mM, 1.0 mM, 1.2 mM, 1.4 mM, 1.6 mM, 1.8 mM, or any value within the range of any of the foregoing values.

[0134] The molar ratio of the first transition metal ion to the antibody and the molar ratio of the second transition metal ion to the antibody may be the same or different.

[0135] In some embodiments, the molar ratio of transition metal ions to antibodies in steps (a) and (d) is independently (1-100):1, (5-100):1, (10-100):1, (30-100):1, (40-100):1, (50-100):1, (20-80):1, (30-70):1, or (30-50):1. In some embodiments, the molar ratio of transition metal ions to antibodies dissolved in the reaction buffer in steps (a) and (d) is independently 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or any value within the range of any of the foregoing values.

[0136] In some embodiments, the first and second reducing agents are each independently including, but not limited to, TCEP (tris(2-carboxyethyl)phosphine), THPP (tris(3-hydroxypropyl)phosphine), TCEPNO, TCEP3, TCEP1, TCEP26, TCEP28 and / or TCEP33, the structures of which are shown below: , , , , , .

[0137] In some implementations, the first reducing agent is TCEP, and the second reducing agent is either TCEP or THPP.

[0138] In some embodiments, the concentrations of the first reducing agent and the second reducing agent are each independently 0.02 mM - 0.2 mM, 0.02 mM - 0.15 mM, 0.02 mM - 0.12 mM, 0.02 mM - 0.1 mM, or 0.04 mM - 0.2 mM. In some embodiments, the concentration of the reducing agent in the reaction buffer is 0.03 mM, 0.04 mM, 0.05 mM, 0.07 mM, 0.09 mM, 0.1 mM, 0.12 mM, 0.15 mM, 0.17 mM, 0.19 mM, 0.2 mM, or any value within the range of any of the foregoing values.

[0139] In some embodiments, to generate an ADC with a DAR of 4, the molar ratio of the first reducing agent to the antibody is (2-8):1, (2-7):1, (2-5):1, or (2-3):1. In some embodiments, the molar ratio is 8, 7.5, 7, 6.5, 6, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, or any value within the range of any of the foregoing values.

[0140] In some embodiments, to generate an ADC with a DAR of 2 via a sulfur-bridged reagent, the molar ratio of the first reducing agent to the antibody is (2-8):1, (2-7):1, (2-5):1, or (2-3):1. In some embodiments, the molar ratio is 8, 7.5, 7, 6.5, 6, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, or any value within the range of any of the foregoing values.

[0141] In some embodiments, to generate an ADC with a DAR of 6, the molar ratio of the first reducing agent to the antibody is (3.5-7):1, (3.5-6):1, (4-5):1, (4-6):1, or (3-5):1. In some embodiments, the molar ratio is 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.5, 6, 6.5, 7, or any value within the range of any of the foregoing values.

[0142] In some embodiments, to generate an ADC with a DAR of 3 via a sulfur-bridged reagent, the molar ratio of the first reducing agent to the antibody is (3-7):1, (3.5-7):1, (3.5-6):1, (4-5):1, (4-6):1, or (3-5):1. In some embodiments, the molar ratio is 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.5, 6, 6.5, 7, or any value within the range of any of the foregoing values.

[0143] In some implementations, to generate an ADC having approximately DAR4+DAR2, the molar ratio of the first reducing agent to the antibody is (2-7):1, (2-6):1, (2-5):1, (2-4):1, or (2-3):1, and the molar ratio of the second reducing agent to the antibody is (1-7):1, (1-5):1, (2-3):1, or (3-5):1.

[0144] In some implementations, to generate an ADC having approximately DAR4+DAR2, the molar ratio of the first reducing agent to the antibody is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, or any value within the range of any of the aforementioned values; and the molar ratio of the second reducing agent to the antibody is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, or any value within the range of any of the aforementioned values.

[0145] In some implementations, to generate an ADC having approximately DAR2+DAR4, the molar ratio of the first reducing agent to the antibody is (2-7):1, (2-6):1, (2-5):1, (2-4):1, or (2-3):1, and the molar ratio of the second reducing agent to the antibody is (2-9):1, (2-7):1, (2-6):1, (2-5):1, (2-4):1, (2-3):1, or (3-5):1.

[0146] In some implementations, to generate an ADC having approximately DAR2+DAR4, the molar ratio of the first reducing agent to the antibody is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, or any value within the range of any of the aforementioned values; and the molar ratio of the second reducing agent to the antibody is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or any value within the range of any of the aforementioned values.

[0147] In some implementations, to generate an ADC having approximately DAR4+DAR4, the molar ratio of the first reducing agent to the antibody is (2-7):1, (2-6):1, (2-5):1, (2-4):1, or (2-3):1, and the molar ratio of the second reducing agent to the antibody is (2-9):1, (2-7):1, (2-6):1, (2-5):1, (2-4):1, (2-3):1, or (3-5):1.

[0148] In some embodiments, to generate an ADC having approximately DAR4+DAR4, the molar ratio of the first reducing agent to the antibody is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, or any value within the range of any of the foregoing values; and the molar ratio of the second reducing agent to the antibody is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or any value within the range of any of the foregoing values. In some implementations, to generate an ADC having approximately DAR6+DAR2, the molar ratio of the first reducing agent to the antibody is (3-7):1, (3.5-7):1, (3.5-6):1, (4-5):1, (4-6):1, (3-5):1, and the molar ratio of the second reducing agent to the antibody is (1-9):1, (1-7):1, (1-5):1, (2-3):1, or (3-5):1.

[0149] In some implementations, to generate an ADC having approximately DAR6+DAR2, the molar ratio of the first reducing agent to the antibody is 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, or any value within the range of any of the aforementioned values; and the molar ratio of the second reducing agent to the antibody is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or any value within the range of any of the aforementioned values.

[0150] In some implementations, the first reaction buffer and the second reaction buffer are each independently PB, PBS, acetate buffer, His buffer, Bis-Tris buffer, PIPES buffer, ADA, HEPES buffer, MOPS buffer, MOBS buffer, DIPSO buffer, MOPSO buffer, TES buffer, TAPSO buffer, ACES buffer, BES buffer, or MES buffer.

[0151] In some embodiments, the pH values ​​of the first reaction buffer and the second reaction buffer are each independently from 5.5 to 8. In some embodiments, the pH values ​​of the first reaction buffer and the second reaction buffer are each independently from 5.8 to 7.4. Exemplary pH values ​​of the reaction buffers are 5.8, 6.4, 6.7, 7.0, and 7.4.

[0152] In some embodiments, the first metal chelating agent and the second metal chelating agent are each independently ethylenediaminetetraacetic acid (EDTA) or pentiformin (DTPA). In some embodiments, the first metal chelating agent and the second metal chelating agent are both EDTA.

[0153] The metal chelating agent is used to remove excess transition metal ions to promote the coupling reaction. In some embodiments, the molar ratio of the first metal chelating agent to the antibody, or the molar ratio of the second metal chelating agent to the antibody, is (10-250):1. In some embodiments, the molar ratio of the first metal chelating agent to the antibody, or the molar ratio of the second metal chelating agent to the antibody, is (30-250):1. In some embodiments, the molar ratio of the first metal chelating agent to the antibody, or the molar ratio of the second metal chelating agent to the antibody, is (80-250):1. In some embodiments, the molar ratio of the first or second metal chelating agent to the antibody is independently (100-250):1, (130-250):1, (150-250):1, (180-250):1, (200-250):1, or (230-250):1.

[0154] In some embodiments, to generate an ADC with a DAR of 4, the molar ratio of linker-loador to antibody is (2-8):1, (2-7):1, (3-7):1, or (4-7):1. In other embodiments, to generate an ADC with a DAR of 4, the molar ratio of linker-loador to antibody is 2:1, 3:1, 4:1, 4.5:1, 4.8:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, or 8:1. The molar ratio of linker-loador to antibody can be any value within the range of the aforementioned values. In some embodiments, to generate an ADC with a DAR of 2 via a sulfur-bridged reagent, the molar ratio of linker-load to antibody is (1-5):1, (1.5-4.5):1, (2-5):1, or (2-4):1. In some embodiments, to generate an ADC with a DAR of 2 via a sulfur-bridged reagent, the molar ratio of linker-load to antibody is 1.2:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1. The first linker-load to antibody molar ratio can be any value within the range of the aforementioned values. In some embodiments, to generate an ADC with a DAR of 6, the molar ratio of the first linker-loader to the antibody is (6-12):1, (7-12):1, or (7-10):1. In some embodiments, to generate an ADC with a DAR of 6, the molar ratio of the first linker-loader to the antibody is 6:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 10:1, 11:1, 12:1, or a value within the range of the foregoing values.

[0155] In some embodiments, to generate an ADC with a DAR of 3, the molar ratio of the first linker-loader to the antibody is (3-6):1 or (3.5-5.5):1. In some embodiments, to generate an ADC with a DAR of 3, the molar ratio of the first linker-loader to the antibody is 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, or a value within the range of the foregoing values.

[0156] In some implementations, to generate an ADC with a DAR of 4+2, the molar ratio of the first linker-load to the antibody is (4-8):1, and the molar ratio of the second linker-load to the antibody is (2-8):1. In the method for preparing an ADC with a DAR of 4+2, the molar ratio of the first linker-loader to the antibody is 4:1, 5:1, 6:1, 7:1, or 8:1. Optionally, the molar ratio of the first linker-loader to the antibody is (4-7):1, (4-6):1, or (4-5):1. The molar ratio of the second linker-loader to the antibody is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1. Optionally, the molar ratio of the second linker-loader to the antibody is (2-8):1, (3-8):1, (4-8):1, or (5-8):1.

[0157] In some implementations, to generate an ADC with a DAR of 2+4, the molar ratio of the first linker-load to the antibody is (2-8):1, and the molar ratio of the second linker-load to the antibody is (4-12):1.

[0158] In the method for preparing an ADC with a DAR of 2+4, the molar ratio of the first linker-loader to the antibody is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1. Optionally, the molar ratio of the first linker-loader to the antibody is (2-7):1, (3-7):1, or (4-7):1. The molar ratio of the second linker-loader to the antibody is 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12:1. Optionally, the molar ratio of the second linker-loader to the antibody is (5-10):1, (6-9):1, or (7-8):1.

[0159] In some implementations, to generate an ADC with a DAR of 4+4, the molar ratio of the first linker-load to the antibody is (4-8):1, and the molar ratio of the second linker-load to the antibody is (4-12):1.

[0160] In the method for preparing an ADC with a DAR of 4+4, the molar ratio of the first linker-loader to the antibody is 4:1, 5:1, 6:1, 7:1, or 8:1. Optionally, the molar ratio of the first linker-loader to the antibody is (5-8):1, (4-7):1, (4-6):1, or (4-5):1. The molar ratio of the second linker-loader to the antibody is 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12:1. Optionally, the molar ratio of the second linker-loader to the antibody is (5-10):1, (6-9):1, or (7-8):1.

[0161] In some implementations, to generate an ADC with a DAR of 6+2, the molar ratio of the first linker-load to the antibody is (6-12):1, and the molar ratio of the second linker-load to the antibody is (2-8):1.

[0162] In the method for preparing an ADC with a DAR of 6+2, the molar ratio of the first linker-loader to the antibody is 6:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 10:1, 11:1, or 12:1. Optionally, the molar ratio of the first linker-loader to the antibody is (6-12):1, (7-11):1, (7-10):1, or (7-9):1. The molar ratio of the second linker-loader to the antibody is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1. Optionally, the molar ratio of the second linker-loader to the antibody is (2-8):1, (3-8):1, (4-8):1, or (5-8):1.

[0163] In some implementations, the incubation temperature in step (a) is 0°C to 37°C, 0°C to 25°C, 0°C to 15°C, or 0°C to 5°C; and the incubation time in step (a) is 10 min to 8 h, or 10 min to 4 h. For example, the incubation temperature in step (a) is 0°C, 4°C, 15°C, or 25°C, and the incubation time is 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, or 8 h.

[0164] In some implementations, the incubation temperature in step (b) is 0°C to 37°C, 0°C to 25°C, 0°C to 15°C, or 0°C to 5°C; and the incubation time in step (b) is 10 min to 8 h, or 10 min to 4 h. For example, the incubation temperature in step (b) is 0°C, 4°C, 15°C, or 25°C, and the incubation time is 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, or 8 h.

[0165] In some implementations, the incubation temperature in step (d) is 0°C to 37°C, 0°C to 25°C, 0°C to 15°C, or 0°C to 10°C; and the incubation time in step (d) is 0.5h to 5h, or 0.5h to 3h. For example, the incubation temperature in step (d) is 0°C, 4°C, or 15°C, and the incubation time is 0.5h, 1h, 2h, 3h, 4h, or 5h.

[0166] In some embodiments, the incubation temperature in step (e) is 0°C to 37°C, 0°C to 25°C, 0°C to 15°C, or 0°C to 10°C; and the incubation time in step (e) is 0.5h to 5h, or 0.5h to 3h. For example, the incubation temperature in step (e) is 0°C, 4°C, or 15°C, and the incubation time is 0.5h, 1h, 2h, 3h, 4h, or 5h.

[0167] The method described herein for preparing antibody-drug conjugates does not use transglutaminase or enzyme-mediated ligation, simplifying the procedure and saving production costs and time. The method provided herein can produce antibody-drug conjugates with improved uniformity.

[0168] Unbound by any particular theory, homogeneity is improved by increasing the selectivity and / or reduction kinetics of the antibody hinge or Fab region. In some embodiments, the homogeneity of antibody-drug conjugates with a DAR of 4 is primarily improved by increasing the selectivity of the antibody Fab region. In some embodiments, the homogeneity of antibody-drug conjugates with a DAR of 6 is primarily improved by increasing reduction kinetics.

[0169] In some implementations, metal chelators, uncoupled linker-loads, or antibodies can be filtered out in a filtration step to further obtain highly homogeneous antibody-drug conjugates, said filtration including but not limited to dialysis, ultrafiltration, and gel filtration.

[0170] Polynucleotides This disclosure provides a polynucleotide encoding the antibody described above.

[0171] The polynucleotide is a polymer of DNA, RNA, a DNA / RNA hybrid, or a modification thereof. In some embodiments, the polynucleotide is a polymer of DNA. The polynucleotide is a polymer of RNA. The DNA or RNA encoding the antibody described above can be readily isolated and sequenced using conventional procedures, e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody. The encoding DNA or RNA can also be obtained by synthetic methods.

[0172] The isolated polynucleotides described above can be inserted into vectors for further cloning (DNA amplification) or for expression, using recombinant techniques known in the art.

[0173] Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer element, promoter (e.g., SV40, CMV, EF-1α), and transcription termination sequence.

[0174] carrier This document provides a vector comprising the polynucleotide described above. Methods for constructing this vector are known to those skilled in the art. For example, the vector can be obtained by in vitro recombinant DNA technology, DNA synthesis technology, or in vivo recombination technology. More specifically, vectorization can be achieved by inserting isolated polynucleotides into the multiple cloning site of the expression vector. The expression vectors in this disclosure generally refer to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell infection viruses, mammalian cell infection viruses such as adenoviruses, retroviruses, or other vectors. The vector may also include one or more regulatory sequences operatively linked to the polynucleotide sequence, wherein the regulatory sequences may include suitable promoter sequences. The promoter sequence is generally operatively linked to a sequence encoding an amino acid sequence to be expressed. The promoter can be any nucleotide sequence that exhibits transcriptional activity in a selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to the host cell. The regulatory sequences may also include suitable transcription terminator sequences, i.e., sequences recognized by the host cell to terminate transcription. The terminator sequence is attached to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in a selected host cell may be used in this disclosure.

[0175] Typically, a suitable vector may contain at least one effective origin of replication from an organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers. For example, these promoters may include, but are not limited to: the *E. coli* lac or *trp* promoter; the *λ* phage PL promoter; and eukaryotic promoters (including the CMV immediate early promoter, the HSV thymidine kinase promoter, the SV40 early and late promoters, and the *Pichia pastoris* methanol oxidase promoter), as well as other promoters known to control gene expression in prokaryotic, eukaryotic, or viral cells. Marker genes may be used to provide phenotypic characteristics for the selection of transformed host cells. For example, marker genes may include, but are not limited to: dihydrofolate reductase, neomycin resistance, and green fluorescent protein for eukaryotic cell culture, or tetracycline resistance or ampicillin resistance for *E. coli*. When the polynucleotide is expressed, the expression vector may also include an enhancer sequence. If an enhancer sequence is inserted into the vector, transcription will be enhanced. An enhancer is a cis-acting factor of DNA and typically contains approximately 10 to 300 base pairs. Enhancers act on promoters to enhance gene transcription.

[0176] host cells This disclosure provides a host cell comprising the vector provided above or integrating the exogenous polynucleotides provided above into its genome. Any cell suitable for the expression vector can be used as the host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, specifically including but not limited to *Escherichia coli*, *Streptomyces*; bacterial cells of *Salmonella typhimurium*; or fungal cells, such as yeast and filamentous fungi; plant cells; insect cells derived from *Drosophila S2* or SF9; animal cells, such as CHO, COS, HEK293 cells, or Bowes melanoma cells, or combinations thereof. Methods for constructing this expression system should be known to those skilled in the art, including but not limited to microinjection, gene gun method, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, or combinations thereof.

[0177] Reagent test kit This disclosure provides a kit comprising the antibodies, antibody-drug conjugates, polynucleotides, vectors, or host cells described herein. If desired, such kits may also include one or more of a variety of conventional pharmaceutical kit components, such as containers having one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily understood by those skilled in the art. Instructions for use, whether as inserts or labels, indicating the amount of components to be administered, administration guidelines, and / or guidelines for mixing components, may also be included in the kit.

[0178] Pharmaceutical Composition This disclosure relates to a pharmaceutical composition comprising the antibodies, antibody-drug conjugates, polynucleotides, vectors or host cells described above, and pharmaceutically acceptable vectors. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is compatible with other components of a pharmaceutical composition and can be safely administered to a subject. The pharmaceutical compositions disclosed herein comprise a safe and effective amount (e.g., 0.001-99% by weight, preferably 0.01-95% by weight, more preferably 0.1-90% by weight) of an antibody or antibody-drug conjugate and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to) saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of this application can be prepared in injectable form, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms to about 100 milligrams per kilogram of body weight per day. Furthermore, modified antibodies may also be used in combination with other therapeutic agents.

[0179] Given the contents of this disclosure, pharmaceutical compositions and their preparation and application techniques are known to those skilled in the art. For detailed descriptions of suitable pharmaceutical compositions and their application techniques, please refer to relevant literature, such as Remington Pharmaceutical Science (17th edition, 1985) and the works of Brunton et al.

[0180] This pharmaceutical composition may be in any form permissible for administration to a subject. For example, the composition may be in solid or liquid form. Typical routes of administration include, but are not limited to, parenteral, ocular, and intratumoral administration. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, or intrasternal injection or infusion techniques. In one aspect, the composition is administered parenterally. In one specific embodiment, the composition is administered intravenously.

[0181] Therapeutic uses This disclosure provides the use of the antibodies, antibody-drug conjugates, polynucleotides, vectors, host cells, kits, or pharmaceutical compositions described above in the preparation of therapeutic agents for the diagnosis, prevention, and treatment of diseases.

[0182] This disclosure provides a method for preventing, diagnosing, or treating a disease in a subject in need, comprising administering to the subject a therapeutically effective amount of the antibody, antibody-drug conjugate, polynucleotide, vector, host cell, kit, or pharmaceutical composition.

[0183] In some implementations, the disease is a neoplastic disease. In some implementations, the tumor is a solid tumor. Generally, tumors include benign tumors and malignant tumors (also known as cancer).

[0184] The "therapeutic effective dose" in this disclosure preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of injury or disability caused by disease or suffering. For example, in the treatment of tumors (including, for example, melanoma, lymphoma, bladder cancer, non-small cell lung cancer, head and neck cancer, and colon cancer), the "therapeutic effective dose" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80% relative to an untreated subject. The ability to inhibit tumor growth can be assessed in an animal model system for predicting the efficacy of treatment for human tumors, or by detecting the ability to inhibit cell growth. This inhibitory effect can be determined in vitro by assays well known to those skilled in the art. Those skilled in the art can select an appropriate therapeutic effective dose based on specific circumstances, such as the size of the subject's tumor, the severity of the subject's symptoms, and the particular composition or route of administration chosen. The treatment regimen (e.g., the determination of dosage, etc.) can be determined by a physician, generally considering factors including but not limited to the disease being treated, the patient's condition, the site of delivery, the route of administration, and other factors.

[0185] The subjects were mammals, such as humans, mice, and cynomolgus monkeys.

[0186] Example The invention is further described in the following examples, which are not intended to limit the scope of the invention as described in the claims.

[0187] Unless otherwise stated, the reagents used in the examples are commercially available.

[0188] Uniformity test The distribution of ADCs was analyzed using HIC-HPLC (Agilent 1200) and a TSK gel butyl-NPR column (4.6 mm × 3.5 cm inner diameter) (purchased from Tosoh Biosciences) at 30°C and a flow rate of 0.5 mL / min. Solvent A consisted of 1.5 M (NH4)2SO4 and 50 mM potassium phosphate, pH 7.0. Solvent B consisted of 75% v / v 50 mM potassium phosphate (pH 7.0) and 25% v / v isopropanol. The washing procedure was as follows:

[0189] The distribution of the ADC is calculated based on the peak area.

[0190] Example 1 Example 1-1 (a) In the buffer system BES (20 mM, pH 7.0), in Zn 2+ TCEP (2.4 equivalents, 0.04 mM) and mutant 04 (0.0167 mM) were incubated at 4°C for 4 h in the presence of (30 equivalents, 0.5 mM). (b) Add EDTA (80 equivalents, 1.34 mM) and drug / linker-loading MC-VA-PAB-MMAE (7 equivalents, 0.1169 mM, purchased from Levena Biopharma) and react with the reduced thiol group generated in step (a) at room temperature (20°C ~ 25°C) for 1 h. (c) The resulting antibody-drug conjugate was then recovered and purified using a desalting column (Thermo, model: 40K, 0.5 mL, catalog number: 87766, batch number: SJ251704).

[0191] Examples 1-2 to 1-16 The methods in Examples 1-2 to 1-8 are similar to those in Example 1-1, except that the reaction conditions are adjusted. See Table 1-1 for details.

[0192] Comparative Examples 1-1 to 1-2: The methods used in Comparative Examples (CE) 1-1 and 1-2 are similar to those in Example (E) 1-1, except that in Comparative Example 1-1, antibody mutant 04 was replaced with wild-type trastuzumab, and in Comparative Example 1-2, antibody mutant 04 was replaced with mutant 11. See Table 1-1 for details.

[0193] Table 1-1

[0194] The uniformity test results of Examples 1-1 to 1-8 and Comparative Examples 1-1 to 1-2 are shown in Table 1-2 below. Table 1-2

[0195] The results showed that the ADC ratio of all DAR4s was improved, demonstrating excellent DAR4 selectivity.

[0196] Compared to CE1-1, the proportion of DAR4ADCs prepared from antibodies containing amino acid substitutions at positions 221, 222, 223, 224, and 225 was significantly increased. Furthermore, the D221H mutation contributes to improved disulfide bond reduction selectivity compared to CE1-2, E1-2, E1-4, E1-5, or E1-8.

[0197] Compared to E1-1, the results of E1-11 to E1-16 show that the proportion of DAR4 ADCs prepared from antibodies containing the amino acid sequences HKTDTCPPC (SEQ ID NO: 17), HKTSDCPPC (SEQ ID NO: 14), HKDSTCPPC (SEQ ID NO: 15), and HKTETCPPC (SEQ ID NO: 16) is at least 90%, with some even reaching 96%. This indicates that the D221H mutation and the mutation at position 224 to D, E, or S significantly improve the reduction selectivity of disulfide bonds, thereby enhancing the homogeneity of the ADC. Furthermore, antibodies containing the D221H mutation and the mutation at position 224 to D or E also exhibit excellent DAR4 selectivity, suggesting that the acidic amino acid residue at position 224 is crucial for producing high DAR4 selectivity.

[0198] As shown in the results of E1-11, E1-12 and E1-15, HKTDTCPPC (SEQ ID NO: 17) is suitable for three different IgG1 antibodies to produce ADCs with excellent and consistent ultra-high DAR4 selectivity.

[0199] Examples 1-17 to 1-21 The methods in Examples 1-17 to 1-21 are similar to those in Example 1-1, except that the reaction conditions are adjusted. See Tables 1-3 and 1-4 for details.

[0200] Table 1-3

[0201] Table 1-4

[0202] According to the results of E1-17, when the antibody contains a mutation at position 229, i.e. -CPPC- becomes -CPPX-, and X is Val, Leu, Ile or Phe, the ultra-high DAR4 selectivity is maintained; however, when X is Ser, the DAR4 selectivity drops to 80%.

[0203] Comparative Examples 1-3 (a) TCEP (2.4 equivalents, 0.04 mM) and mutant 04 (0.0167 mM) were incubated in a BES buffer system (20 mM, pH 7.0) at 4°C for 4 h. (b) Add EDTA (2 equivalents, 0.0334 mM) and drug / linker-loading MC-VA-PAB-MMAE (7 equivalents, 0.1169 mM), and react with the reduced thiol group generated in step (a) at room temperature for 1 h. (c) The resulting antibody-drug conjugate was then recovered and purified using a desalting column (Thermo, model: 40K, 0.5 mL, catalog number: 87766, batch number: SJ251704).

[0204] The results of the homogeneity test are shown in Table 1-5 below.

[0205] Table 1-5

[0206] Compared to E1-1, the Zn in the buffer solution 2+ This is beneficial for improving reduction selectivity and ADC uniformity. However, the DAR4 ADC ratio of CE1-1 was only 44.53%, lower than that of CE1-3, indicating that antibodies containing HKTHTCPPC (SEQ ID NO: 11) help improve the reduction selectivity and uniformity of antibody-drug conjugates, even in the absence of Zn. 2+ The same applies to the buffer solution. Examples 1-22 Preparation of mutant 25-D2 (bismaleimide-PEG4-VC-MMAE) (a) TCEP (2.1 equivalent, 0.03 mM) and mutant 25 (0.0135 mM) were mixed in Zn 2+ Incubate at 25°C for 10 min in a BES buffer system (20 mM, pH 7.0) in the presence of (30 equivalents, 0.4 mM); (b) Add EDTA (80 equivalents, 1.08 mM) and bismaleimide-PEG4-VC-MMAE (7 equivalents, 0.095 mM), and react with the reduced thiol groups generated in step (a) at room temperature for 1 h. (c) The resulting antibody-drug conjugate was then recovered and purified using a desalting column (Thermo, model: 40K, 0.5 mL, catalog number: 87766, batch number: SJ251704). The results of the homogeneity test are shown in Table 1-6 below.

[0207] Table 1-6

[0208] This antibody is suitable for producing ADCs using a sulfur-bridged reagent containing MMAE, and produces highly homogeneous ADCs.

[0209] Example 2 Example 2-1 (a) TCEP (2.3 equivalents, 0.038 mM) and mutant 04 (0.0167 mM) were incubated in a BES buffer system (20 mM, pH 7.0) in the presence of ZnCl2 (30 equivalents, 0.5 mM) for 10 min at 4°C. (b) Add EDTA (80 equivalents, 1.34 mM) and drug / linker-loading MC-VA-PAB-MMAE (7 equivalents, 0.1169 mM), and react with the reduced thiol group generated in step (a) at room temperature for 1 h. (c) The resulting antibody-drug conjugate was then recovered and purified using a desalting column (Thermo, model: 40K, 0.5 mL, catalog number: 87766, batch number: SJ251704).

[0210] Examples 2-2 to 2-28 The methods in Examples 2-2 to 2-28 are similar to those in Examples 1-7, except that the reaction conditions are adjusted. See Table 2-1 for details.

[0211] Table 2-1

[0212] The results of the homogeneity test are shown in Table 2-2 below.

[0213] Table 2-2

[0214] As shown in the table, the proportion of ADCs with a DAR of 4 reached a maximum of 70% within 10 minutes, and then plateaued after 10 minutes. These results demonstrate that the method for producing antibody-drug conjugates is simple, time-saving, and efficient.

[0215] In addition, Zn 2+ A Zn / mAb ratio in the range of 1 to 100 is beneficial for antibody-drug conjugates with a DAR of 4. 2 + When the / mAb ratio is as high as 30:1 and 100:1, the ADC ratio of DAR4 is significantly higher than that of Zn. 2+ When the / mAb ratio is 1:1 and 10:1, these results indicate that higher Zn content is possible within a certain range. 2+ The / mAb ratio will improve the ADC ratio and reduction selectivity of DAR4.

[0216] In addition, the reaction temperature of this method is 0°C to 25°C, which helps to reduce production costs.

[0217] Example 3 Examples 3-1 to 3-18 The methods in Examples 3-1 to 3-12 are similar to those in Example 1-1, except that the reaction buffer in step (a) is adjusted. The methods in Examples 3-13 to 3-18 are similar to those in Example 1-1, except that the TCEP / mAb ratio is adjusted to 2.3, and the reaction buffer in step (a) is adjusted. See Table 3-1 for details.

[0218] Comparative Example 3-1 The method of Comparative Example 3-1 is similar to that of Example 1-1, except that the reaction buffer in step (a) is adjusted. Table 3-1

[0219] The results showed that the proportion of ADCs with a DAR of 4 was greater than 60%, and could reach as high as 80%, indicating good reducing selectivity in most types of biological buffers.

[0220] Examples 3-19 to 3-24 The methods in Examples 3-19 to 3-24 are similar to those in Examples 1-7, except that the pH of the buffer solution was adjusted. See Table 3-2 for details.

[0221] Table 3-2

[0222] The results of the homogeneity test are shown in Table 3-3 below.

[0223] Table 3-3

[0224] When the pH range of the MES buffer was between 5.8 and 6.7 or the pH range of the BES buffer was between 6.4 and 7.4, the ADC ratio of DAR4 was observed to be 75% or higher; at pH 6.4, the ADC ratio of DAR4 reached as high as 91%. These results indicate that an appropriate pH range of the reaction buffer helps to improve the ADC ratio and reduction selectivity of DAR4, thereby improving homogeneity.

[0225] Example 4 The methods in Examples 4-1 to 4-7 are similar to those in Example 1-1, except that the reducing agent in step (a) and the molar ratio of the reducing agent to antibody mutant O4 are adjusted. See Table 4-1 for details.

[0226] Table 4-1

[0227] The results of the homogeneity test are shown in Table 4-2 below.

[0228] Table 4-2

[0229] As shown in the table, all reducing agents TCEPNO, TCEP, and TCEP3 produced antibody-drug conjugates with improved homogeneity. When TCEP and TCEP3 were used as reducing agents, the proportion of ADCs with a DAR of 4 was significantly higher than when TCEPNO was used.

[0230] Example 5 The methods in Examples 5-1 to 5-2 are similar to those in Example 1-1, except that the transition metal salt and its molar equivalent relative to the antibody in step (a) are adjusted. The transition metal salt is replaced with Mn(OAc)2. See Table 5-1 for details.

[0231] Table 5-1

[0232] The results of the homogeneity test are shown in Table 5-2 below.

[0233] Table 5-2

[0234] The results show that Mn 2+ It also helps improve the uniformity of antibody-drug conjugates.

[0235] Example 6 Example 6-1 Preparation of mutant 4-D4(MC-VC-PAB-MMAE)+D2(MC-GGFG-DXd) (a) In Zn 2+ In the presence of 30 equivalents (0.5 mM), TCEP (2.3 equivalents, 0.0384 mM) and mutant 04 (0.0167 mM) were incubated at 4°C for 4 h in a BES buffer system (20 mM, pH 7.0). (b) Add EDTA (80 equivalents, 1.34 mM) and drug / linker-loading MC-VC-PAB-MMAE (7 equivalents, 0.1169 mM, purchased from Levena Biopharma), and react with the reduced thiol group generated in step (a) at room temperature for 1 h. (c) The resulting antibody-drug conjugate was then recovered and purified using a desalting column (Thermo, model: 40K, 0.5 mL, catalog number: 87766, batch number: SJ251704).

[0236] (d) The product from step (c) is reacted with TCEP (4.5 equivalents, 0.075 mM) in Zn2+ Incubation at 4°C for 4 h in a BES buffer system (20 mM, pH 7.0) in the presence of (30 equivalents, 0.3 mM) (e) Add EDTA (80 equivalents, 1.34 mM) and drug / linker-loading MC-GGFG-DXd (8 equivalents, 0.135 mM, purchased from Levena Biopharma), and react with the reduced thiol group generated in step (a) at room temperature for 1 h. (f) The resulting antibody-drug conjugate was then recovered and purified using a desalting column (Thermo, model: 40K, 0.5 mL, catalog number: 87766, batch number: SJ251704).

[0237] The homogeneity test results of the products in steps (c) and (f) are shown in Tables 6-1 and 6-2, respectively.

[0238] Table 6-1

[0239] Table 6-2

[0240] The results showed that the proportion of antibody-drug conjugate mutant 04-[MC-VC-PAB-MMAE]4 was as high as 79%, and the proportion of dual-load ADCs with DAR 4+2 was typically as high as 76%. Antibodies containing HKTHTCPPC (SEQ ID NO:11) are suitable for preparing dual-load ADCs and produce highly homogeneous dual-load ADCs.

[0241] Example 6-2 Preparation of mutant 4-D2 (dibromomaleimide-PEG4-VC-PAB-MMAE)+D4 (MC-GGFG-DXd) (a) In Zn 2+ In the presence of 30 equivalents (0.5 mM), TCEP (2.3 equivalents, 0.0384 mM) and mutant 04 (0.0167 mM) were incubated at 4°C for 4 h in a BES buffer system (20 mM, pH 7.0). (b) Add EDTA (80 equivalents, 1.34 mM) and drug / linker-loaded dibromomaleimide-PEG4-VC-PAB-MMAE (7 equivalents, 0.1169 mM), and react with the reduced thiol group generated in step (a) at room temperature for 1 h. (c) The resulting antibody-drug conjugate was then recovered and purified using a desalting column (Thermo, model: 40K, 0.5 mL, catalog number: 87766, batch number: SJ251704).

[0242] (d) The product from step (c) is reacted with TCEP (4 equivalents, 0.067 mM) in Zn 2+ In the presence of (30 equivalents, 0.3 mM), incubated at 37°C for 3.5 h in a BES buffer system (20 mM, pH 7.0). (e) Add EDTA (80 equivalents, 1.34 mM) and drug / load MC-GGFG-DXd (8 equivalents, 0.134 mM, purchased from Levena Biopharma), and react with the reduced thiol group generated in step (a) at room temperature for 1 h. (f) The resulting antibody-drug conjugate was then recovered and purified using a desalting column (Thermo, model: 40K, 0.5 mL, catalog number: 87766, batch number: SJ251704).

[0243] Dibromomaleimide-PEG4-VC-PAB-MMAE was prepared according to PCT patent applications WO2022167689 or WO2021144314. The homogeneity determination results of the products in steps (c) and (f) are shown in Tables 7-1 and 7-2, respectively.

[0244] Table 7-1

[0245] Table 7-2

[0246] The results showed that when the reducing agent was TCEP, the proportion of ADCs with a DAR of 2+4 was typically as high as 75%. Antibodies containing HKTHTCPPC (SEQ ID NO:11) are suitable for preparing dual-load ADCs with different DAR values.

[0247] Example 7 Preparation of mutant 25-D4(MC-VC-PAB-MMAE)+D4(MC-GGFG-DXd) (a) In Zn 2+ In the presence of 30 equivalents (0.5 mM), TCEP (3.0 equivalents, 0.0501 mM) and mutant 25 (0.0167 mM) were incubated at 4°C for 2 h in a BES buffer system (20 mM, pH 7.0). (b) Add EDTA (80 equivalents, 1.34 mM) and drug / linker-loading MC-VC-PAB-MMAE (8 equivalents, 0.135 mM), and react with the reduced thiol group generated in step (a) at room temperature for 1 h. (c) The resulting antibody-drug conjugate was then recovered and purified using a desalting column (Thermo, model: 40K, 0.5 mL, catalog number: 87766, batch number: SJ251704).

[0248] (d) The product from step (c) is reacted with TCEP (3.0 equivalent, 0.0501 mM) in Zn 2+ In the presence of (100 equivalents, 1.67 mM), incubated at 37°C for 4 h in a BES buffer system (20 mM, pH 7.0). (e) Add EDTA (230 equivalents, 3.84 mM) and drug / load MC-GGFG-DXd (8 equivalents, 0.134 mM), and react with the reduced thiol group generated in step (a) at room temperature for 1 h. (f) The resulting antibody-drug conjugate was then recovered and purified using a desalting column (Thermo, model: 40K, 0.5 mL, catalog number: 87766, batch number: SJ251704).

[0249] The results of the homogeneity test are shown in Table 7-3 below.

[0250] Table 7-3

[0251] The results showed that when the reducing agent was TCEP, the proportion of ADCs with D4+D4 was typically as high as 87%. Antibodies containing HKTDTCPPC (SEQ ID NO:17) are suitable for preparing dual-load ADCs.

[0252] Example 8 Example 8-1: (a) In a BES buffer system (20 mM, pH 7.0), at 4°C, TCEP (4.5 equivalents, 0.0752 mM) and mutant O4 (0.0167 mM) were added to Zn... 2+ Incubate for 1 hour in the presence of (30 equivalents, 0.5 mM) (b) Add EDTA (80 equivalents, 1.34 mM) and drug / linker-loading MC-VC-PAB-MMAE (7 equivalents, 0.1169 mM), and react with the reduced thiol group generated in step (a) at room temperature for 1 h. (c) The resulting antibody-drug conjugate was then recovered and purified using a desalting column (Thermo, model: 40K, 0.5 mL, catalog number: 87766, batch number: SJ251704).

[0253] Examples 8-2 to 8-17 The methods in Examples 8-2 to 8-17 are similar to those in Example 8-1, except that the antibody and reduction conditions are adjusted. See Table 8-1 for details.

[0254] Comparative Example 8-1 The method for Comparative Example 8-1 is similar to that of Example 8-1, except that the antibody and reduction conditions were adjusted. See Table 8-1 for details.

[0255] Table 8-1

[0256] The results of the homogeneity test are shown in Table 9-2 below.

[0257] Table 9-2

[0258] Compared to CE8-1, the proportion of all ADCs with a DAR of 6 was increased. Antibodies containing the amino acid sequences HKTHTCPPC (SEQ ID NO:11), DKTHRCPPC (SEQ ID NO:28), SKTHTCPPC (SEQ ID NO:27), or HSTHTCPPC (SEQ ID NO:13) help improve reduction selectivity, thereby enhancing the homogeneity of the ADC.

[0259] The results of E8-1 to E8-9 show that when the reaction time of step (1) increases from 1 h to 4 h, the proportion of ADCs with a DAR of 6 increases accordingly, and reaches a plateau after 4 h at 4°C or 25°C, thereby shortening the production time of the ADC. As shown in the results of E8-1, E8-12, and E8-13, the amino acid sequence HKTHTCPPC (SEQ ID NO:11) is suitable for three different IgG1 antibodies to produce ADCs with good and consistent high DAR6 selectivity.

[0260] The results from E8-15 to E8-17 indicate that, in BES buffer, antibody-based Zn 2+ Molar equivalents can affect the proportion of ADCs with a DAR of 6. When antibody-based Zn... 2+ When the molar equivalent is 10, the proportion of ADCs with a DAR of 6 can reach a maximum of 85%. Subsequently, the proportion of ADCs with a DAR of 6 decreases with increasing Zn content. 2+ The decrease is due to the reduction.

[0261] While specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are currently unforeseeable or potentially unforeseeable may arise to the applicant or others skilled in the art. Therefore, the appended claims, both submitted and possibly modified, are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. An antibody comprising a hinge region, wherein the hinge region comprises the amino acid sequence X1X2X3X4X5CPPX6, wherein X1 is D or H; X2 is E, D, K, or S; X3 is E, T, H, or D; X4 is T, H, D, S, or E; X5 is E, H, T, or D; and X6 is G, A, H, D, E, Y, C, S, V, L, I, or F; or The hinge region contains the amino acid sequence X. 11 X 12 THX 15 CPPC (SEQ ID NO: 30), where X 11 For H, D, or S; X 12 For K or S, X 15 For T or R; The X 11 X 12 THX 15 The amino acid sequence of CPPC or X1X2X3X4X5CPPX6 is not DKTHTCPPC (SEQ ID NO: 26).

2. The antibody according to claim 1, wherein... The X1X2X3X4X5CPPX6 is selected from any one of the following groups: (1) X1 is D or H; X2 is D, K or S; X3 is T, H or D; X4 is T, H, D, S or E; X5 is H, T or D; X6 is C, S, V, L, I or F; (2) X1 is H; X2 is D, K or S; X3 is T, H or D; X4 is T, H, D, S or E; X5 is H, T or D; X6 is C, S, V, L, I or F; (3) X1 is H; X2 is D, K or S; X3 is T, H or D; X4 is D; X5 is H, T or D; X6 is G, A, H, D, E, Y, C, S, V, L, I or F; (4) X1 is H; X2 is D, K or S; X3 is T, H or D; X4 is D; X5 is H, T or D; X6 is C, S, V, L, I or F; (5) X1 is H; each X2, X3 and X5 is independently E; (6) X1 is H; X4 is D; X6 is G, A, H, D, E, Y, C, S, V, L, I or F; (7) X1 is D; X2 is K; X3 is T or H; X4 is T or H; X5 is H, T or D; X6 is C; The X 11 X 12 THX 15 CPPC is selected from any of the following groups: (1) X 11 For H or S; X 15 Let T be the value of T. (2) X 11 For D; X 15 Let R be the value.

3. The antibody according to claim 1 or 2, wherein the X1X2X3X4X5CPPX6 is selected from any one of the following groups: X1 is H; X4 is D, S or E; Optionally, X1 is H; X3 is T or D; X4 is D, S or E; X5 is T or D; Optionally, X1 is H; X2 is K; X3 is T or D; X4 is D, S or E; X5 is T or D; Optionally, X1 is H; X2 is K; X3 is T; X4 is D, S, or E; X5 is T or D; Optionally, X1 is H; X2 is K; X3 is T; X4 is D or E; X5 is T; Optionally, X1 is H; X2 is K; X3 is T; X4 is D or E; X5 is T; X6 is S, V, L, I or F; Optionally, X1 is H; X2 is K; X3 is T; X4 is D or E; X5 is T; X6 is V, L, I or F; Optionally, X1 is H; X2 is D; and X3 is S.

4. The antibody according to any one of claims 1-3, wherein the X1X2X3X4X5CPPX6 has an amino acid sequence of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29 or 31; optionally, the X1X2X3X4X5CPPX6 has an amino acid sequence of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 29, 31 or a conserved substituted amino acid sequence thereof; The X 11 X 12 THX 15 CPPC has the amino acid sequence of SEQ ID NO: 11, 13, 27, 28 or its conservatively substituted amino acids.

5. The antibody according to any one of claims 1-4, wherein the antibody comprises a heavy chain constant region comprising one or more amino acid substitutions selected from the group consisting of: K218H, D221H, K222S, K222D, T223H, T223D, H224S, H224D, H224E, H224T, T225H, T225R, T225D, C229S, C229V, C229L, C229I, C229E or combinations thereof, wherein the first amino acid at the N-terminus of SEQ ID NO: 1 is defined as position 118 compared to SEQ ID NO: 1; Optionally, the heavy chain constant region comprises amino acid substitutions from the following groups: (1) D221H; (2) D221H and T225D; (3) D221H and K222S; (4) D221S, H224S and T225D; (5) D221H, T223D and H224S; (6) D221H and H224E; (7) D221H and H224D; (8) D221H, H224D and C229S; (9) D221H, H224D and C229V; (10) D221H, H224D and C229L; (11) D221H, H224D and C229I; (12) D221H, H224D and C229E; (13) K218H, D221H and H224S; (14) T225R; (15) D221H and K222D; (16) D221H and H224S; or (17) D221H, K222D and H224S.

6. The antibody according to any one of claims 1-4, wherein the antibody comprises an antigen-binding fragment capable of binding to one or more target antigens. Optionally, the target antigen is expressed on tumor cells or is an immune checkpoint; Optionally, the target antigen is selected from the group consisting of: EGFR, HER2, CD3, calcium channels, PDL1, CD19, VEGFR, GLP1R, DRD2, NMDAR, bacterial DNA gyrase, PD1, VGSC, COX2, TOP2, VSIG4, B7-H3, B7-H4, ROR1, ROR2, HER3, c-MET, CEACAM5, CD25, CD71, CD70, CD74, CD38, BCMA, TROP2, CD79b, CLDN6, Claudin18.2, mesothelin, 5T4, LIV-1, PSMA, PD-L1, CD142, MUC-1, MUC-16. DLL3, CDH6, CDH3, FGFR2, NaPi2b, FGFR3, GPRC5D, LRRC15, PTK7, STn, CD117, CD45, Ly6E, BTN3A1, GPR65, GABA, DDR1, ART1, ARIH1, KDM5B, CD155, CD200R1, LIF, ESCRT, LILRB, CD161, SETB1, COP1, CLIP1-LTK, LSD1, METTL3, TAM, BCAM, NSD3, PSGL-1, IL8, PTPN2, CCR8, SOX4, ELANE, FOLR, CDH17, Nectin 4, TNK1 or combinations thereof; Optionally, the antigen-binding fragment targets HER2, FOLR, CDH17, DLL3, or a combination thereof.

7. An antibody-drug conjugate comprising an antibody according to any one of claims 1-6 and at least one linker-loader, optionally having a drug-antibody ratio (DAR) of about 2 to 8, or 2 to 6.

8. The antibody-drug conjugate of claim 7, wherein the linker-load comprises a first linker-load and / or a second linker-load, the first linker-load and the second linker-load each independently comprising at least one thiol reactive group or a thiobridging agent.

9. The antibody-drug conjugate according to claim 7 or 8, wherein the first linker-load and the second linker-load each independently comprise a load, the load comprising a cytotoxic agent, a marker, a nucleic acid, a radionuclide, a hormone, an immunomodulator, a prodrug converting enzyme, a ribonuclease, an agonist antibody, an antagonist antibody and fragments thereof, a fusion protein or a derivative thereof, or a combination thereof; Optionally, the first connector-load and the second connector-load each independently contain a cytotoxic agent.

10. The antibody-drug conjugate according to any one of claims 7-9, wherein the first linker-loader and the second linker-loader are each independently MC-VC-PAB-MMAE, MC-VA-PAB-MMAE, MC-GGFG-DXd, MC-VC-PAB-MMAD, MC-VC-PAB-Eribulin, MC-MMAF or MC-VC-PAB-MMAF, dibromomaleimide-PEG4-VC-PAB-MMAE, dibromomaleimide-PEG4-VC-PAB-MMAF, bismaleimide-PEG2-VC-MMAE, bismaleimide-PEG4-VC-MMAE, bismaleimide-PEG4-VC-DX8951f, bismaleimide-PEG4-VC-Eribulin, bismaleimide-PEG2-VC-Eribulin, bismaleimide-PEG2-MMAF or bismaleimide-PEG4-MMAF; Optionally, the first connector-load and the second connector-load are each independently MC-VC-PAB-MMAE, MC-GGFG-DXd, or dibromomaleimide-PEG4-VC-PAB-MMAE.

11. The antibody-drug conjugate according to any one of claims 7-10, wherein the DAR of the antibody-drug conjugate is about 4, and the proportion of antibody-drug conjugates with a DAR of about 4 is up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or higher; or The DAR of the antibody-drug conjugate is approximately 2, meaning that the proportion of ADCs with a DAR of 2 carrying the linker-load via the sulfur-bridged reagent can reach up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 90%, or higher; or The DAR of the antibody-drug conjugate is approximately 6, and the proportion of antibody-drug conjugates with a DAR of approximately 6 can reach up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90% or higher; or The antibody-drug conjugate has a DAR of approximately 3, meaning that the proportion of antibody-drug conjugates with a DAR of approximately 3 via a sulfur-bridged reagent can reach up to 60%, optionally up to 65%, 70%, 72%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, or higher; or The antibody-drug conjugate is a dual-load type with a DAR of approximately 2+4, i.e., carrying a first linker-load with a DAR of approximately 2 and a second linker-load with a DAR of approximately 4. The proportion of antibody-drug conjugates with a DAR of 2+4 can be up to 50%, optionally up to 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or higher; or The antibody-drug conjugate is a dual-load type with a DAR of approximately 4+2, i.e., carrying a first linker-load with a DAR of approximately 4 and a second linker-load with a DAR of approximately 2. The proportion of antibody-drug conjugates with a DAR of 4+2 can reach up to 60%, optionally up to 65%, 66%, 67%, 68%, 69%, 70%, 75% or higher; or The antibody-drug conjugate is a dual-load type with a DAR of approximately 4+4, meaning it carries a first linker-load with a DAR of approximately 4 and a second linker-load with a DAR of approximately 4. The proportion of antibody-drug conjugates with a DAR of 4+4 can reach up to 60%, optionally up to 65%, 70%, 75%, 80%, 85%, 90%, or higher; or The antibody-drug conjugate is a dual-load type with a DAR of approximately 6+2, that is, carrying a first linker-load with a DAR of approximately 6 and a second linker-load with a DAR of approximately 2. The proportion of antibody-drug conjugates with a DAR of 6+2 can be up to 60%, and optionally up to 65%, 70%, 75%, 80%, 85%, 90% or higher.

12. A method for producing an antibody-drug conjugate (ADC) according to any one of claims 7-11, comprising the following steps: (a) In a first reaction buffer, in the presence of a first transition metal ion, the antibody according to any one of claims 1-6 is incubated with a first reducing agent to reduce at least one interchain disulfide bond of the antibody. (b) Introducing a first metal chelating agent and a first linker-loading agent to react with the reduced thiol group generated in step (a). (c) The mixture from step (b) is purified to obtain the product ADC.

13. The method of claim 12, further comprising: (d) In a second reaction buffer, in the presence of a second transition metal ion, the ADC from step (c) is incubated with a second reducing agent to reduce at least one remaining interchain disulfide bond of the ADC. (e) Introduce a second linker-load to react with the reduced thiol group generated in step (d). (f) The mixture from step (e) is purified to obtain the dual-loaded ADC product.

14. The method according to claim 12 or 13, wherein the first transition metal ion and the second transition metal ion are each independently Zn. 2+ and / or Mn 2+ ; Optionally, the Zn 2+ and / or Mn 2+ Salts or complexes derived from the transition metal ions, optionally including lactates, chlorides, nitrates, sulfates, acetates, iodides, bromides, formates, and tetrafluoroborates; or The concentration of the first transition metal ion in the reaction buffer is 0.01 mM - 2 mM, optionally 0.1 mM - 2 mM; and / or, the concentration of the second transition metal ion in the reaction buffer is 0.01 mM - 2 mM, optionally 0.1 mM - 2 mM; or The molar ratio of the first transition metal ion to the antibody is (1-100):1, optionally (10-100):1; and / or, the molar ratio of the second transition metal ion to the antibody is (1-100):1, optionally (10-100):

1.

15. The method according to any one of claims 12-14, wherein the first reducing agent and the second reducing agent are each independently TCEP, THPP, TCEPNO, TCEP3, TCEP1, TCEP26, TCEP28 or TCEP33, and the structure of the reducing agent is as follows: 、 、 、 、 、 ； Optionally, the first reducing agent is TCEP, and the second reducing agent is either TCEP or THPP. Optionally, the concentrations of the first reducing agent and the second reducing agent are each independently 0.02 mM - 0.2 mM, or optionally 0.02 mM - 0.1 mM.

16. The method according to any one of claims 12-15, wherein the molar ratio of the first reducing agent to the antibody is (2-8):1 to produce an ADC with a DAR of 4, optionally the ratio being (2-7):1, (2-5):1, or (2-3):1; or The molar ratio of the first reducing agent to the antibody is (2-8):1, to produce an ADC with a DAR of 2 via the sulfur-bridged reagent. Optionally, the ratio is (2-7):1, (2-5):1, or (2-3):

1. The molar ratio of the first reducing agent to the antibody is (3.5-7):1 to produce an ADC with a DAR of 6; optionally, the ratio is (4-6):1 or (4-5):1; or The molar ratio of the first reducing agent to the antibody is (3.5-7):1, to produce an ADC with a DAR of 3 via the sulfur-bridged reagent; optionally, the ratio is (4-6):1 or (4-5):1; or The molar ratio of the first reducing agent to the antibody is (2-7):1 or (2-5):1, and the molar ratio of the second reducing agent to the antibody is (1-7):1 or (1-5):1, to produce an ADC with a DAR of approximately 4+2; The molar ratio of the first reducing agent to the antibody is (2-7):1 or (2-5):1, and the molar ratio of the second reducing agent to the antibody is (2-9):1 or (2-7):1, to produce an ADC with a DAR of approximately 2+4; The molar ratio of the first reducing agent to the antibody is (2-7):1, and the molar ratio of the second reducing agent to the antibody is (2-9):1 or (2-7):1, to produce an ADC with a DAR of approximately 4+4; or The molar ratio of the first reducing agent to the antibody is (3-7):1, and the molar ratio of the second reducing agent to the antibody is (1-9):1 or (1-7):1, to produce an ADC with a DAR of approximately 6+2.

17. The method according to any one of claims 12-16, wherein the first reaction buffer and the second reaction buffer are each independently PB, PBS, acetate buffer, His buffer, Bis-Tris buffer, PIPES buffer, ADA, HEPES buffer, MOPS buffer, MOBS buffer, DIPSO buffer, MOPSO buffer, TES buffer, TAPSO buffer, ACES buffer, BES buffer, or MES buffer. Optionally, the pH values ​​of the first reaction buffer and the second reaction buffer are each independently 5.5 to 8, and optionally 5.8 to 7.

4.

18. The method according to any one of claims 12-17, wherein the first metal chelating agent and the second metal chelating agent are each independently EDTA or DTPA. Optionally, the molar ratio of the first metal chelating agent to the antibody is (30-250):1 or (80-250):1; and / or, the molar ratio of the second metal chelating agent to the antibody is (30-250):1 or (80-250):

1.

19. The method according to any one of claims 12-18, wherein The molar ratio of the linker-load to the antibody is (2-8):1, (2-7):1, or (3-7):1 to produce an ADC with a DAR of 4; The molar ratio of the linker-load to the antibody is (1-5):1, (1.5-4.5):1, or (2-5):1, to produce an ADC with a DAR of 2 using the sulfur-bridged reagent; The molar ratio of the linker-load to the antibody is (6-12):1 to produce an ADC with a DAR of 6; The molar ratio of the linker-load to the antibody is (3-6):1 or (3.5-5.5):1, to produce an ADC with a DAR of 3 using the sulfur-bridged reagent; The molar ratio of the first linker-load to the antibody is (4-8):1, and the molar ratio of the second linker-load to the antibody is (2-8):1, to produce an ADC with a DAR of 4+2; The molar ratio of the first linker-load to the antibody is (2-8):1, and the molar ratio of the second linker-load to the antibody is (4-12):1, to produce an ADC with a DAR of 2+4; The first linker-load to antibody molar ratio is (4-8):1, and the second linker-load to antibody molar ratio is (4-12):1, to produce an ADC with a DAR of 4+4; and / or The first linker-load to antibody molar ratio is (6-12):1, and the second linker-load to antibody molar ratio is (2-8):1, to produce an ADC with a DAR of 6+2.

20. The method according to any one of claims 12-19, wherein The incubation temperature in step (a) or (b) is independently 0°C to 37°C, 0°C to 25°C, 0°C to 15°C, or 0°C to 5°C; and / or, the incubation time in step (a) or (b) is independently 10 min to 8 h, or 10 min to 4 h; or The incubation temperature in step (d) or (e) is independently 0°C to 37°C, 0°C to 25°C, 0°C to 15°C, or 0°C to 10°C; and / or the incubation time in step (d) or (e) is independently 0.5h to 5h, or 0.5h to 3h.

21. The method according to any one of claims 12-20, wherein the homogeneity of the antibody-drug conjugate is improved. Optionally, the homogeneity of the antibody-drug conjugate is improved by increasing the selectivity of the antibody Fab region and / or reducing reaction kinetics.

22. A polynucleotide encoding an antibody according to any one of claims 1-6.

23. A vector comprising the polynucleotide according to claim 22.

24. A host cell comprising the polynucleotide of claim 22 or the vector of claim 23.

25. A kit comprising an antibody according to any one of claims 1-6, an antibody-drug conjugate according to any one of claims 7-11, a polynucleotide according to claim 22, a vector according to claim 23, or a host cell according to claim 24.

26. A pharmaceutical composition comprising an antibody according to any one of claims 1-6, an antibody-drug conjugate according to any one of claims 7-11, an antibody-drug conjugate prepared by the method according to any one of claims 12-21, and at least one pharmaceutically acceptable carrier.

27. Use of the antibody according to any one of claims 1-6, the antibody-drug conjugate according to any one of claims 7-11, the antibody-drug conjugate prepared by the method according to any one of claims 12-21, the polynucleotide according to claim 22, the carrier according to claim 23, the host cell according to claim 24, the kit according to claim 25, and the pharmaceutical composition according to claim 26 in the preparation of therapeutic agents for the diagnosis, prevention, and treatment of diseases.

28. A method for preventing, diagnosing, or treating a disease in a subject in need, comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1-6, an antibody-drug conjugate according to any one of claims 7-11, an antibody-drug conjugate prepared by the method according to any one of claims 12-21, a polynucleotide according to claim 22, a carrier according to claim 23, a host cell according to claim 24, a kit according to claim 25, or a pharmaceutical composition according to claim 26.

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