Method for selectively preparing antibody-drug conjugate

Abstract

Provided is a method for selectively preparing an antibody-drug conjugate, comprising a step of selectively reducing the interchain disulfide bond of one of the two Fab regions of an antibody to a sulfhydryl group. The molar quantity of antibody-drug conjugates in which the obtained conjugation site is located in one of the two Fab regions of the antibody accounts for at least 70% of the total molar quantity of a mixture of antibody-drug conjugates.

Description

A method for selectively preparing antibody-drug conjugates Technical Field

[0001] This application relates to a method for selectively preparing antibody-drug conjugates (ADCs). Specifically, this application relates to a method for selectively preparing antibody-drug conjugates in which the coupling site is located in one of the two Fab regions of the antibody. Background Technology

[0002] An antibody-drug conjugate (ADC) contains an antibody for targeting, a linker for drug attachment, and a highly potent payload (e.g., a drug) as the effector. Since the US FDA approved Adcetris (brentuximab vedotin) in 2011, the development of ADC drugs has accelerated, and their use in cancer treatment has expanded significantly. Current examples include Adcetris, Kadcyla (trastuzumab-metazine conjugate), Besponsa (oligotuzumab), Mylotarg (gem-tuzumab), Polivy (polovtuzumab), Blenrep (mabetuzumab), Enhertu (detrastuzumab), Padcev (enrofloxacin), Trodelvy (goxatusuzumab), Tivdak (teisosuzumab), Zynlonta (ronutuzumab), Akalux, Lumoxicillin (pakumomab), and Adrenaline, among others. Meanwhile, many more antibody-drug conjugates are in the clinical trial stage.

[0003] The most widely used existing method for preparing ADCs is based on cysteine ​​coupling. The coupling process consists of two steps: First, a reducing agent is used to reduce the disulfide bonds on the cysteine ​​residues between the antibody chains, making them two cysteine ​​residues. Second, an organic solvent and a linker-drug are added to the reaction system, causing the thiol groups of the cysteine ​​residues to react with the linker, generating a mixed solution of ADCs, which is then purified to obtain the final product.

[0004] Since the reduction of each disulfide bond releases two reactive thiol groups, which can couple two linkers-loaders, the drug-antibody ratio (DAR value) of the resulting ADC is always an even number when using conventional linkers-loaders. For example, a conventional IgG1 antibody has four pairs of interchain disulfide bonds, so the DAR value of an ADC using thiol-coupled components is typically 0, 2, 4, 6, 8, or higher, representing a mixture of components in any proportion. A DAR of 0 indicates that the antibody molecule is not conjugated with any drug; a DAR of 2 indicates an ADC molecule with two drug molecules coupled to one antibody molecule; a DAR of 4 indicates an ADC molecule with four drug molecules coupled to one antibody molecule; a DAR of 6 indicates an ADC molecule with six drug molecules coupled to one antibody molecule; and a DAR of 8 indicates an ADC molecule with eight drug molecules coupled to one antibody molecule, and so on. Furthermore, depending on the structure and properties of the linkers or linker-loaders involved in the reaction, mixtures of components in any proportion with DAR values ​​of 1, 3, 5, 7, or higher can also be prepared.

[0005] To obtain a relatively pure compound, the resulting ADC mixture needs to be purified, or the antibody needs to be reduced using a selective method.

[0006] WO / 2024 / 251196 describes a method for selectively reducing an antibody to obtain an ADC with high purity and a DAR value of 2. The method is characterized by selectively reducing the antibody in the presence of transition metal ions, followed by selective oxidation of the antibody, to obtain an intermediate in which a pair of disulfide bonds in the antibody hinge region are selectively reduced, thereby obtaining an ADC with high purity and a DAR value of 2.

[0007] However, the drug conjugation site of the ADC obtained in WO / 2024 / 251196 is located in the hinge region of the antibody. Selective interchain disulfide bond conjugation in the Fab region of the antibody remains a significant scientific challenge. This is because if the substrate is a monoclonal antibody, its two Fab regions are chemically or biologically equivalent. Therefore, when modifying the thiol groups of the antibody, it is impossible to selectively reduce the interchain disulfide bonds in one of the two Fab regions using any conventional method. Typically, only after conjugation can the difference in DAR values ​​of the resulting ADC be used to extract the portion with a DAR value of 2.

[0008] Therefore, there is an urgent need to develop a method for preparing ADCs with the coupling site located in one of the two Fab regions of the antibody. Summary of the Invention

[0009] The inventors have unexpectedly discovered that by incubating the reaction solution at a certain temperature for a period of time before the antibody to be coupled with the linker or linker-loader undergoes a coupling reaction, the interchain disulfide bond of one of the two Fab regions of the antibody can be selectively reduced to a thiol group, thereby obtaining a high-purity ADC with the coupling site in the antibody Fab region.

[0010] According to the present invention, "antibody" encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a specific antigen. A natural, intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR") 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 as α, δ, ε, γ, and μ, and light chains as λ or κ. Antibodies are "Y"-shaped, with the stem of the Y formed by the second and third constant regions of the two heavy chains linked by disulfide bonds. Each arm of the Y-shape contains a variable region and a first constant region of one heavy chain, as well as a variable region connected to a variable region and a constant region of one light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in two chains typically contain three highly variable loops called complementarity-determining regions (CDRs) (light chain (L) CDRs include LCDR1, LCDR2, and LCDR3, and heavy chain (H) CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundary of an antibody can be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3):651-63(1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83(1989); Kabat EA et al., National Institutes of Health, Bethesda, Md.(1991)). Three CDRs are located between flanking regions called framework regions (FRs), which are more conserved than CDRs and form a scaffold supporting the hypervariable ring. 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 into several classes 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 further divided 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).

[0011] According to the present invention, the term "hinge region" of an antibody refers to a portion comprising the linking CH1 and CH2 domains of the heavy chain molecule. The hinge region comprises approximately 25 amino acid residues and is flexible, thereby allowing independent movement of the two N-terminal antigen-binding regions. The interchain disulfide bond in the hinge region refers to the disulfide bond between the heavy chains. A "disulfide bond" refers to a covalent bond having an RSS-R' structure. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond with another thiol group (e.g., a thiol group from another cysteine ​​residue). This disulfide bond can form between the thiol groups of two cysteine ​​residues located on the two polypeptide chains, thereby forming an interchain bridge or interchain bond.

[0012] According to the present invention, the term "Fab region" of an antibody refers to the following portion of the antibody, which consists of a single light chain (both the variable and constant regions) bonded to a variable region and a first constant region of a single heavy chain via disulfide bonds. The interchain disulfide bonds in the Fab region refer to the disulfide bonds between the heavy and light chains.

[0013] According to the present invention, the term "DAR" refers to the molar ratio of drug load to antibody.

[0014] According to the present invention, the term "D0 component" refers to an ADC component with a DAR of 0, that is, the antibody molecule is not conjugated with any drug payload.

[0015] According to the present invention, the term "D0%" refers to the percentage of the moles of the D0 component relative to the total moles of the antibody-drug conjugate mixture.

[0016] According to the present invention, the term "D2 component" refers to an ADC component with a DAR of 2.

[0017] According to the present invention, the term "D2%" refers to the percentage of the moles of the D2 component relative to the total moles of the antibody-drug conjugate mixture.

[0018] According to the present invention, the term "D4 component" refers to an ADC component with a DAR of 4.

[0019] According to the present invention, the term "D4%" refers to the percentage of the moles of the D4 component relative to the total moles of the antibody-drug conjugate mixture.

[0020] According to the present invention, the term "HIC DAR" refers to the molar ratio of the average drug load to the antibody in an antibody-drug conjugate mixture as determined by hydrophobic interaction chromatography.

[0021] According to the present invention, the term "L0%" refers to the percentage of the total number of moles of the antibody light chain portion carrying 0 drug loads.

[0022] According to the present invention, the term "L1%" refers to the percentage of the total number of moles of the antibody light chain portion carrying one drug load.

[0023] According to the present invention, the term "DAR2(Fab)%" refers to the percentage of the total number of moles of the antibody-drug conjugate mixture in which the DAR is 2 and the coupling site is located in the Fab region of the antibody.

[0024] According to the present invention, the term "DAR2 (hinge)%" refers to the percentage of the total number of moles of antibody-drug conjugate mixture in which the DAR is 2 and the coupling site is located in the hinge region of the antibody.

[0025] Therefore, according to a first aspect, this application provides a method for selectively preparing antibody-drug conjugates, comprising the step of selectively reducing an interchain disulfide bond in one of the two Fab regions of the antibody to a thiol group.

[0026] In some embodiments, the selective reduction can be achieved by incubating the antibody reaction solution at a certain temperature for a period of time before the antibody-linker-load coupling reaction or the antibody-linker coupling reaction. Before the incubation step begins, a pair of disulfide bonds in the hinge region of the antibody remains in a reduced state; at the end of the incubation step, a pair of disulfide bonds in the Fab region of the antibody remains in a reduced state.

[0027] In some embodiments, the incubation can be carried out at temperatures ranging from about -10°C to 37°C, for example, about -10°C, about -5°C, about 0°C, about 5°C, about 10°C, about 12°C, about 15°C, about 20°C, about 25°C, about 30°C, or about 37°C, for example, at temperatures ranging from about 0°C to 25°C, for example, for at least 0.1 hours, at least 0.5 hours, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or at least 5 hours. For example, the incubation can be carried out at temperatures ranging from about -10°C to 37°C, for example, about -10°C, about -5°C, about 0°C, about 5°C, about 10°C, about 12°C, about 15°C, about 20°C, about 25°C, about 30°C, or about 37°C, for example, at temperatures ranging from about 0°C to 25°C, for 1 to 1.5 hours, for example, about 1 hour, about 1.1 hours, about 1.2 hours, about 1.3 hours, about 1.4 hours, or about 1.5 hours. The incubation time and temperature are determined based on the specific antibody and are within the capabilities of a person skilled in the art.

[0028] In some embodiments, the method for selectively preparing antibody-drug conjugates may include the following steps:

[0029] (a) Incubate the antibody to be conjugated and the reducing agent in a buffer system to reduce the interchain disulfide bonds of the antibody to thiol groups;

[0030] (b) Add an effective amount of transition metal ions to the reaction solution at the beginning or after step (a);

[0031] (c) Add an effective amount of oxidant to the reaction solution to oxidize the excess thiol groups, so that a pair of disulfide bonds in the hinge region of the antibody remain reduced;

[0032] (d) Add an effective amount of metal chelating agent to the reaction solution to remove the transition metal ions that are bound to the antibody;

[0033] (e) The reaction solution is incubated at a certain temperature for a period of time to selectively reduce the interchain disulfide bond of one of the two Fab regions of the antibody to a thiol group; and

[0034] (f) The antibody obtained in step (e) is coupled with a linker-load that can react with thiol groups, or the antibody obtained in step (e) is first coupled with a linker that can react with thiol groups to obtain an antibody-linker, and then the antibody-linker is coupled with a load to obtain an antibody-drug conjugate with the coupling site located in one of the two Fab regions of the antibody.

[0035] Optionally, by adding an effective amount of metal ions to the reaction solution at the beginning of reaction step (a) above, the reduction reaction between the antibody to be conjugated and the reducing agent can be controlled to the extent of incomplete reduction of the four disulfide bonds on the antibody. In this case, steps (a) and (b) can be combined into one step.

[0036] Optionally, after the above reaction step (a) is completed, adding an effective amount of transition metal ions to the reaction solution can completely reduce the four pairs of disulfide bonds on the antibody.

[0037] In some implementations, depending on the combination of the above-mentioned constraints, those skilled in the art may choose appropriate buffer systems for the reaction in step (a), including but not limited to Hepes, histidine buffer, PBS, MES, Tris, and BES.

[0038] In some implementations, the pH of the buffer system in step (a) will typically be from about 5 to about 9, for example from about 5 to 8, such as about 5.0, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, or about 9. The optimal reaction conditions will, of course, depend on the specific reactants used.

[0039] In some embodiments, based on the specific antibody to be conjugated, those skilled in the art can determine the incubation time and temperature for step (a). The optimal temperature for the reaction can typically be between approximately -10°C and 37°C, for example, approximately -10°C, approximately -5°C, approximately 0°C, approximately 5°C, approximately 10°C, approximately 12°C, approximately 15°C, approximately 20°C, approximately 25°C, approximately 30°C, or approximately 37°C. For example, the reaction can be carried out at a temperature between approximately 0°C and 25°C.

[0040] Regarding the selection of reducing agents, the reducing agents applicable in this application must have an electrode potential sufficient to reduce the antibody disulfide bonds, and the nucleophilicity of the reducing agent itself or its reduction product should be significantly different from that of the thiol groups generated by the antibody. Therefore, in some embodiments, the reducing agent in step (a) includes, but is not limited to, phosphine reducing agents, such as: tris(2-carboxyethyl)phosphine (TCEP), diphenylphosphinoacetic acid, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(diphenylphosphino)propionic acid, 2-(diphenylphosphino)ethyl ...propionic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propionic acid, 2-(diphenylphosphino)propionic acid, 2-(diphenylphosphino)propionic acid, 2-(diphenylphosphino)propionic acid, 2-(diphenylphosphino)propionic acid, 2-(diphenylphosphino)propionic acid, 2-(diphenylphosphino)propionic acid, Isopropylphosphinoethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, 1,3,5-triaza-7-phosphatricyclo[3.3.1.13.7]decane and n-butyldi(1-adamantyl)phosphine, etc.; and sulfur-based reducing agents, such as dithiothreitol, dithioerythritol, ethanethiol, sodium sulfide, glutathione, cysteine, N-acetylcysteine ​​and cysteamine, etc.; are all acceptable reducing agents.

[0041] In some embodiments, the equivalent ratio of the reducing agent to the antibody in step (a) can be 3-300, corresponding to a concentration of 0.09 mM to 9 mM, for example 0.09-1.0 mM, 0.5-1.5 mM, 1.0-2.0 mM, 1.5-2.5 mM, 2.0-3.0 mM, 2.5-3.5 mM, 3.0-4.0 mM, 3.5-4.5 mM, 4.0-5.0 mM, 4.5-5.5 mM, 5.0-6.0 mM, 5.5-6.5 mM, 6.0-7 mM. 0.0mM, 6.5-7.5mM, 7.0-8.0mM, 7.5-8.5mM, 8.0-9.0mM, approx. 0.09mM, approx. 0.1mM, approx. 0.5mM, approx. 1.0mM, approx. 1.5mM, approx. 2.0mM, approx. 2.5mM, approx. 3.0mM, approx. 3.5mM, approx. 4.0mM, approx. 4.5mM, approx. 5.0mM, approx. 5.5mM, approx. 6.0mM, approx. 6.5mM, approx. 7.0mM, approx. 7.5mM, approx. 8.0mM, approx. 8.5mM, or approx. 9.0mM.

[0042] In this application, transition metal ions provide additional selectivity in the reduction / oxidation of disulfide bonds. In the presence of transition metal ions, the interchain disulfide bonds of the reduced antibody are selectively oxidized, leaving only one pair of disulfide bonds in the antibody hinge region in a reduced state. In some embodiments, the interchain disulfide bonds of the antibody are selectively reduced in the presence of transition metal ions. Regarding the selection of transition metal ions, since the binding site of the metal ion is a thiol group, and thiol groups are broad-spectrum metal complexing agents with good binding effects on metal ions other than alkali metals, in some embodiments, the transition metal ions in step (b) include, but are not limited to, metal element ions numbered 12-13, 20-32, 38-50, or 56-84 in the periodic table. Preferably, the metal element can be a first transition series and gallium, germanium, indium, tin, or bismuth, as they have lower toxicity. Preferably, the transition metal element can include zinc, manganese, nickel, iron, copper, mercury, chromium, or calcium. In principle, the counter ion of the metal ion only needs to remain inert in the reaction system, and there are no restrictions on this. For example, a suitable transition metal salt or transition metal complex can be added in step (b). In some embodiments, the transition metal salt or transition metal complex is selected from one or more of the following: hydrochlorides, sulfates, nitrates, halides, acetates, and transition metal complexes disclosed in WO2022078524A2 of zinc, manganese, nickel, iron, copper, mercury, chromium, and calcium.

[0043] In some embodiments, the equivalent ratio of the transition metal ion to the antibody in step (b) is 0.1-200. For example, about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200.

[0044] As described above, in step (c), an effective amount of oxidant is added to the reaction solution to oxidize the excess thiol groups. At this point, a pair of disulfide bonds in the hinge region of the antibody remains in a reduced state and binds to transition metal ions.

[0045] In some embodiments, the oxidant in step (c) includes, but is not limited to, hydrogen peroxide (H2O2), nitrate compounds, potassium chlorate (KClO3), persulfate (H2S2O8), persulfate (H2SO5), NaClO, sodium dichromate (Na2Cr2O7), permanganate compounds, sodium perborate, nitrous oxide, sodium bismuthate (NaBiO3), cerium sulfate, dehydroascorbic acid (DHAA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), and 2-aminophenyl disulfide (DDD).

[0046] In some embodiments, the reaction solution may optionally be purified after the oxidation in step (c) to remove excess oxidizing / reducing agents.

[0047] In some embodiments, the metal chelating agent in step (d) includes, but is not limited to, EDTA, DTPA, ethylenediamine, 2,2'-bipyridine, and 1,10-phenanthroline. The chelating agent serves to remove transition metal ions, which may optionally be filtered out in subsequent dialysis, ultrafiltration, or gel filtration.

[0048] In the selective preparation method of antibody-drug conjugates of this application, the incubation step (e) is crucial. Before the start of incubation step (e), a pair of disulfide bonds in the hinge region of the antibody remains in a reduced state. During step (e), the reduced disulfide bonds in the hinge region of the antibody act as a reducing agent to reduce the disulfide bonds in the Fab region of the same antibody. Since only one pair of disulfide bonds in the hinge region is reduced, only one pair of disulfide bonds in the Fab region can be reduced, thereby achieving the separation of disulfide bonds in two completely chemically equivalent Fab regions. Therefore, at the end of incubation step (e), a pair of disulfide bonds in the Fab region of the antibody remains in a reduced state.

[0049] Based on the specific antibody to be conjugated, those skilled in the art can determine the incubation time and temperature. Therefore, in some embodiments, the incubation in step (e) can be performed at temperatures ranging from about -10°C to 37°C, for example, about -10°C, about -5°C, about 0°C, about 5°C, about 10°C, about 12°C, about 15°C, about 20°C, about 25°C, about 30°C, or about 37°C, for example, at temperatures ranging from about 0°C to 25°C, for example, for at least 0.1 hours, at least 0.5 hours, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or at least 5 hours. For example, the incubation can be carried out at temperatures ranging from about -10°C to 37°C, for example, about -10°C, about -5°C, about 0°C, about 5°C, about 10°C, about 12°C, about 15°C, about 20°C, about 25°C, about 30°C, or about 37°C, for example, at temperatures ranging from about 0°C to 25°C, for 1 to 1.5 hours, for example, about 1 hour, about 1.1 hours, about 1.2 hours, about 1.3 hours, about 1.4 hours, or about 1.5 hours. The incubation time and temperature are determined based on the specific antibody and are within the capabilities of a person skilled in the art.

[0050] There are no particular limitations on antibodies that can be conjugated to the linker-loader using the conjugation method of this application. The choice of antibody depends on the disease or condition (e.g., tumor or cancer) to be treated by the antibody-drug conjugate. The antibody may specifically bind to the corresponding antigen (also known as tumor-associated antigen (TAA)), viral antigen, or microbial antigen expressed on cancer cells, have antibody-dependent cell-mediated phagocytosis (ADCP) activity, and have in vivo antitumor, antiviral, or antimicrobial activity. The interchain SS bond in the antibody is the site for linking the linker-loader complex. In some embodiments, the tumor or cancer is selected from breast cancer, lymphoma, leukemia, gastric cancer, colorectal cancer, pancreatic cancer, colon cancer, ovarian cancer, liver cancer, lung cancer, endometrial cancer, prostate cancer, head and neck cancer, multiple myeloma, cholangiocarcinoma, and kidney cancer.

[0051] In some implementations, the antibody may include, but is not limited to, monoclonal or polyclonal antibodies, or it may be a monospecific or multispecific antibody, such as a bispecific antibody. Specific examples of antibodies include human antibodies, humanized antibodies, or chimeric antibodies.

[0052] "Human antibody" refers to an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source using a human antibody library or other human antibody coding sequences. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0053] "Humanized antibody" refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise substantially all or at least one (typically two) variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Optionally, the humanized antibody may comprise at least a portion of the antibody constant region derived from the human antibody. The "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0054] "Chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species, such as a molecule having a variable region derived from a mouse monoclonal antibody and a constant region derived from a human immunoglobulin. (See, for example, U.S. Patent No. 4,816,567 to Cabilly et al.; and U.S. Patent No. 4,816,397 to Boss et al., the entire contents of which are incorporated herein by reference.) Chimeric and humanized monoclonal antibodies can be produced using recombinant DNA techniques known in the art, for example using International Publication No. WO 87 / 02671; European Patent Publication No. 184,187; European Patent Publication No. 171,496; European Patent Publication No. 173,494; International Publication No. WO 86 / 01533; U.S. Patent No. 4,816,567; European Patent Publication No. 125,023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, Journal of Immunology. 139:3521-3526; Sun et al., 1987, Proc. National. Arkad. Science. USA 84:214-218; Nishimura et al., 1987, Canc. Resources. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Institute. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, Biotechnology 4:214; USA, Pat. References 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060; each of these is incorporated into this paper in its entirety through citation.

[0055] In some embodiments, the antibody is a monoclonal antibody, such as a human antibody or a humanized antibody. Useful monoclonal antibodies are homogeneous groups of antibodies against specific antigens (e.g., cancer cell antigens, viral antigens, microbial antigens covalently linked to a second molecule). Monoclonal antibodies (mAbs) against a target antigen can be prepared using any technique known in the art, such as generating antibody molecules by culturing serial cell lines. These techniques include, but are not limited to, hybridoma techniques originally described by Kohler and Milstein (1975, Nature 256, 495-497), human-B cell hybridoma techniques (Kozbor et al., 1983, Immunology Today 4:72), and EBV-hybridoma techniques (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies can be any class of immunoglobulins, including IgG, IgM, IgE, IgA, and IgD, and any of their subclasses. Hybridomas that produce the monoclonal antibodies used in this invention can be cultured in vitro or in vivo. Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies or human-mouse (or other species) chimeric monoclonal antibodies. Human monoclonal antibodies can be prepared by a variety of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

[0056] Examples of isotypes of the antibodies disclosed herein include IgG (IgG1, IgG2, IgG3, or IgG4). Preferably, the antibody in the method of this application can be an IgG1 type antibody.

[0057] In some embodiments, the antibody is a polyclonal antibody. A useful polyclonal antibody is a heterogeneous group of antibody molecules derived from the serum of an immunized animal. Polyclonal antibodies against a target antigen can be produced using various methods well known in the art. For example, to produce polyclonal antibodies, various host animals, including but not limited to rabbits, mice, rats, and guinea pigs, can be immunized by injecting a target antigen or a derivative thereof.

[0058] In some implementations, the antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, a biantibody, or an F(ab')2 fragment.

[0059] In some embodiments, the antibody is selected from trastuzumab, rituximab, cetuximab, or a monoclonal antibody, bispecific or multispecific antibody, or an antigen-binding fragment thereof specifically binding to any of the following antigens, including but not limited to Fab, Fab', Fv, scFv, or VHH: BMPR1B, E16, MPF, Napi2b, Sema. 5b, PSCAhlg, ETBR, MSG783, TrpM4, CRIPTO, CD21, FcRH2, NCA, MDP, IL20Ra, Brevican, EphB2R, AS LG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, FcRH5, TENB2, PMEL1 7. TMEFF1, GDNF-Ra1, TMEM46, Ly6G6D, LGR5, RET, Ly6K, GPR19, GPR54, ASPHD1, tyrosinase, TMEM118, GPR 172A, HER2, HER3, CLDN18.2, TROP2, c-Met, PSMA, BCMA, B7H3, PDL1, CD19, CD20, CD33, CD30, CD22, CD25, CD37, CD38, CD44, CD46, CD51, CD56, CD70, CD74, CD79b, CD123, CD138, CD205, CD228, CDCP1 , DLL3, Nectin-4, FRα, ROR1, MSLN, TNF-α, ENPP3, MUC1, Axl, ROR2, GPNMB, CEACAM5, CEACAM6, GC-C , LIV-1, GPC3, CA9, FUT3, B7H4, CTLA4, RNF43, CDH3, CDH6, CDH17, CLDN6, DPEP3, 5T4, ITGB6, EFNA4 , Notch-3, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, IGF-1R, PDL1, FCRL5, TIM1, sTn, ETB, Globo H, Ly6E, SLITRK6, GPR20, FGFR2, MUC13, MUC16, MUC18, SLAMF7, LAMP-1, CCR7, PTK7, SEZ6, SLC34A2, c-kit, LYPD 3. PRLR, FGFR3, KAAG1, STEAP1, STEAP2, Flt3, LRRC15, P-Cadherin, Integrinαvβ6, Integrinαvβ3, EphA2 or EGFR.

[0060] The payload carrying a reactive group to be conjugated to the selected antibody typically takes the form of a linker-load.

[0061] Regarding the selection of the payload, for the preparation of antibody-drug conjugates, the payload, as the toxic component, does not participate in the chemical reaction, and therefore should not be restricted in principle. The payload can include, but is not limited to, cytotoxic agents such as chemotherapeutic agents, immunotherapeutic agents, antiviral agents, or antimicrobial agents. The payload includes, but is not limited to, a wide variety of diagnostic agents, therapeutic agents, and labeling agents known in the art. For example, in the broadest sense, the payload or drug to be conjugated can include diagnostic agents, drug molecules such as cytotoxic agents (e.g., chemotherapeutic agents, immunotherapeutic agents, antiviral agents, or antibacterial agents), toxins, radionuclides, fluorescent agents, etc. (e.g., amine-derived fluorescent probes, such as 5-dimethylaminonaphthalene-1-(N-(2-aminoethyl))sulfonamide-dansyl ethylenediamine, etc.). 488 cadaverine (catalog number O-10465, Molecular Probes), dansyl cadaverine, N-(2-aminoethyl)-4-amino-3,6-disulfon-1,8-naphthalenediamine, dipotassium salt (fluorescent yellow ethylenediamine), or rhodamine B ethylenediamine (catalog number L-2424, Molecular Probes), or thiol-derived fluorescent probes, such as FLL-cystine (catalog number B-20340, molecular probe). In some embodiments, the drug to be conjugated with the antibody may be selected from, but is not limited to, MMAE (monomethyloprestatin E), MMAD (monomethyloprestatin D), MMAF (monomethyloprestatin F), etc. In addition, besides traditional small molecule cytotoxic drugs, this invention includes therapeutic peptides and nucleic acid molecules (such as oligonucleotides, siRNA, ASO, etc.) within the scope of ADC loading. In particular, DAR1 or DAR2 structures have wide applications in antibody-peptide conjugates and antibody-oligonucleotide conjugates.

[0062] In some embodiments, the payload is selected from drugs used to treat tumors, such as chemotherapy drugs (e.g., doxorubicin, cyclophosphamide, taxanes, vincristine); microtubule inhibitors (e.g., MMAE, MMAF, eribulin); DNA damaging agents (e.g., PBD drugs); RNA polymerase II inhibitors (e.g., triptolide); anti-apoptotic pathway inhibitors (e.g., BCL family inhibitors, IAP inhibitors); immune agonists (e.g., Sting agonists, TLR agonists); and kinase inhibitors (e.g., PI3K inhibitors, JAK inhibitors, JNK inhibitors, cMet inhibitors). Inhibitors include: BTK inhibitors, EGFR inhibitors; protein degraders (e.g., PROTACs, molecular glue); epigenetic pathway inhibitors (e.g., BET inhibitors, HDAC inhibitors); cell cycle pathway inhibitors (e.g., WEE1 inhibitors, PLK1 inhibitors, CDK inhibitors); cell metabolism pathway inhibitors (e.g., NAMPT inhibitors); DNA damage repair pathway inhibitors (e.g., PARP inhibitors, CHK1 / 2 inhibitors, ATR inhibitors, ATM inhibitors); and upstream oncogenic pathway inhibitors (e.g., RAS inhibitors, RAF inhibitors, MAPK inhibitors).

[0063] The linker portion of the load needs to react with an antibody containing a nucleophilic thiol group, therefore requiring a group capable of reacting with the thiol, such as maleimide, organic bromides, iodides, sulfones, and alkenes and alkynes with electron-deficient groups. Mono- or di-disulfide derivatives, sulfones, bicyclic [1.1.0]butane derivatives, sulfonyl fluorides, pentafluorophenol esters, palladium oxidative addition complexes, iodoxone, and highly electron-deficient aromatics are also commonly used. Those skilled in the art will understand that any other group capable of selectively reacting with the thiol group may be used.

[0064] In some embodiments, the linker in step (f) may contain at least two reactive groups, one of which can covalently bind the load molecule and the other of which can covalently couple the antibody.

[0065] The antibody to be coupled obtained in step (e) can be reacted with any suitable linker-loader that can react with thiol groups, or it can be reacted with any suitable linker that can react with thiol groups to obtain an antibody-linker, and then the antibody-linker is reacted with the loader to obtain the crude ADC product.

[0066] In some embodiments, optionally, after completing the coupling in step (f), a quencher (e.g., N-acetylcysteine, cysteine, cysteamine, dithiothreitol, or a salt of the above compounds) may be added to the reaction solution to inactivate excess linker-load.

[0067] In some embodiments, the method described in this application may further include step (g): purifying the antibody-drug conjugate obtained in step (f). For step (g) above, those skilled in the art can select an appropriate purification method to recover the obtained antibody-drug conjugate. Many ADC purification methods are known in the art. For example, the obtained antibody-drug conjugate can be purified using any suitable method, such as desalting column chromatography, size exclusion chromatography, etc., to obtain the final ADC product.

[0068] According to the second aspect, this application provides an antibody-drug conjugate mixture obtained according to the method of the first aspect, wherein the molar number of the antibody-drug conjugate with the coupling site located in one of the two Fab regions of the antibody accounts for at least 70%, preferably at least 80%, and more preferably at least 88% of the total molar number of the antibody-drug conjugate mixture (i.e., DAR2(Fab)%).

[0069] According to a third aspect, the present invention provides an antibody-drug conjugate mixture, wherein the number of moles of the antibody-drug conjugate mixture in which the coupling site is located in one of the two Fab regions of the antibody accounts for at least 70% of the total number of moles of the antibody-drug conjugate mixture, and the antibody Fab is unengineered, preferably at least 80%, more preferably at least 88%.

[0070] According to a fourth aspect, the present invention provides an antibody intermediate having the following formula (I):

[0071] The interchain disulfide bond in one of the two Fab regions of the antibody intermediate is reduced to two thiol groups, while the remaining interchain disulfide bonds remain intact.

[0072] According to a fifth aspect, the present invention provides a method for preparing the above-mentioned antibody intermediate, comprising the following steps:

[0073] (i) Incubate the antibody to be treated and the reducing agent in a buffer system to reduce the interchain disulfide bonds of the antibody to thiol groups;

[0074] (ii) Add an effective amount of transition metal ions to the reaction solution at the beginning or end of step (i);

[0075] (iii) Add an effective amount of oxidant to the reaction solution to oxidize the excess thiol groups, so that a pair of disulfide bonds in the hinge region of the antibody remain reduced;

[0076] (iv) Add an effective amount of a metal chelating agent to the reaction solution to remove the transition metal ions bound to the antibody; and

[0077] (v) The reaction solution is incubated at a certain temperature for a period of time to selectively reduce the interchain disulfide bond of one of the two Fab regions of the antibody to a thiol group to obtain the antibody intermediate of formula (I).

[0078] The buffer system, reducing agent, transition metal ion, oxidizing agent and metal chelating agent are as defined above.

[0079] Compared with the prior art, the beneficial technical effects of the method in this application are as follows:

[0080] 1) By using the selective preparation method of antibody-drug conjugates of this application, the homogeneity of antibody-drug conjugates is higher than that produced by conventional conjugation methods. Specifically, in the ADCs prepared by the methods of Examples 1-9 of this application, the proportion of the ADC component with a DAR of 2 (referred to as the D2 component) is typically higher than 70%, and even as high as 90% or more. Among them, the component with the conjugation site located in the Fab region accounts for more than 80% of the D2 component. In contrast, the D2 content in ADCs prepared by conventional conjugation methods is typically less than 40 wt%.

[0081] 2) The method of this application avoids any need for protein modification or enzyme catalysis, and is based on natural interchain disulfide bonds and only requires transition metal ions. Therefore, compared with conventional methods for preparing ADCs, the antibody-drug conjugates obtained by the method of this application have significantly improved homogeneity and are significantly different from the selection of conjugation sites in existing methods.

[0082] 3) The method of this application uses the reduced disulfide bonds in the antibody hinge region as a reducing agent, and selectively reduces the disulfide bonds in the antibody Fab region through an intramolecular quantitative reaction, achieving the separation of two chemically equivalent sites. This method can yield high-purity ADCs with coupling sites in the antibody Fab region. Furthermore, this method does not have a higher cost than existing methods for selectively preparing ADCs. Attached Figure Description

[0083] Figure 1 is a schematic flowchart of the method for selectively preparing antibody-drug conjugates according to this application, where M represents a transition metal ion.

[0084] Figure 2 shows the hydrophobic interaction chromatogram of Hercceptin-MMAE-D2 prepared in the comparative example.

[0085] Figure 3 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2 prepared in Example 1 of this application.

[0086] Figure 4 shows the PLRP chromatograms of Herceptin-MMAE-D2 prepared in the comparative example and Example 1 of this application.

[0087] Figure 5 shows the hydrophobic interaction chromatogram of Herceptin-DL001-D2 prepared according to the comparative example in Example 2 of this application.

[0088] Figure 6 is a PLRP chromatogram of Herceptin-DL001-D2 prepared according to the comparative example in Example 2 of this application.

[0089] Figure 7 shows the hydrophobic interaction chromatogram of Herceptin-DL001-D2 prepared according to the method of Example 1 in Example 2 of this application.

[0090] Figure 8 is a PLRP chromatogram of Herceptin-DL001-D2 prepared according to the method of Example 1 in Example 2 of this application.

[0091] Figure 9 shows the hydrophobic interaction chromatogram of Herceptin-DL002-D2 prepared according to the comparative example in Example 2 of this application.

[0092] Figure 10 is a PLRP chromatogram of Herceptin-DL002-D2 prepared according to the comparative example in Example 2 of this application.

[0093] Figure 11 shows the hydrophobic interaction chromatogram of Herceptin-DL002-D2 prepared according to the method of Example 1 in Example 2 of this application.

[0094] Figure 12 is a PLRP chromatogram of Herceptin-DL002-D2 prepared according to the method of Example 1 in Example 2 of this application.

[0095] Figure 13 is a native MS chromatogram of Herceptin-Doxorubicin-D2 prepared according to the comparative example in Example 2 of this application.

[0096] Figure 14 is a PLRP chromatogram of Herceptin-Doxorubicin-D2 prepared according to the comparative example in Example 2 of this application.

[0097] Figure 15 is a native MS diagram of Herceptin-Doxorubicin-D2 prepared according to the method of Example 1 in Example 2 of this application.

[0098] Figure 16 is a PLRP chromatogram of Herceptin-Doxorubicin-D2 prepared according to the method of Example 1 in Example 2 of this application.

[0099] Figure 17 is a Native MS chromatogram of Herceptin-APN-PEG4-DBCO-D2 prepared according to the comparative example in Example 2 of this application.

[0100] Figure 18 is a PLRP chromatogram of Herceptin-APN-PEG4-DBCO-D2 prepared according to the comparative example in Example 2 of this application.

[0101] Figure 19 is a Native MS image of Herceptin-APN-PEG4-DBCO-D2 prepared according to the method of Example 1 in Example 2 of this application.

[0102] Figure 20 is a PLRP chromatogram of Herceptin-APN-PEG4-DBCO-D2 prepared according to the method of Example 1 in Example 2 of this application.

[0103] Figure 21 shows the hydrophobic interaction chromatogram of Rituxan-MMAE-D2 prepared in Example 3 of this application.

[0104] Figure 22 is a PLRP chromatogram of Rituxan-MMAE-D2 prepared in Example 3 of this application.

[0105] Figure 23 shows the hydrophobic interaction chromatogram of Erbitux-MMAE-D2 prepared in Example 3 of this application.

[0106] Figure 24 is a PLRP chromatogram of Erbitux-MMAE-D2 prepared in Example 3 of this application.

[0107] Figure 25 shows the hydrophobic interaction chromatogram of BsAb1-MMAE-D2 prepared in Example 4 of this application.

[0108] Figure 26 is a PLRP mass spectrum of BsAb1-MMAE-D2 prepared in Example 4 of this application.

[0109] Figure 27 shows the hydrophobic interaction chromatogram of BsAb2-MMAE-D2 prepared in Example 4 of this application.

[0110] Figure 28 is a PLRP mass spectrum of BsAb2-MMAE-D2 prepared in Example 4 of this application.

[0111] Figure 29 shows the hydrophobic interaction chromatogram of Herceptin-DXd-D2 prepared in Example 5 of this application.

[0112] Figure 30 is a PLRP chromatogram of Herceptin-DXd-D2 prepared in Example 5 of this application.

[0113] Figure 31 shows the hydrophobic interaction chromatogram of Herceptin-MMAF-D2 prepared in Example 5 of this application.

[0114] Figure 32 is a PLRP chromatogram of Herceptin-MMAF-D2 prepared in Example 5 of this application.

[0115] Figure 33 shows the hydrophobic interaction chromatogram of Herceptin-tesirine-D2 prepared in Example 5 of this application.

[0116] Figure 34 is a PLRP chromatogram of Herceptin-tesirine-D2 prepared in Example 5 of this application.

[0117] Figure 35 shows the hydrophobic interaction chromatogram of Herceptin-DM21-D2 prepared in Example 5 of this application.

[0118] Figure 36 is a PLRP chromatogram of Herceptin-DM21-D2 prepared in Example 5 of this application.

[0119] Figure 37 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2 (0℃) prepared in Example 6 of this application.

[0120] Figure 38 is a PLRP chromatogram of Herceptin-MMAE-D2 (0°C) prepared in Example 6 of this application.

[0121] Figure 39 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2 (37°C) prepared in Example 6 of this application.

[0122] Figure 40 is a PLRP chromatogram of Herceptin-MMAE-D2 (37°C) prepared in Example 6 of this application.

[0123] Figure 41 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2 (pH5) prepared in Example 7 of this application.

[0124] Figure 42 is a PLRP chromatogram of Herceptin-MMAE-D2 (pH5) prepared in Example 7 of this application.

[0125] Figure 43 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2 (pH6) prepared in Example 7 of this application.

[0126] Figure 44 is a PLRP chromatogram of Herceptin-MMAE-D2 (pH6) prepared in Example 7 of this application.

[0127] Figure 45 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2 (pH8) prepared in Example 7 of this application.

[0128] Figure 46 is a PLRP chromatogram of Herceptin-MMAE-D2 (pH8) prepared in Example 7 of this application.

[0129] Figure 47 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2 (pH9) prepared in Example 7 of this application.

[0130] Figure 48 is a PLRP chromatogram of Herceptin-MMAE-D2 (pH9) prepared in Example 7 of this application.

[0131] Figure 49 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2 (Hepes) prepared in Example 8 of this application.

[0132] Figure 50 is a PLRP chromatogram of Herceptin-MMAE-D2 (Hepes) prepared in Example 8 of this application.

[0133] Figure 51 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2 (histidine) prepared in Example 8 of this application.

[0134] Figure 52 is a PLRP chromatogram of Herceptin-MMAE-D2 (histidine) prepared in Example 8 of this application.

[0135] Figure 53 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2 (PBS) prepared in Example 8 of this application.

[0136] Figure 54 is a PLRP chromatogram of Herceptin-MMAE-D2 (PBS) prepared in Example 8 of this application.

[0137] Figure 55 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2(Tris) prepared in Example 8 of this application.

[0138] Figure 56 is a PLRP chromatogram of Herceptin-MMAE-D2(Tris) prepared in Example 8 of this application.

[0139] Figure 57 shows the hydrophobic interaction chromatogram of Herceptin-MMAE-D2 prepared in Example 9 of this application.

[0140] Figure 58 is a PLRP chromatogram of Herceptin-MMAE-D2 prepared in Example 9 of this application. Detailed Implementation

[0141] The method of this application will now be described in detail with reference to the following embodiments. However, those skilled in the art should understand that the following embodiments are merely illustrative and are not intended to limit the method of this application in any way. The percentages mentioned in Tables 1-4 of the following embodiments are all molar percentages.

[0142] Comparative example: Herceptin-MC-VC-PAB-MMAE conjugate (drug-antibody ratio approximately 2) was prepared using the method of Example 2 of WO / 2024 / 251196.

[0143] (1) Add TCEP (0.15 mM) and ZnCl2 (0.06 mM) to a solution of Herceptin (prepared by WuXi Biologics according to the published corresponding protein sequence, using the standard method for preparing monoclonal antibodies, 0.03 mM, in BES buffer, pH 7, 20 mM) and allow the reaction mixture to react at 4°C for 18 hours.

[0144] (2) Add DHAA (0.6 mM) to the reaction solution and react at 22°C for 2 hours;

[0145] (3) Purify the reaction solution using an ultrafiltration tube (type: 30K, 0.5mL, REF: UFC503096, manufacturer: Thermo);

[0146] (4) Add MC-VC-PAB-MMAE dissolved in DMA (dimethylacetamide, commercially available from Aldrich Sigma) to the reaction solution. The reagents (0.3 mM) and EDTA (0.9 mM) are commercially available from WuXi AppTec and reacted at 22°C for 1 hour.

[0147] (5) Add N-acetylcysteine ​​(0.24 mM) to deplete excess small molecule drug;

[0148] (6) The reaction mixture was purified using a desalting column (type: 40K, 0.5mL, REF: 87766, Lot#SJ251704, manufacturer: Thermo).

[0149] Example 1: Preparation of Herceptin-MC-VC-PAB-MMAE conjugate

[0150] (1) Add TCEP (0.15 mM) and ZnCl2 (0.06 mM) to a solution of Herceptin (prepared by WuXi Biologics according to the published corresponding protein sequence, using the standard method for preparing monoclonal antibodies, 0.03 mM, in BES buffer, pH 7, 20 mM) and allow the reaction mixture to react at 4°C for 18 hours.

[0151] (2) Add DHAA (0.6mM) to the reaction solution and react at 22°C for 2 hours;

[0152] (3) Purify the reaction solution using an ultrafiltration tube (type: 30K, 0.5mL, REF: UFC503096, manufacturer: Thermo);

[0153] (4) Add EDTA (0.9 mM) to the reaction solution and incubate at 22°C for 2 hours;

[0154] (5) Add MC-VC-PAB-MMAE (0.3mM) dissolved in DMA (dimethylacetamide, commercially available from Aldrich Sigma) to the reaction solution and react at 22°C for 1 hour;

[0155] (6) Add N-acetylcysteine ​​(0.24 mM) to deplete excess small molecule drug;

[0156] (7) The reaction mixture was purified using a desalting column (type: 40K, 0.5mL, REF: 87766, manufacturer: Thermo).

[0157] Homogeneity tests were performed on all conjugates prepared in the comparative examples and Example 1 to compare their quality differences. Drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification analysis of various components was performed at ambient temperature using hydrophobic interaction chromatography (HIC) at a flow rate of 0.6 mL / min on a Tosoh TSKgel Butyl-NPR 4.6 mm ID x 3.5 cm, 2.5 μm spectrometer. The injection volume was 30 μg, with solvent A consisting of 1.5 M Na₂SO₄ and 50 mM sodium phosphate at pH 7. Solvent B consisted of 75% v / v 50 mM sodium phosphate at pH 7 and 25% v / v isopropanol. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0158] Site-selectivity assays were performed on all conjugates prepared in the comparative examples and Example 1 to compare their quality differences. The distribution of each component of the completely reduced drug was analyzed using PLRP-HPLC. Purification analysis of each component was performed by reversed-phase chromatography at an Agilent PLRP-S1000A, 8 μm, 50 x 2.1 mm microscope at ambient temperature using a flow rate of 0.6 mL / min. The injection volume was 30 μg, and solvent A was an aqueous solution of 0.5% TFA. Solvent B was an acetonitrile solution of 0.5% TFA. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0159] The final result is calculated by combining the results of homogeneity detection and site selectivity detection.

[0160] The results are shown in Table 1 and Figures 2-4.

[0161] Table 1

[0162] It can be observed that the ADC prepared by the method of Example 1 of this application has a similar HIC spectrum to the ADC prepared by the comparative method, proving that the two methods have similar DAR values ​​and a high proportion of DAR2 species. However, in the ADC obtained by the comparative method, DAR2(Fab) accounts for only 22.7% of the total DAR2; while in the method of Example 1 of this application, DAR2(Fab) accounts for as high as 93.1% of the DAR2 species. Therefore, the method of Example 1 of this application has significant advantages over the comparative method.

[0163] Example 2: Explanation of the practical advantages of the method of this application

[0164] Herceptin and the linker-load conjugate were prepared using the comparative example method and the method of this application in Example 1. The preparation method was the same as steps (1)-(6) of the comparative example and steps (1)-(7) of Example 1, except that the linker-load MC-VC-PAB-MMAE in step (1) of the comparative example and step (1) of Example 1 were replaced with DL001. Synthesized with reference to WO2025209545), DL002( (Design and synthesis based on WO2024227439) N-(Iodoacetamido)-Doxorubicin, commercially available from Broadpharm, ( DBCO-PEG4-APN (commercially available from CONJU-PROBE) should be used to monitor the reaction during conjugation until the load linker and antibody have completely reacted.

[0165] The conjugates obtained in Example 2 were subjected to homogeneity testing to compare their quality differences. Drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification analysis of various components was performed on a Tosoh TSKgel Butyl-NPR 4.6mm IDx 3.5cm, 2.5μm spectrometer at ambient temperature using hydrophobic interaction chromatography (HIC). The injection volume was 30μg, with solvent A consisting of 1.5M Na₂SO₄ and 50mM sodium phosphate at pH 7. Solvent B consisted of 75% v / v 50mM sodium phosphate at pH 7 and 25% v / v isopropanol. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0166] If the prepared conjugate exhibits poor separation on HIC-HPLC, making it difficult to meet quantitative requirements, then SEC-HPLC-MS should be used to analyze the drug / antibody ratio (DAR) and product distribution. (AdvanceBio SEC) Purification and analysis of various components were performed by mass spectrometry (MS) at a flow rate of 0.35 mL / min on a 1.9 μm, 4.6 × 150 mm lens at ambient temperature. The injection volume was 20 μg, and the solvent was NH4OAc at pH 7.

[0167] All conjugates obtained in Example 2 were subjected to site-selective assays to compare their quality differences. The distribution of each component of the completely reduced drug was analyzed using PLRP-HPLC. Purification analysis of each component was performed by reversed-phase chromatography at an Agilent PLRP-S1000A, 8 μm, 50 x 2.1 mm microscope at ambient temperature using a flow rate of 0.6 mL / min. The injection volume was 30 μg, and solvent A was an aqueous solution of 0.5% TFA. Solvent B was an acetonitrile solution of 0.5% TFA. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0168] The final result is calculated by combining the results of homogeneity detection and site selectivity detection.

[0169] The results are shown in Table 2 and Figure 5-20.

[0170] Table 2

[0171] It can be observed that the site distribution of the ADC synthesized by the comparative method varies with the type of connector-loador. In the comparative examples and the scope of this embodiment, the proportion of DAR2(Fab) in the DAR2 species in the comparative examples ranges from 22.7% to 85.5%, while the proportion of DAR2(Fab) in the DAR2 species of the ADC prepared according to the method of Example 1 is generally above 85%, proving that the ADC prepared by the method of this application is superior to the comparative method in terms of site selectivity.

[0172] Example 3: Verifying the general applicability of the monoclonal antibody produced by the method of this application.

[0173] Rituxan-MC-VC-PAB-MMAE conjugate and Erbitux-MC-VC-PAB-MMAE conjugate were prepared using the method described in Example 1. The preparation method was the same as steps (1)-(7) of Example 1, except that the antibody Herceptin in step (1) was replaced with antibodies Rituxan and Erbitux, respectively.

[0174] All conjugates prepared in Example 3 were subjected to homogeneity testing to compare their quality differences. Drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification analysis of various components was performed on a Tosoh TSKgel Butyl-NPR 4.6mm IDx 3.5cm, 2.5μm spectrometer at ambient temperature using hydrophobic interaction chromatography (HIC). The injection volume was 30μg, with solvent A consisting of 1.5M Na₂SO₄ and 50mM sodium phosphate at pH 7. Solvent B consisted of 75% v / v 50mM sodium phosphate at pH 7 and 25% v / v isopropanol. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0175] All conjugates obtained in Example 3 were subjected to site-selective assays to compare their quality differences. The distribution of each component of the completely reduced drug was analyzed using PLRP-HPLC. Purification analysis of each component was performed by reversed-phase chromatography at an Agilent PLRP-S1000A, 8 μm, 50 x 2.1 mm microscope at ambient temperature using a flow rate of 0.6 mL / min. The injection volume was 30 μg, and solvent A was an aqueous solution of 0.5% TFA. Solvent B was an acetonitrile solution of 0.5% TFA. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0176] The final result is calculated by combining the results of homogeneity detection and site selectivity detection.

[0177] The results are shown in Table 3 and Figures 21-24.

[0178] Table 3

[0179] It can be seen that this method is universally applicable to common antibodies on the market and can prepare ADCs with high Fab selectivity and high DAR2 ratio without additional optimization.

[0180] Example 4: Verifying the universality of the application of the method of this application and existing methods in non-monoclonal antibodies.

[0181] Using the two conjugation schemes used in Example 1, BsAb1-MC-VC-PAB-MMAE conjugates and BsAb2-MC-VC-PAB-MMAE conjugates were prepared, respectively. The preparation method was the same as steps (1)-(7) of Example 1, except that the antibody Herceptin in Example 1 was replaced with BsAb1 and BsAb2, respectively. BsAb1 was synthesized according to antibody A in CN119488608A, and BsAb2 was synthesized according to antibody B in CN119488608A. The complete amino acid sequences of antibody A and antibody B are disclosed in CN119488608A, the entire contents of which are incorporated herein by reference.

[0182] All conjugates prepared in Example 4 were subjected to homogeneity testing to compare their quality differences. Drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification analysis of various components was performed on a Tosoh TSKgel Butyl-NPR 4.6mm IDx 3.5cm, 2.5μm spectrometer at ambient temperature using hydrophobic interaction chromatography (HIC). The injection volume was 30μg, with solvent A consisting of 1.5M Na₂SO₄ and 50mM sodium phosphate at pH 7. Solvent B consisted of 75% v / v 50mM sodium phosphate at pH 7 and 25% v / v isopropanol. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0183] All conjugates prepared in Example 4 were subjected to site-selective assays to compare their quality differences. Since the bispecific antibody had many components, making site-selective assays difficult, PLRP-HPLC-MS was used to analyze the distribution of each component after complete drug reduction. Purification analysis of each component was performed by mass spectrometry (MS) at an Agilent PLRP-S1000A, 8μm, 50x2.1mm spectrometer at ambient temperature using a flow rate of 0.6mL / min. The injection volume was 30μg, and solvent A was an aqueous solution of 0.5% TFA. Solvent B was an acetonitrile solution of 0.5% TFA. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0184] The final result is calculated by combining the results of homogeneity detection and site selectivity detection.

[0185] The results are shown in Table 4 and Figures 25-28.

[0186] Table 4

[0187] It can be seen that the method of this application is also universally applicable to bispecific antibodies.

[0188] Example 5: Verifying the universal applicability of the common connector-load method of this application

[0189] Herceptin-MC-GGFG-DXd conjugate, Herceptin-MC-MMAF conjugate, Herceptin-tesirine conjugate, and Herceptin-DM21 conjugate were prepared using the method described in Example 1. The preparation method was the same as steps (1)-(7) of Example 1, except that the linker-load MC-VC-PAB-MMAE in step (5) was replaced with MC-GGFG-DXd. Available commercially from WuXi AppTec, 0.3mM), MC-MMAF ( Available commercially from WuXi AppTec, 0.3mM), tesirine ( Available commercially from WuXi AppTec (0.3mM) and DM21 ( Available commercially from WuXi AppTec (0.3 mM).

[0190] All conjugates prepared in Example 5 were subjected to homogeneity testing to compare their quality differences. Drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification analysis of various components was performed on a Tosoh TSKgel Butyl-NPR 4.6mm IDx 3.5cm, 2.5μm spectrometer at ambient temperature using hydrophobic interaction chromatography (HIC). The injection volume was 30μg, with solvent A consisting of 1.5M Na₂SO₄ and 50mM sodium phosphate at pH 7. Solvent B consisted of 75% v / v 50mM sodium phosphate at pH 7 and 25% v / v isopropanol. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0191] All conjugates obtained in Example 5 were subjected to site-selective assays to compare their quality differences. The distribution of each component of the completely reduced drug was analyzed using PLRP-HPLC. Purification analysis of each component was performed by reversed-phase chromatography at an Agilent PLRP-S1000A, 8 μm, 50 x 2.1 mm microscope at ambient temperature using a flow rate of 0.6 mL / min. The injection volume was 30 μg, and solvent A was an aqueous solution of 0.5% TFA. Solvent B was an acetonitrile solution of 0.5% TFA. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0192] The final result is calculated by combining the results of homogeneity detection and site selectivity detection.

[0193] The results are shown in Table 5 and Figures 29-36.

[0194] Table 5

[0195] It can be seen that this method is universally applicable to common connector-load devices on the market, and can prepare ADCs with high Fab selectivity and high DAR2 ratio without additional optimization.

[0196] Example 6: Verification of the incubation conditions for the method of this application

[0197] Herceptin-MC-VC-PAB-MMAE conjugate was prepared using the method described in Example 1 of this application. The preparation method was the same as steps (1)-(7) of Example 1, except that the incubation conditions in step (4) were modified to incubate at 0°C for 6 hours and at 37°C for 10 minutes.

[0198] All conjugates prepared in Example 6 were subjected to homogeneity testing to compare their quality differences. Drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification analysis of various components was performed on a Tosoh TSKgel Butyl-NPR 4.6mm IDx 3.5cm, 2.5μm spectrometer at ambient temperature using hydrophobic interaction chromatography (HIC). The injection volume was 30μg, with solvent A consisting of 1.5M Na₂SO₄ and 50mM sodium phosphate at pH 7. Solvent B consisted of 75% v / v 50mM sodium phosphate at pH 7 and 25% v / v isopropanol. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0199] All conjugates obtained in Example 6 were subjected to site-selective assays to compare their quality differences. The distribution of each component of the completely reduced drug was analyzed using PLRP-HPLC. Purification analysis of each component was performed by reversed-phase chromatography at an Agilent PLRP-S1000A, 8 μm, 50 x 2.1 mm microscope at ambient temperature using a flow rate of 0.6 mL / min. The injection volume was 30 μg, and solvent A was an aqueous solution of 0.5% TFA. Solvent B was an acetonitrile solution of 0.5% TFA. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0200] The final result is calculated by combining the results of homogeneity detection and site selectivity detection.

[0201] The results are shown in Table 6 and Figures 37-40.

[0202] Table 6

[0203] It can be seen that the method of this application is applicable to a variety of different incubation conditions.

[0204] Example 7: Verification of pH conditions for the method of this application

[0205] Herceptin-MC-VC-PAB-MMAE conjugate was prepared using the method described in Example 1 of this application. The preparation method was the same as steps (1)-(7) of Example 1, except that the pH value of the solvent in step (1) was modified to pH 5, 6, 8 and 9, and the reaction was incubated until equilibrium was reached.

[0206] All conjugates prepared in Example 7 were subjected to homogeneity testing to compare their quality differences. Drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification analysis of various components was performed on a Tosoh TSKgel Butyl-NPR 4.6mm IDx 3.5cm, 2.5μm spectrometer at ambient temperature using hydrophobic interaction chromatography (HIC). The injection volume was 30μg, with solvent A consisting of 1.5M Na₂SO₄ and 50mM sodium phosphate at pH 7. Solvent B consisted of 75% v / v 50mM sodium phosphate at pH 7 and 25% v / v isopropanol. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0207] All conjugates obtained in Example 7 were subjected to site-selective assays to compare their quality differences. The distribution of each component of the completely reduced drug was analyzed using PLRP-HPLC. Purification analysis of each component was performed by reversed-phase chromatography at an Agilent PLRP-S1000A, 8 μm, 50 x 2.1 mm microscope at ambient temperature using a flow rate of 0.6 mL / min. The injection volume was 30 μg, and solvent A was an aqueous solution of 0.5% TFA. Solvent B was an acetonitrile solution of 0.5% TFA. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0208] The final result is calculated by combining the results of homogeneity detection and site selectivity detection.

[0209] The results are shown in Table 7 and Figures 41-48.

[0210] Table 7

[0211] The above experiments demonstrate that the method described in this application is applicable to a relatively wide pH range.

[0212] Example 8: Verification of solvent type and conditions for the method of this application

[0213] Herceptin-MC-VC-PAB-MMAE conjugate was prepared using the method described in Example 1 of this application. The preparation method was the same as steps (1)-(7) of Example 1, except that the BES buffer in step (1) was replaced with Hepes, histidine, phosphate buffer and Tris, respectively.

[0214] All conjugates prepared in Example 8 were subjected to homogeneity testing to compare their quality differences. Drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification analysis of various components was performed on a Tosoh TSKgel Butyl-NPR 4.6mm IDx 3.5cm, 2.5μm spectrometer at ambient temperature using hydrophobic interaction chromatography (HIC). The injection volume was 30μg, with solvent A consisting of 1.5M Na₂SO₄ and 50mM sodium phosphate at pH 7. Solvent B consisted of 75% v / v 50mM sodium phosphate at pH 7 and 25% v / v isopropanol. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0215] All conjugates obtained in Example 8 were subjected to site-selective assays to compare their quality differences. The distribution of each component of the completely reduced drug was analyzed using PLRP-HPLC. Purification analysis of each component was performed by reversed-phase chromatography at an Agilent PLRP-S1000A, 8 μm, 50 x 2.1 mm microscope at ambient temperature using a flow rate of 0.6 mL / min. The injection volume was 30 μg, and solvent A was an aqueous solution of 0.5% TFA. Solvent B was an acetonitrile solution of 0.5% TFA. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0216] The final result is calculated by combining the results of homogeneity detection and site selectivity detection.

[0217] The results are shown in Table 8 and Figures 49-56.

[0218] Table 8

[0219] The above experiments demonstrate that the method described in this application is not limited by the type of solvent.

[0220] Example 9: Verifying the general applicability of the method of this application

[0221] The method described in this application can be applied to various preprocessing workflows, as follows:

[0222] I. Experimental Methods Using a Total Reduction-Oxidation Pretreatment Process

[0223] (1) Add TCEP (0.18 mM) to a solution of Herceptin (prepared by WuXi Biologics according to the published corresponding protein sequence, using the standard method for preparing monoclonal antibodies, 0.03 mM, in BES buffer, pH 7, 40 mM) and allow the reaction mixture to react at 12°C for 17 hours to allow the interchain disulfide bonds of the protein to be completely reduced.

[0224] (2) Add ZnCl2 (0.24 mM) to the reaction solution and incubate the solution at 4 °C for 1 h;

[0225] (3) Add DHAA (0.36 mM) to the reaction solution and react at 22°C for 2 hours;

[0226] (4) Purify the reaction solution using an ultrafiltration tube (type: 30K, 0.5mL, REF: UFC503096, manufacturer: Thermo);

[0227] (5) Add EDTA (0.9 mM) to the reaction solution and incubate at 22°C for 2 hours;

[0228] (6) Add MC-VC-PAB-MMAE (0.3mM) dissolved in DMA (dimethylacetamide, commercially available from Aldrich Sigma) to the reaction solution and react at 22°C for 1 hour;

[0229] (7) Add N-acetylcysteine ​​(0.36 mM) to deplete excess small molecule drug;

[0230] (8) The reaction mixture was purified using a desalting column (type: 40K, 0.5mL, REF: 87766, manufacturer: Thermo).

[0231] The conjugates obtained in Example 9 were subjected to homogeneity testing to compare their quality differences. Drug / antibody ratio (DAR) and product distribution were analyzed using HIC-HPLC. Purification analysis of various components was performed on a Tosoh TSKgel Butyl-NPR 4.6mm IDx 3.5cm, 2.5μm spectrometer at ambient temperature using hydrophobic interaction chromatography (HIC). The injection volume was 30μg, with solvent A consisting of 1.5M Na₂SO₄ and 50mM sodium phosphate at pH 7. Solvent B consisted of 75% v / v 50mM sodium phosphate at pH 7 and 25% v / v isopropanol. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0232] The conjugates obtained in Example 9 were subjected to site-selective assays to compare their quality differences. The distribution of each component of the completely reduced drug was analyzed using PLRP-HPLC. Purification analysis of each component was performed by reversed-phase chromatography at an Agilent PLRP-S1000A, 8 μm, 50 x 2.1 mm microscope at ambient temperature using a flow rate of 0.6 mL / min. The injection volume was 30 μg, and solvent A was an aqueous solution of 0.5% TFA. Solvent B was an acetonitrile solution of 0.5% TFA. Different drug-loaded substances were eluted using a sequential fractional gradient.

[0233] The results are shown in Table 9 and Figures 57-58.

[0234] Table 9

[0235] It can be seen that this method can be applied to any preprocessing method that can form the initial state in Figure 1, and has a wide range of applications.

Claims

1. A method for selectively preparing antibody-drug conjugates, comprising the step of selectively reducing an interchain disulfide bond in one of the two Fab regions of the antibody to a thiol group.

2. The method according to claim 1, wherein the selective reduction is achieved by incubating the antibody reaction solution at a certain temperature for a period of time before the antibody-linker-load coupling reaction or the antibody-linker coupling reaction.

3. The method according to claim 1, comprising the following steps: (a) Incubate the antibody to be conjugated and the reducing agent in a buffer system to reduce the interchain disulfide bonds of the antibody to thiol groups; (b) Add an effective amount of transition metal ions to the reaction solution at the beginning or after step (a); (c) Add an effective amount of oxidant to the reaction solution to oxidize the excess thiol groups, so that a pair of disulfide bonds in the hinge region of the antibody remain reduced; (d) Add an effective amount of metal chelating agent to the reaction solution to remove the transition metal ions bound to the antibody; (e) The reaction solution is incubated at a certain temperature for a period of time to selectively reduce the interchain disulfide bond of one of the two Fab regions of the antibody to a thiol group; and (f) The antibody obtained in step (e) is coupled with a linker-load that can react with thiol groups, or the antibody obtained in step (e) is first coupled with a linker that can react with thiol groups to obtain an antibody-linker, and then the antibody-linker is coupled with a load to obtain an antibody-drug conjugate with the coupling site located in one of the two Fab regions of the antibody.

4. The method according to claim 3, wherein the buffer system in step (a) is selected from Hepes, histidine buffer, PBS, MES, Tris or BES.

5. The method of claim 3, wherein the pH of the buffer system in step (a) is about 5 to about 9, for example about 5 to 8.

6. The method according to claim 3, wherein the reducing agent in step (a) is selected from tris(2-carboxyethyl)phosphine (TCEP), diphenylphosphinoacetic acid, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(diphenylphosphino)propionic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, 1,3,5-triaza-7-phosphatricyclo[3.3.1.13.7]decane, n-butyldi(1-adamantyl)phosphine, dithiothreitol, dithioerythritol, ethanethiol, sodium sulfide, glutathione, cysteine, N-acetylcysteine, or cystamine.

7. The method according to claim 3, wherein the equivalent ratio of the reducing agent to the antibody in step (a) is 3-300.

8. The method according to claim 3, wherein the transition metal ion in step (b) is selected from metal element ions numbered 12-13, 20-32, 38-50 or 56-84 in the periodic table.

9. The method according to claim 8, wherein the transition metal ion in step (b) is selected from a first transition metal system and gallium, germanium, indium, tin or bismuth.

10. The method according to claim 3, wherein the equivalent ratio of the transition metal ion to the antibody in step (b) is 0.1-200.

11. The method according to claim 3, wherein the oxidant in step (c) is selected from hydrogen peroxide (H2O2), nitrate compounds, potassium chlorate (KClO3), persulfate (H2S2O8), persulfate (H2SO5), NaClO, sodium dichromate (Na2Cr2O7), permanganate compounds, sodium perborate, nitrous oxide, sodium bismuthate (NaBiO3), cerium sulfate, dehydroascorbic acid (DHAA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTN B), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), or 2-aminophenyl disulfide (DDD).

12. The method according to claim 3, wherein the metal chelating agent in step (d) is selected from EDTA, DTPA, ethylenediamine, 2,2'-bipyridine or 1,10-phenanthroline.

13. The method of claim 3, wherein the incubation in step (e) is carried out at a temperature of about -10°C to 37°C, for example, about 0°C to 25°C, for at least 0.1 hours.

14. The method of claim 3, wherein the load in step (f) is selected from diagnostic agents, therapeutic agents, or labeling agents.

15. The method of claim 3, wherein the linker in step (f) contains at least two reactive groups, one of which can bind the load and the other of which can couple the antibody.

16. The method of claim 3, further comprising (g): purifying the antibody-drug conjugate obtained in step (f).

17. The antibody-drug conjugate mixture obtained by the method of any one of claims 1-16, wherein the number of moles of the antibody-drug conjugate located at one of the two Fab regions of the antibody accounts for at least 70% of the total number of moles of the antibody-drug conjugate mixture.

18. An antibody-drug conjugate mixture, wherein, The antibody-drug conjugate mixture contains at least 70% of the total molar amount of the antibody-drug conjugate mixture, with the conjugation site located in one of the two Fab regions of the antibody, and the antibody Fab is not engineered.

19. An antibody intermediate having the following formula (I): The interchain disulfide bond in one of the two Fab regions of the antibody intermediate is reduced to two thiol groups, while the remaining interchain disulfide bonds remain intact.

20. A method for preparing the antibody intermediate according to claim 19, comprising the following steps: (i) Incubate the antibody to be treated and the reducing agent in a buffer system to reduce the interchain disulfide bonds of the antibody to thiol groups; (ii) Add an effective amount of transition metal ions to the reaction solution at the beginning or end of step (i); (iii) Add an effective amount of oxidant to the reaction solution to oxidize the excess thiol groups, so that a pair of disulfide bonds in the hinge region of the antibody remain reduced; (iv) Add an effective amount of a metal chelating agent to the reaction solution to remove the transition metal ions bound to the antibody; and (v) The reaction solution is incubated at a certain temperature for a period of time to selectively reduce the interchain disulfide bond of one of the two Fab regions of the antibody to a thiol group to obtain the antibody intermediate of formula (I); The buffer system, reducing agent, transition metal ion, oxidizing agent and metal chelating agent are as defined above.

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