A method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6

Abstract

The present invention relates to a method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6, and the method comprises the following steps: (1) mixing a solution containing a bispecific antibody, a reducing agent and a metal ion solution for reaction; (2) adding a metal ion chelating agent and a drug molecule connected with a linker, and obtaining the bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 after the reaction. The present invention controls the reduction of disulfide bonds with the assistance of metal ions, and the prepared antibody-drug conjugate has high uniformity, and the proportion of ADC molecules with a DAR value of 6 exceeds 60wt%.

Description

Technical Field

[0001] The present invention relates to the field of antibody drugs, and in particular to a method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6. Background Art

[0002] Antibodies are highly homogeneous immunoglobulins produced by B lymphocytes and targeting only specific antigenic epitopes. They have a wide range of mechanisms of action, including inhibition of receptor and ligand binding, direct cytotoxic killing, and the use of antibody Fc fragments to mediate immune effector functions. Therefore, antibody-mediated tumor immunotherapy has become an important means of cancer treatment. With the development of technology, bispecific antibodies (Bispecificantibody, BsAb) with multiple antibody binding fragments have been developed based on natural antibodies with only one antigen-binding fragment (Antigen-binding fragment, Fab). It can target multiple tumor antigens or different antigenic epitopes on tumor cells to enhance the binding specificity of BsAb, while reducing the adverse reactions caused by off-target toxicity. On this basis, multivalent bispecific antibodies and multispecific antibodies with three or more multiple or repeated Fab fragments will further enhance the therapeutic effect.

[0003] Antibody drug conjugations (ADCs) have received extensive attention in recent years. ADCs direct cytotoxic drugs to antigen-expressing cells or other target cells through antibody-antigen interactions. They are composed of antibodies against tumor antigens, linkers, and payloads with cytotoxic activity. After entering the body, ADCs recognize and bind to target antigens on the surface of tumor cells. After the ADC-antigen complex is internalized into the lysosome through receptor-mediated endocytosis, the complex releases toxic substances during lysosomal degradation, and the payload damages DNA or inhibits cell division in other ways, ultimately killing tumor cells. The improved targeting of cytotoxic substances improves the therapeutic efficiency of ADCs.

[0004] The development of bispecific antibody technology also brings new ideas to ADC drugs. For example, CN118085101A discloses a bivalent bispecific antibody, antibody-coupled nuclide drug and its application, which has good targeting affinity, and its antibody-coupled nuclide drug has excellent SPECT imaging results and can continue to accumulate in tumors over time. Currently, 10 bispecific antibody ADCs have entered the clinical stage, most of which use the introduction of engineered reactive cysteine ​​residues or random coupling using interchain disulfide bonds to achieve site-specific coupling of small molecule drugs on bispecific antibodies.

[0005] The drawback of the engineering reactive cysteine ​​residue technology is that the thiol introduction step may cause the formation of an incorrect disulfide bond between the two Fab regions in the antibody, affecting the subsequent coupling, while the random coupling method will lead to the production of heterogeneous ADCs. Products with low DAR values ​​have insufficient efficacy, and products with high DAR values ​​have high toxicity and unstable PK in the body, so the heterogeneity of ADCs hinders the expansion of the therapeutic window.

[0006] In summary, how to improve the homogeneity of ADC products has become one of the urgent problems to be solved in this field. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6, incubating a reducing agent, a metal ion and a bispecific antibody to be conjugated in a buffer system, and then adding a metal ion chelator and a thiol-reactive load to generate a conjugate with a conjugate-antibody ratio of 6.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing a bispecific multivalent antibody-drug conjugate having a conjugate-antibody ratio of 6, the method for preparing the bispecific multivalent antibody-drug conjugate comprising the following steps:

[0010] (1) mixing a solution containing a bispecific antibody, a reducing agent, and a metal ion solution for reaction;

[0011] (2) adding a metal ion chelator and a drug molecule connected with a linker, and obtaining a bispecific multivalent antibody-drug conjugate with a conjugate antibody ratio of 6 after reaction.

[0012] The present invention incubates a reducing agent and an effective amount of metal ions with a bispecific antibody to be coupled in a buffer system, wherein the metal ions selectively affect the reduction of disulfide bonds, selectively reduce the interchain disulfide bonds in the antibody, and then add an effective amount of a metal chelator and an excess of a thiol-reactive load (such as a maleimide-linked drug, etc.) to react and generate a conjugate with a DAR value (Drug-to-Antibody Ratio, the ratio of drug to antibody in an antibody-drug conjugate) of 6.

[0013] The ADC molecules prepared by the method provided by the present invention have a higher uniformity than those produced by conventional random coupling processes, and the content of the D6 component (six molecules of drug coupled to one molecule of antibody) is usually more than 65wt%, and the best is more than 70wt%. In the ADC prepared by conventional reduction and adding a certain amount of thiol-reactive loading linkers, the content of D6 is usually less than 50wt%.

[0014] The method provided by the present invention can be used to couple various diagnostic, therapeutic and labeling agents to antibody molecules. The most common reactive group that can couple with thiol 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, iodides are often used, and any other group that selectively reacts with thiol can be 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:(8-16):(2-6), preferably 1:(11-13):(3-5).

[0022] The specific point values ​​(8-16) mentioned above can be selected from 8, 9, 10, 11, 12, 13, 14, 15 or 16, 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 (11-13) can be selected as 11, 11.5, 11.8, 12, 12.2, 12.5 or 13, 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 metal ion chelator comprises EDTA.

[0028] Preferably, the molar ratio of the metal ions to the metal ion chelating agent in the metal ion solution is 1:(25-50), preferably 1:(35-40).

[0029] The specific point values ​​of the above (25-50) can be selected as 25, 30, 35, 40, 45 or 50, etc.

[0030] 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.

[0031] Preferably, the linker in the linker-attached drug molecule has a thiol-reactive load.

[0032] Preferably, the molar ratio of the bispecific antibody to the linker-linked drug molecule in the solution containing the bispecific antibody is 1:(6-40), preferably 1:(15-25).

[0033] The specific point values ​​(6-40) mentioned above can be selected from 6, 8, 10, 15, 20, 25, 30, 35 or 40, etc.

[0034] 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.

[0035] Preferably, the reaction temperature in step (2) 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.).

[0036] Preferably, before obtaining the bispecific multivalent antibody-drug conjugate having a conjugate-antibody ratio of 6 in step (2), the process further comprises: ultrafiltration replacing the buffer in the product with a pH of 5 to 6 (e.g., 5, 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.

[0037] 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.

[0038] Preferably, the light chain constant region and the heavy chain constant region of the second Fab domain are connected by a disulfide bond.

[0039] Preferably, the light chain constant region and the heavy chain constant region of the third Fab domain are connected by a disulfide bond.

[0040] Preferably, the hinge region contains at least two interchain disulfide bonds.

[0041] Preferably, the Fc domain contains a first CH2, a second CH2, a first CH3 and a second CH3.

[0042] Preferably, the Fc domain has a knob-in-hole structure.

[0043] Preferably, the first CH3 and the second CH3 in the Fc domain are connected via a knob-and-hole structure.

[0044] Preferably, the heavy chain constant region of the first Fab domain is connected to the first CH2 via a hinge region.

[0045] Preferably, the heavy chain constant region of the second Fab domain is connected to the second CH2 via a hinge region.

[0046] 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.

[0047] Preferably, the first Fab domain, the second Fab domain and the third Fab domain are T cell receptor constant domains.

[0048] The present invention utilizes the T cell receptor (TCR) constant domain to replace the antibody Fab domain, thereby ensuring homologous pairing and maintaining the heterodimerization of the bispecific antibody through the knob-in-hole structure. At the same time, the antigen binding is divided into two parts, and the two parts bind to different antigens respectively, one of which is designed as a repeated antibody Fab fragment, thereby constructing a bispecific trivalent antigen binding complex.

[0049] Preferably, the composition of the bispecific antibody includes the amino acid sequence shown in SEQ ID NO.1 to SEQ ID NO.4.

[0050] SEQ ID NO.1:DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKP GKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0051] SEQ ID NO.2:QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWV KQAPGQGLKWMGWNTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

[0052] SEQ ID NO.3: DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQK PGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT PPTFGQGTKVEIKPDIQNPDCAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKD SDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPECTFFPS.

[0053] SEQ ID NO.4: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWV KQAPGQGLKWMGWNTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0054] 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.

[0055] 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.

[0056] In a second aspect, the present invention provides a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6, wherein the bispecific multivalent antibody-drug conjugate is prepared by the method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 described in the first aspect.

[0057] Preferably, the bispecific multivalent antibody-drug conjugate contains a bispecific antibody, a linker and a drug, and the molar ratio of the drug to the bispecific antibody is 6.

[0058] Preferably, the drug is connected to six thiol groups on the bispecific antibody through a linker, and the six thiol groups are respectively located in the light chain constant region of the first Fab domain, the heavy chain constant region of the first Fab domain, the light chain constant region of the third Fab domain, the heavy chain constant region of the third Fab domain, the connection between the first Fab domain and the Fc domain, and the connection between the third Fab domain and the Fc domain, and the six thiol groups exist in the form of disulfide bonds when not connected.

[0059] According to the relative positions of the thiol groups and the structure of the antibody itself, it can be known which thiol groups will form disulfide bonds when the thiol groups are not connected to the linker drug structure. That is, the thiol groups located in the light chain constant region of the second Fab domain and the heavy chain constant region of the second Fab domain will form disulfide bonds, the thiol groups located in the light chain constant region of the third Fab domain and the heavy chain constant region of the third Fab domain will form disulfide bonds, and the thiol groups located at the junction of the first Fab domain and the Fc domain and at the junction of the second Fab domain and the Fc domain will form disulfide bonds.

[0060] In a third aspect, the present invention provides a method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 as described in the first aspect, or use of the bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 as described in the second aspect in preparing anti-tumor drugs.

[0061] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] (1) The present invention provides a method for preparing a DAR6 antibody-drug conjugate. The metal ion-assisted drug conjugate controls the reduction of disulfide bonds so that the sulfhydryl group is selectively coupled to the drug payload, and six molecules of drug payload are coupled to three pairs 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 DAR6 is usually more than 60wt%, preferably more than 65wt%. No protein engineering, enzymatic reaction or additional payload modification is required, and the process is simple.

[0064] (2) The present invention prepares a DAR6 bispecific antibody-drug conjugate, which uses the interchain disulfide bonds of the bispecific antibody itself to couple the drug molecules without introducing any new amino acid sequences, thereby reducing the immunogenicity caused by the antibody in the body, 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

[0065] Figure 1 The figure is a schematic diagram of the preparation process of the antibody-drug conjugate of the present invention.

[0066] Figure 2 This is the hydrophobic interaction chromatogram of the antibody-drug conjugate prepared in Example 1. DETAILED DESCRIPTION

[0067] 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.

[0068] 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.

[0069] Example 1

[0070] This embodiment provides a method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6. The schematic diagram of the preparation process is shown in FIG. Figure 1 As shown, the following steps are included:

[0071] (1) TCEP (C4706-2G, Sigma-Aldrich), ZnCl 2 (STBK6550, Sigma-Aldrich) was mixed with a solution containing a bispecific antibody, 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, a bispecific antibody, TCEP and Zn 2+ The molar ratio of is 1:12:4, 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.

[0072] (2) 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.

[0073] (3) The product buffer was replaced with its storage buffer (20 mM histidine buffer, pH 5.5) using an ultrafiltration tube (30K, UFC803024, Thermo Fisher) to remove excess small molecule impurities in the reaction system.

[0074] Test Example 1

[0075] In this test example, the DAR value and drug distribution of the antibody-drug conjugate prepared in Example 1 were determined by HIC-HPLC. The results of the determination of the mass percentage of each component are shown in Table 1, and the hydrophobic interaction chromatogram is shown in Figure 2 As shown. Wherein D0 indicates that there is no drug coupling on the antibody, D1 indicates that one molecule of drug is coupled to one molecule of antibody, D2 indicates that two molecules of drug are coupled to one molecule of antibody, D3 indicates that three molecules of drug are coupled to one molecule of antibody, D4 indicates that four molecules of drug are coupled to one molecule of antibody, D5 indicates that five molecules of drug are coupled to one molecule of antibody, D6 indicates that six molecules of drug are coupled to one molecule of antibody, D8 indicates that eight molecules of drug are coupled to one molecule of antibody, and HIC DAR indicates the DAR value result calculated after hydrophobic interaction chromatography detection. It can be seen from Table 1 that the antibody-drug conjugate prepared by the method provided by the present invention has a content of D6 component exceeding 60wt%, preferably exceeding 65wt%, and its uniformity is higher than that produced by conventional random coupling process, and the content of D6 obtained by conventional method is usually less than 50wt%.

[0076] Table 1

[0077]

[0078]

[0079] In summary, the present invention provides a method for preparing a DAR6 antibody-drug conjugate, wherein the metal ion-assisted drug conjugation controls the reduction of disulfide bonds so that the sulfhydryl groups are selectively coupled to the drug payload, and six molecules of the drug payload are coupled to three pairs of open disulfide bonds of the antibody. The uniformity of the bispecific ADC is higher than that produced by conventional conjugation processes.

[0080] 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 multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6, characterized in that: The method for preparing the bispecific multivalent 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 a metal ion chelator and a drug molecule connected with a linker, and obtaining a bispecific multivalent antibody-drug conjugate with a conjugate antibody ratio of 6 after reaction.

2. The method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 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:(8-16):(2-6), preferably 1:(11-13):(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 multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 according to claim 1 or 2, 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:(25-50), 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 linker-linked drug molecule in the solution containing the bispecific antibody is 1:(6-40), preferably 1:(15-25); Preferably, the reaction temperature in step (2) is 8 to 16° C. and the reaction time is 0.5 to 1.5 h; Preferably, before obtaining the bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 in step (2), 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.

4. The method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 according to any one of claims 1 to 3, 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.

5. The method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 according to claim 4, 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.

6. The method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 according to claim 4 or 5, characterized in that: The first Fab domain, the second Fab domain and the third Fab domain are T cell receptor constant domains.

7. The method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 according to any one of claims 4 to 6, characterized in that: The composition of the bispecific antibody includes the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.

4.

8. A bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6, characterized in that: The bispecific multivalent antibody-drug conjugate is prepared by the method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 according to any one of claims 1 to 7.

9. The bispecific multivalent antibody-drug conjugate according to claim 8, characterized in that: The bispecific multivalent antibody-drug conjugate comprises a bispecific antibody, a linker and a drug, and the molar ratio of the drug to the bispecific antibody is 6; Preferably, the drug is connected to six thiol groups on the bispecific antibody through a linker, and the six thiol groups are respectively located in the light chain constant region of the second Fab domain, the heavy chain constant region of the second Fab domain, the light chain constant region of the third Fab domain, the heavy chain constant region of the third Fab domain, the junction between the first Fab domain and the Fc domain, and the junction between the second Fab domain and the Fc domain, and the six thiol groups exist in the form of disulfide bonds when not connected.

10. A method for preparing a bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 according to any one of claims 1 to 7, or use of the bispecific multivalent antibody-drug conjugate with a conjugate-antibody ratio of 6 according to claim 8 or 9 in preparing anti-tumor drugs.

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