Preparation method of bispecific antibody coupling medicine with conjugate-antibody ratio of 2

Abstract

The invention relates to a preparation method of a bispecific antibody coupling medicine with a conjugate-antibody ratio of 2. The preparation method of the antibody coupling medicine comprises the following steps: (1) mixing a solution containing a bispecific antibody, a reducing agent and a metal ion solution for reaction; (2) adding an oxidizing agent for oxidation reaction, and adding a quenching agent for quenching the oxidation reaction; and (3) adding a metal ion chelating agent and a medicine molecule connected with a joint, and reacting to obtain the bispecific antibody coupling medicine with the conjugate-antibody ratio of 2. The antibody-coupled drug prepared by the invention is high in homogeneity, the content of the antibody-coupled drug with the DAR value of 2 is higher than 70wt%, protein engineering, enzymatic reaction or additional payload modification are not required to be introduced, and the process method is simple and convenient.

Description

Technical Field

[0001] The present invention relates to the field of antibody drugs, and in particular to a method for preparing a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2. Background Art

[0002] Antibodies refer to proteins with protective effects produced by the body due to the stimulation of antigens. Currently, antibodies are playing a vital role in the treatment of various diseases. However, natural monoclonal antibodies only have one antigen-binding fragment (Fab), that is, only a single antigen-binding epitope, which limits their role in disease treatment. Therefore, bispecific antibodies (BsAbs) that can specifically bind to two antigen molecules or two different epitopes of the same antigen at the same time have advantages that monoclonal antibodies cannot match. At present, bispecific antibodies are gradually becoming a new type of therapeutic antibodies and produce additive or synergistic effects that are superior to the effects of single antibodies.

[0003] Antibody-drug conjugates (ADCs) are a type of targeted biological agents composed of antibodies, linkers, and cytotoxic payloads. They combine the target selectivity of antibodies with the lethality of highly cytotoxic drugs. They are known as "biological missiles" and are one of the fastest-growing cancer treatments. At the same time, because ADCs can synergistically exert "specific" targeting effects and "efficient" killing effects on cancer cells, such drugs are like precision-guided "biological missiles" that can accurately destroy cancer cells, increase the treatment window, and reduce off-target side effects.

[0004] The development of bispecific antibody technology also brings new ideas to ADC drugs. By designing a bispecific ADC (BsADC), one Fab fragment binds to tumor cell-related antigens, and the other Fab fragment is used to promote the endocytosis and degradation of molecules, so that tumor targets with weak internalization can be more fully utilized. At present, 10 bispecific antibody ADCs have entered the clinical stage, most of which use the introduction of engineered reactive cysteine ​​residues or the use of interchain disulfide bonds for random coupling to achieve site-specific coupling of small molecule drugs on bispecific antibodies. For example, CN117659203A discloses an anti-MET / EGFR bispecific antibody and its drug conjugate, which uses the cysteine ​​method to couple antibodies and drugs. The defect of the engineering reactive cysteine ​​residue technology is that the wrong disulfide bond will be formed between the two Fab regions in the antibody during the introduction of the thiol group, which will affect the subsequent coupling. The random conjugation method will lead to the production of heterogeneous ADCs. Products with low DAR values ​​have insufficient efficacy, while products with high DAR values ​​have higher toxicity and unstable PK in vivo. Therefore, the heterogeneity of ADCs hinders the expansion of the therapeutic window.

[0005] In summary, how to improve the homogeneity of bispecific antibodies when preparing ADC products has become one of the urgent problems to be solved in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for preparing a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2, wherein the reduction and oxidation of disulfide bonds are selectively affected by metal ions, and then a metal chelator and a thiol-reactive load are added to generate a conjugate with a conjugate-antibody ratio of 2.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a bispecific antibody-drug conjugate having a conjugate-antibody ratio of 2, the method comprising the following steps:

[0009] (1) mixing a solution containing a bispecific antibody, a reducing agent, and a metal ion solution for reaction;

[0010] (2) adding an oxidant to carry out an oxidation reaction, and adding a quencher to quench the oxidation reaction;

[0011] (3) adding a metal ion chelator and a drug molecule connected with a linker, and obtaining a bispecific antibody-drug conjugate having a conjugate-antibody ratio of 2 after reaction.

[0012] The present invention incubates antibodies, reducing agents and metal ions in a buffer system, wherein the metal ions selectively affect the reduction and oxidation of disulfide bonds, the reducing agent selectively reduces the interchain disulfide bonds in the antibody, and then an oxidant is added to selectively oxidize the reduced sulfhydryl groups. A metal chelator and a sulfhydryl-reactive load (such as maleimide-linked drugs, etc.) are then added to react and generate a conjugate with a DAR value (Drug-to-Antibody Ratio, the ratio of drug to antibody in antibody-conjugated drugs) of 2.

[0013] The present invention provides a novel bioconjugation process for preparing a multivalent bivalent antibody ADC with improved homogeneity, which is simple and efficient, does not require protein engineering, linker payload modification and / or enzyme catalysis, and has improved homogeneity. Compared with the conventional thiol random conjugation process, the homogeneity of the DAR2 multivalent bivalent antibody ADC product produced by the bioconjugation process of the present invention can be significantly improved, and the content of DAR2 is usually more than 70wt%, preferably more than 80wt%, while in the ADC prepared by the conventional thiol random conjugation process, the content of DAR2 is usually less than 50wt%.

[0014] The process of the present invention can be used to couple various diagnostic, therapeutic and labeling reagents to antibody molecules. The most common reactive group that can couple to thiol groups in the preparation of ADC is maleimide. In addition, other electron-deficient alkenyl or alkynyl derivatives, mono- or di-derivatives of disulfides, sulfones, bicyclobutane derivatives, sulfonyl fluorides, pentafluorophenol esters, palladium oxide addition complexes, benzyl iodide, highly electron-deficient aromatic hydrocarbons, organic bromides, and iodides are also often used.

[0015] Preferably, the reducing agent comprises tris(2-carboxyethyl)phosphine (TCEP).

[0016] Preferably, the metal ions in the metal ion solution include transition metal ions.

[0017] Preferably, the transition metal ions include Zn 2+ .

[0018] Preferably, the concentration of the bispecific antibody in the solution containing the bispecific antibody is 0.02-0.04 mM (for example, it may be 0.02 mM, 0.025 mM, 0.03 mM, 0.035 mM or 0.04 mM, etc.).

[0019] Preferably, the solution containing the bispecific antibody further contains a 30-50 mM (e.g., 30 mM, 35 mM, 40 mM, 45 mM or 50 mM, etc.) buffer solution with a pH of 6-8 (e.g., 6, 6.5, 7, 7.5 or 8, etc.).

[0020] Preferably, the buffer comprises any one of HEPES, histidine buffer, PBS or MES, or a combination of at least two thereof.

[0021] Preferably, the molar ratio of the bispecific antibody in the solution containing the bispecific antibody, the reducing agent, and the metal ion in the metal ion solution is 1:(5-15):(2-6), preferably 1:(9-11):(3-5).

[0022] The specific point values ​​(5-15) mentioned above can be selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, etc.

[0023] The specific point values ​​(2 to 6) mentioned above can be selected from 2, 3, 4, 5 or 6, etc.

[0024] The specific point values ​​of the above (9-11) can be selected from 9, 9.5, 9.8, 10, 10.2, 10.5 or 11, etc.

[0025] The specific point values ​​of the above (3-5) can be selected from 3, 3.5, 3.8, 4, 4.2, 4.5 or 5, etc.

[0026] Preferably, the mixed reaction in step (1) is carried out at 8-16°C (for example, 8°C, 10°C, 12°C, 14°C or 16°C, etc.) and allowed to stand for 10-20h (for example, 10h, 12h, 14h, 16h, 18h or 20h, etc.).

[0027] Preferably, the oxidant comprises any one or a combination of at least two of dehydroascorbic acid (DHAA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP).

[0028] Preferably, the molar ratio of the bispecific antibody to the oxidant in the solution containing the bispecific antibody is 1:(8-15), preferably 1:(9-11).

[0029] The specific point values ​​(8-15) mentioned above can be selected from 8, 9, 10, 11, 12, 13, 14 or 15, etc.

[0030] The specific point values ​​of the above (9-11) can be selected from 9, 9.5, 9.8, 10, 10.2, 10.5 or 11, etc.

[0031] Preferably, the temperature of the oxidation reaction in step (2) is 8-16°C (for example, 8°C, 10°C, 12°C, 14°C or 16°C, etc.), and the time is 0.5-1.5h (for example, 0.5h, 0.7h, 0.9h, 1h, 1.1h, 1.3h or 1.5h, etc.).

[0032] Preferably, the quencher comprises triphenylphosphine tris-sulfonic acid (DiPPBS3).

[0033] Preferably, the molar ratio of the oxidant to the quencher is (1-10):1, preferably (4-6):1.

[0034] The specific point values ​​(1 to 10) mentioned above can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0035] The specific point values ​​of the above (4-6) can be selected as 4, 4.5, 4.8, 5, 5.2, 5.5 or 6, etc.

[0036] Preferably, the temperature of quenching the oxidation reaction in step (2) is 8-16°C (for example, 8°C, 10°C, 12°C, 14°C or 16°C, etc.), and the time is 0.3-0.7h (for example, 0.3h, 0.4h, 0.5h, 0.6h or 0.7h).

[0037] Preferably, the metal ion chelator comprises EDTA.

[0038] Preferably, the molar ratio of the metal ions to the metal ion chelating agent in the metal ion solution is 1:(20-50), preferably 1:(35-40).

[0039] The specific point values ​​of the above (20-50) can be selected as 20, 25, 30, 35, 40, 45 or 50, etc.

[0040] The specific point values ​​of the above (35-40) can be selected as 35, 36, 36.5, 37, 37.5, 38, 38.5, 39 or 40, etc.

[0041] Preferably, the linker in the linker-attached drug molecule has a thiol-reactive load.

[0042] Preferably, the molar ratio of the bispecific antibody to the drug molecule connected with a linker in the solution containing the bispecific antibody is 1:(10-50), preferably 1:(15-25).

[0043] The specific point values ​​(10-50) mentioned above can be selected from 10, 15, 20, 25, 30, 35, 40, 45 or 50, etc.

[0044] The specific point values ​​(15-25) mentioned above can be selected as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, etc.

[0045] Preferably, the reaction temperature in step (3) is 8-16°C (for example, 8°C, 10°C, 12°C, 14°C or 16°C, etc.), and the reaction time is 0.5-1.5h (for example, 0.5h, 0.7h, 0.9h, 1h, 1.1h, 1.3h or 1.5h, etc.).

[0046] Preferably, before obtaining the bispecific antibody-drug conjugate having a conjugate-antibody ratio of 2 in step (3), the process further comprises: ultrafiltration replacing the buffer in the product with a pH of 5 to 6 (e.g., 5.2, 5.4, 5.5, 5.6, 5.8 or 6, etc.) 10 to 30 mM (e.g., 10 mM, 15 mM, 20 mM, 25 mM or 30 mM, etc.) histidine buffer to remove excess small molecule impurities in the reaction system.

[0047] Preferably, the bispecific antibody contains a first Fab domain, a second Fab domain, a third Fab domain and an Fc domain, the first Fab domain and the second Fab domain are connected to the Fc domain through a hinge region, the first Fab domain and the third Fab domain are covalently connected, and the amino acid sequences of the second Fab domain and the third Fab domain are the same.

[0048] Preferably, the light chain constant region and the heavy chain constant region of the second Fab domain are connected by a disulfide bond.

[0049] Preferably, the light chain constant region and the heavy chain constant region of the third Fab domain are connected by a disulfide bond.

[0050] Preferably, the hinge region contains at least two interchain disulfide bonds.

[0051] Preferably, the Fc domain contains a first CH2, a second CH2, a first CH3 and a second CH3.

[0052] Preferably, the Fc domain has a knob-in-hole structure.

[0053] Preferably, the first CH3 and the second CH3 in the Fc domain are connected via a knob-and-hole structure.

[0054] Preferably, the heavy chain constant region of the first Fab domain is connected to the first CH2 via a hinge region.

[0055] Preferably, the heavy chain constant region of the second Fab domain is connected to the second CH2 via a hinge region.

[0056] Preferably, the heavy chain variable region of the first Fab domain is covalently linked to the heavy chain constant region of the third Fab domain.

[0057] Preferably, the first Fab domain, the second Fab domain and the third Fab domain are T cell receptor constant domains.

[0058] The present invention utilizes the T cell receptor (TCR) constant domain to replace the antibody Fab domain, thereby ensuring homologous pairing, adopts an asymmetric form, and ensures heterodimerization through a knob-and-hole structure.

[0059] Preferably, the composition of the bispecific antibody includes the amino acid sequence shown in SEQ ID NO.1 to SEQ ID NO.4.

[0060] SEQ ID NO.1: DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0061] SEQ ID NO.2:QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWNTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

[0062] SEQ ID NO.3:DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKPDIQNPDCAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPECTFFPS。

[0063] SEQ ID NO.4: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWV KQAPGQGLKWMGWNTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0064] In the present invention, SEQ ID NO.1 contains the light chain of the first Fab domain, SEQ ID NO.2 contains the heavy chain of the first Fab domain and the third Fab domain, the first CH2 and the first CH3, SEQ ID NO.3 contains the light chain of the second Fab domain and the third Fab domain, and SEQ ID NO.4 contains the heavy chain of the second Fab domain, the second CH2 and the second CH3.

[0065] The first Fab domain provided by the present invention can specifically bind to the TROP2 antigen, and the second Fab and third Fab domains can specifically bind to the HER2 antigen.

[0066] In a second aspect, the present invention provides a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2, wherein the antibody-drug conjugate is prepared by the method for preparing a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2 described in the first aspect.

[0067] Preferably, the antibody-drug conjugate contains a bispecific antibody, a linker and a drug, and the molar ratio of the drug to the bispecific antibody is 2.

[0068] Preferably, the drug is connected to the hinge region of the bispecific antibody via a linker.

[0069] Preferably, the drug is connected to two sulfhydryl groups in the hinge region of the bispecific antibody respectively through a linker, and the two sulfhydryl groups form a disulfide bond when not connected.

[0070] In a third aspect, the present invention provides a method for preparing a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2 as described in the first aspect, or use of the bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2 as described in the second aspect in preparing an anti-tumor drug.

[0071] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] (1) The present invention provides a method for preparing a DAR2 antibody-drug conjugate, which is a metal ion-assisted drug conjugate. By controlling the reduction of disulfide bonds and the oxidation of sulfhydryl groups, the sulfhydryl groups are selectively coupled to the drug payload, and two molecules of drug payload are coupled to a pair of open disulfide bonds of the antibody. The uniformity of the bispecific ADC is higher than that produced by conventional conjugation processes. The content of DAR2 is usually more than 70wt%, preferably more than 80wt%. No protein engineering, enzymatic reaction or additional payload modification is required, and the process is simple.

[0074] (2) The present invention prepares a DAR2 bispecific antibody-drug conjugate, which uses the interchain disulfide bonds of the bispecific antibody itself to couple the drug molecule without introducing any new amino acid sequence, thereby reducing the immunogenicity of the antibody in vivo, being able to specifically bind to two antigen molecules at the same time, and having a repeated Fab fragment, thereby improving the antibody's ability to specifically bind to antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 The figure is a schematic diagram of the preparation process of the antibody-drug conjugate of the present invention.

[0076] Figure 2 This is the hydrophobic interaction chromatogram of the antibody-drug conjugate prepared in Example 1.

[0077] Figure 3 This is the hydrophobic interaction chromatogram of the antibody-drug conjugate prepared in Example 2. DETAILED DESCRIPTION

[0078] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0079] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0080] Example 1

[0081] This embodiment provides a method for preparing a bispecific antibody-drug conjugate of DAR2. The schematic diagram of the preparation process is shown in FIG. Figure 1 As shown, the following steps are included:

[0082] (1) TCEP (C4706-2G, Sigma-Aldrich), ZnCl2 (STBK6550, Sigma-Aldrich) and a solution containing a bispecific antibody were mixed, wherein the concentration of the bispecific antibody in the solution was 0.032 mM, and the solution also contained a 40 mM phosphate buffer at pH 7. The bispecific antibody, TCEP and ZnCl2 were mixed. 2+ The molar ratio of is 1:10:4, and the reaction mixture is allowed to stand at 12°C for 16 hours. The bispecific antibody is composed of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.4, and is prepared by WuXi Biologics. The bispecific antibody provided by the present invention is prepared by referring to the prior art method.

[0083] (2) DTNB (D8130-500MG, Sigma-Aldrich) was added at a molar ratio of 1:10 between the bispecific antibody and DTNB, and the reaction was continued at 12°C for 1 h. DiPPBS3 (prepared by WuXi Biologics) was added at a molar ratio of 5:1 between DTNB and DiPPBS3, and the reaction was continued at 12°C for 0.5 h to quench the oxidation reaction.

[0084] (3) Add EDTA to capture Zn 2+ , Zn 2+ The molar ratio of MC-VC-PAB-MMAE (prepared by WuXi AppTec) in DMA (dimethylacetamide, Sigma-Aldrich) was 1:37.5, and the molar ratio of bispecific antibody to MC-VC-PAB-MMAE was 1:20. The reaction was continued at 12°C for 1 h.

[0085] (4) Use an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to replace the product buffer with its storage buffer (20 mM histidine buffer, pH 5.5) to remove excess small molecule impurities in the reaction system.

[0086] Example 2

[0087] This example provides a method for preparing a bispecific antibody-drug conjugate of DAR2, which is different from Example 1 only in that in step (2), the molar ratio of the bispecific antibody to DTNB is 1:11, and the molar ratio of DTNB to DiPPBS3 is 6:1.

[0088] Test Example 1

[0089] In this test example, the antibody-drug conjugate prepared in Example 1-2 was used to measure the DAR value and drug distribution using HIC-HPLC. The results of the mass percentage content of each component are shown in Table 1. The hydrophobic interaction chromatogram of Example 1 is shown in Figure 2 As shown, the hydrophobic interaction chromatogram of Example 2 is as shown Figure 3 As shown. Wherein D0 means no drug is coupled to the antibody, D1 means one molecule of drug is coupled to one molecule of antibody, D2 means two molecules of drug are coupled to one molecule of antibody, D4 means four molecules of drug are coupled to one molecule of antibody, and HIC DAR means the DAR value result calculated after hydrophobic interaction chromatography detection. As can be seen from Table 1, the antibody-drug conjugate prepared by the method provided by the present invention has a content of D2 component exceeding 70wt%, preferably exceeding 80wt%, and its uniformity is higher than that produced by conventional random coupling process, and the content of D2 obtained by conventional method is usually less than 50wt%.

[0090] Table 1

[0091] Group D0(％) D1(％) D2(％) D4(％) HIC DAR Example 1 14.0 2.2 82.8 1.0 1.72 Example 2 26.2 3.0 70.8 NA 1.45

[0092] In summary, the present invention provides a method for preparing a DAR2 antibody-drug conjugate, by controlling the reduction of disulfide bonds and the oxidation of thiol groups, two molecules of drug payload are coupled to a pair of open disulfide bonds of the antibody, the bispecific ADC has high uniformity and the process is simple.

[0093] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2, characterized in that: The method for preparing the antibody-drug conjugate comprises the following steps: (1) mixing a solution containing a bispecific antibody, a reducing agent, and a metal ion solution for reaction; (2) adding an oxidant to carry out an oxidation reaction, and adding a quencher to quench the oxidation reaction; (3) adding a metal ion chelator and a drug molecule connected with a linker, and obtaining a bispecific antibody-drug conjugate having a conjugate-antibody ratio of 2 after reaction.

2. The method for preparing a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2 according to claim 1, characterized in that: The reducing agent includes tris(2-carboxyethyl)phosphine; Preferably, the metal ions in the metal ion solution include transition metal ions; Preferably, the transition metal ions include Zn 2+ ; Preferably, the concentration of the bispecific antibody in the solution containing the bispecific antibody is 0.02 to 0.04 mM; Preferably, the solution containing the bispecific antibody further contains 30-50 mM buffer at a pH of 6-8; Preferably, the buffer comprises any one or a combination of at least two of HEPES, histidine buffer, PBS or MES; Preferably, the molar ratio of the bispecific antibody, the reducing agent, and the metal ion in the metal ion solution in the solution containing the bispecific antibody is 1:(5-15):(2-6), preferably 1:(9-11):(3-5); Preferably, the mixing reaction in step (1) is carried out at 8 to 16° C. for 10 to 20 hours.

3. The method for preparing a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2 according to claim 1 or 2, characterized in that: The oxidant includes any one of dehydroascorbic acid, 5,5'-dithiobis(2-nitrobenzoic acid), nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, or a combination of at least two thereof; Preferably, the molar ratio of the bispecific antibody to the oxidant in the solution containing the bispecific antibody is 1:(8-15), preferably 1:(9-11); Preferably, the temperature of the oxidation reaction in step (2) is 8 to 16° C., and the time is 0.5 to 1.5 h; Preferably, the quencher comprises triphenylphosphine tris-sulfonic acid; Preferably, the molar ratio of the oxidant to the quencher is (1-10):1, preferably (4-6):1; Preferably, the temperature for quenching the oxidation reaction in step (2) is 8 to 16° C. and the time is 0.3 to 0.7 h.

4. The method for preparing a bispecific antibody-drug conjugate having a conjugate-antibody ratio of 2 according to any one of claims 1 to 3, characterized in that: The metal ion chelating agent includes EDTA; Preferably, the molar ratio of the metal ion to the metal ion chelating agent in the metal ion solution is 1:(20-50), preferably 1:(35-40); Preferably, the linker in the linker-attached drug molecule has a thiol-reactive load; Preferably, the molar ratio of the bispecific antibody to the drug molecule connected with a linker in the solution containing the bispecific antibody is 1:(10-50), preferably 1:(15-25); Preferably, the reaction temperature in step (3) is 8 to 16° C. and the reaction time is 0.5 to 1.5 h; Preferably, before obtaining the bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2 in step (3), the process further comprises: ultrafiltration replacing the buffer in the product with a 10-30 mM histidine buffer at a pH of 5-6 to remove excess small molecule impurities in the reaction system.

5. The method for preparing a bispecific antibody-drug conjugate having a conjugate-antibody ratio of 2 according to any one of claims 1 to 4, characterized in that: The bispecific antibody contains a first Fab domain, a second Fab domain, a third Fab domain and an Fc domain, wherein the first Fab domain and the second Fab domain are connected to the Fc domain via a hinge region, the first Fab domain and the third Fab domain are covalently connected, and the amino acid sequences of the second Fab domain and the third Fab domain are identical.

6. The method for preparing a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2 according to claim 5, characterized in that: The light chain constant region and the heavy chain constant region of the second Fab domain are connected by a disulfide bond; Preferably, the light chain constant region and the heavy chain constant region of the third Fab domain are connected by a disulfide bond; Preferably, the hinge region contains at least two interchain disulfide bonds; Preferably, the Fc domain contains a first CH2, a second CH2, a first CH3 and a second CH3; Preferably, the Fc domain has a knob-in-hole structure; Preferably, the first CH3 and the second CH3 in the Fc domain are connected by a knob-and-hole structure; Preferably, the heavy chain constant region of the first Fab domain is connected to the first CH2 via a hinge region; Preferably, the heavy chain constant region of the second Fab domain is connected to the second CH2 via a hinge region; Preferably, the heavy chain variable region of the first Fab domain is covalently linked to the heavy chain constant region of the third Fab domain.

7. The method for preparing a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2 according to claim 5 or 6, characterized in that: The first Fab domain, the second Fab domain and the third Fab domain are T cell receptor constant domains; Preferably, the composition of the bispecific antibody includes the amino acid sequence shown in SEQ ID NO.1 to SEQ ID NO.

4.

8. A bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2, characterized in that: The antibody-drug conjugate is prepared by the method for preparing a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2 according to any one of claims 1 to 7.

9. The antibody-drug conjugate according to claim 8, characterized in that: The antibody-drug conjugate contains a bispecific antibody, a linker and a drug, and the molar ratio of the drug to the bispecific antibody is 2; Preferably, the drug is connected to the hinge region of the bispecific antibody via a linker; Preferably, the drug is respectively connected to two sulfhydryl groups in the hinge region of the bispecific antibody through a linker, and the two sulfhydryl groups form a disulfide bond when not connected.

10. A method for preparing a bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2 according to any one of claims 1 to 7, or use of the bispecific antibody-drug conjugate with a conjugate-antibody ratio of 2 according to claim 8 or 9 in preparing an anti-tumor drug.

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