Preparation method of bispecific antibody coupling medicine

Abstract

The invention relates to a preparation method of a bispecific antibody coupling drug. An engineered bispecific antibody comprises a first Fab structural domain, a second Fab structural domain, a hinge structural domain and an Fc structural domain, the second Fab structural domain comprises a TCR constant structural domain; the Fc domain comprises a KiH mutation. According to the engineering bispecific antibody disclosed by the invention, the homogeneity of a bispecific bispecific antibody coupling medicine can be remarkably improved. According to the invention, a metal-assisted coupling technology is adopted, disulfide bond reduction is controlled, so that sulfydryl is selectively coupled with the drug load, and the four-molecule drug load is coupled to two pairs of opened disulfide bonds of the antibody, so that the homogeneity of the bispecific antibody coupling drug is remarkably improved.

Description

Technical Field

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

[0002] Antibodies are immunoglobulins with multiple functions, but 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. Bispecific antibodies (BsAbs) are artificial antibodies that can specifically bind to two antigens or antigen epitopes at the same time. It can simultaneously target different antigens or epitopes on the cell surface, enhance its specific binding to tumor cells and directly kill tumor cells. Compared with monoclonal antibody combination therapy, it can also effectively reduce treatment costs.

[0003] Antibody-drug conjugates (ADCs) direct cytotoxic drugs to antigen-expressing cells or other target cells through antibody-antigen interactions. Therefore, compared with the use of antibodies or small molecule drugs alone, ADCs have the dual advantages of small molecules and antibody drugs. Currently, 10 bispecific 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. The defect of the engineering reactive cysteine ​​residue technology is that the thiol introduction step may cause the formation of incorrect disulfide bonds between the two Fab regions in the antibody, affecting subsequent coupling. The random coupling method will lead to the production of heterogeneous ADCs. The mixing of products with different DAR values ​​will hinder the expansion of the therapeutic window. Therefore, improving the homogeneity of ADC products is an urgent problem to be solved in the current ADC construction. Summary of the invention

[0004] To solve the above technical problems, the present invention provides a method for preparing a bispecific antibody-drug conjugate. The engineered bispecific antibody in the present invention can significantly improve the uniformity of the bispecific antibody-drug conjugate and increase the content of the bispecific antibody-drug conjugate with an antibody ratio of 4.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an engineered bispecific antibody for preparing a bispecific antibody-drug conjugate, wherein the engineered bispecific antibody comprises a first Fab domain, a second Fab domain, a hinge domain and an Fc structural region;

[0007] The second Fab domain comprises a TCR constant domain;

[0008] The Fc domain includes a KiH mutation.

[0009] In the present invention, the engineered bispecific antibody utilizes the TCR constant domain to replace the CH1 / CL constant domain in the antibody Fab domain, thereby ensuring homologous HC-LC pairing, and through KiH mutation ("knob-in-hole" technology), the heterodimerization of the bispecific antibody can be ensured.

[0010] Preferably, the hinge region contains at least two interchain disulfide bonds.

[0011] Preferably, the KiH mutation site is located in the Fc structural region.

[0012] Preferably, the Fc domain contains a first CH2, a second CH2, a first CH3 and a second CH3.

[0013] Preferably, the first CH3 and the second CH3 in the Fc domain are connected via a KiH mutation.

[0014] In the present invention, KiH is mutated into a knob-hole structure, which is a mechanism to ensure high heterodimerization. It means that a steric effect can also be selectively produced by amino acid engineering to favor heterodimer formation and disfavor homodimer formation. The specific structure is that the first Fc region, i.e., the Fc region in the first monomer containing the antibody Fab domain, contains a hole mutation, and the second Fc region, i.e., the Fc region in the second monomer containing the second Fab domain replaced with the TCR constant domain, contains a knob mutation. This further enhances heterodimerization and avoids mismatching between the two monomers.

[0015] Preferably, the antibody targeting antigen includes any one of HER2, ANG2, VEGFRA or Trop2, or a combination of at least two of them.

[0016] Preferably, the engineered bispecific antibody comprises any one of antibody A, antibody B or antibody C.

[0017] Preferably, the amino acid sequence of the first heavy chain of antibody A is as shown in SEQ ID NO:1.

[0018] SEQ ID NO: 1:

[0019] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0020] Preferably, the amino acid sequence of the first light chain of antibody A is as shown in SEQ ID NO:2.

[0021] SEQ ID NO:2:

[0022] DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0023] Preferably, the amino acid sequence of the second heavy chain of antibody A is as shown in SEQ ID NO:3.

[0024] SEQ ID NO:3:

[0025] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSPNPYYYDSSGYYYPGAFDIWGQ GTMVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAK PVTQIVSAEAAWGRASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0026] Preferably, the amino acid sequence of the second light chain of antibody A is as shown in SEQ ID NO:4.

[0027] SEQ ID NO:4:

[0028] QPGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFG TGTKVTVLPDIQNPDCAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPECTFFPS.

[0029] Preferably, the amino acid sequence of the first heavy chain of antibody B is as shown in SEQ ID NO:5.

[0030] SEQ ID NO:5:

[0031] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVL EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ IVSAEAWGRASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0032] Preferably, the amino acid sequence of the first light chain of antibody B is as shown in SEQ ID NO:6.

[0033] SEQ ID NO:6:

[0034] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFG QGTKVEIKPDIQNPDCAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPECTFFPS.

[0035] Preferably, the amino acid sequence of the second heavy chain of antibody B is as shown in SEQ ID NO:7.

[0036] SEQ ID NO:7:

[0037] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0038] Preferably, the amino acid sequence of the second light chain of antibody B is as shown in SEQ ID NO:8.

[0039] SEQ ID NO:8:

[0040] DIQMTQSPSSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0041] Preferably, the amino acid sequence of the first heavy chain of antibody C is as shown in SEQ ID NO:9.

[0042] SEQ ID NO:9:

[0043] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVL EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ IVSAEAWGRASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0044] Preferably, the amino acid sequence of the first light chain of antibody C is as shown in SEQ ID NO:10.

[0045] SEQ ID NO:10:

[0046] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFG QGTKVEIKPDIQNPDCAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPECTFFPS.

[0047] Preferably, the amino acid sequence of the second heavy chain of antibody C is as shown in SEQ ID NO:11.

[0048] SEQ ID NO:11:

[0049] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWNTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQ GSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0050] Preferably, the amino acid sequence of the second light chain of antibody C is as shown in SEQ ID NO:12.

[0051] SEQ ID NO:12:

[0052] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0053] In a second aspect, the present invention provides an antibody-drug conjugate having a conjugate-antibody ratio of 4, wherein the antibody-drug conjugate is an engineered bispecific antibody-loaded drug for preparing a bispecific antibody-drug conjugate as described in the first aspect.

[0054] Preferably, the drug comprises any one of paclitaxel, methotrexate, 5-fluorouracil, cytarabine, DM1, DM2, DM3, DM4, monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin D, Dxd, SN38 or MC-VC-PAB-MMAE, or a combination of at least two thereof.

[0055] The Chinese name of the MC-VC-PAB-MMAE is maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E.

[0056] The present invention includes cytotoxic drugs, especially cytotoxic drugs used in cancer treatment. Such drugs include but are not limited to DNA damaging agents, DNA binding agents, enzyme inhibitors (such as thymidylate synthase inhibitors and topoisomerase inhibitors) and tubulin inhibitors. Specific drugs include: paclitaxel, methotrexate, 5-fluorouracil, cytarabine, maytansinoids (including DM1, DM2, DM3, DM4) and auristatins (including monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD)), camptothecin topoisomerase I inhibitors (including Dxd and SN38). In a specific embodiment, the drug is coupled in the form of an intermediate linker-drug load compound, such as "MC-VC-PAB-MMAE".

[0057] Preferably, the location of drug loading includes the hinge domain and / or the first Fab domain.

[0058] Preferably, the drug is linked to four thiol groups on the bispecific antibody.

[0059] Preferably, the four thiol groups are respectively located at the junction of the first Fab domain and the Fc domain, the junction of the second Fab domain and the Fc domain, the heavy chain constant region of the first Fab domain and the light chain constant region of the first Fab domain.

[0060] Preferably, the four sulfhydryl groups exist in the form of disulfide bonds when not linked.

[0061] In the present invention, the bispecific antibody-drug conjugate constructed based on bispecific antibodies combines the advantages of bispecific antibodies and antibody-drug conjugates: compared with monoclonal antibodies, the bispecific antibodies in the combination can target tumor cells more specifically, overcome drug resistance, and increase drug safety. In addition, by promoting the coordinated endocytosis of the two targets through cross-linking, while improving the efficiency of toxins entering tumor cells, it can also further inhibit tumor cell growth signals by reducing the expression of receptor proteins on the cell membrane, achieving better therapeutic effects.

[0062] In the present invention, there are three pairs of disulfide bonds that can be opened, namely two pairs of disulfide bonds in the hinge region and the disulfide bonds in the first Fab domain, while the disulfide bonds in the second Fab domain cannot be opened. The second Fab domain has been engineered and there are no disulfide bonds that can be opened.

[0063] In a third aspect, the present invention provides a method for preparing an antibody-drug conjugate having a conjugate-antibody ratio of 4 as described in the second aspect, the preparation method comprising adding a reducing agent and a salt solution of transition metal ions to an engineered bispecific antibody to form a mixed solution, allowing the solution to stand, adding a chelating agent and a drug to react, and purifying the solution to obtain the drug.

[0064] In the present invention, metal-assisted coupling technology is used, and metal ions are contained in the salt solution, which produces selectivity in the disulfide bond reduction. After adding an effective amount of a metal chelator and an excess of a load that can react with a thiol group, an antibody-drug conjugate with a conjugate-antibody ratio of 4 is formed. By using the disclosed process to produce antibody-drug conjugates, the uniformity of the antibody-drug conjugates is higher than that produced by conventional coupling processes. Since the interchain disulfide bonds of the bispecific antibodies themselves are used for coupling without introducing any new amino acid sequences, the resulting engineered antibodies will cause less immunogenicity in vivo.

[0065] Preferably, the concentration of the engineered bispecific antibody in the mixed solution is 0.02-0.05 mM, for example, it can be 0.02 mM, 0.022 mM, 0.024 mM, 0.025 mM, 0.026 mM, 0.028 mM, 0.03 mM, 0.032 mM, 0.034 mM, 0.036 mM, 0.038 mM, 0.04 mM, 0.042 mM, 0.044 mM, 0.046 mM, 0.048 mM or 0.05 mM, etc.

[0066] Preferably, the reducing agent comprises tris(2-carboxyethyl)phosphine and / or dithiothreitol.

[0067] Preferably, the concentration of the reducing agent in the mixed solution is 0.3-0.6 mM, for example, it can be 0.3 mM, 0.32 mM, 0.34 mM, 0.36 mM, 0.38 mM, 0.4 mM, 0.42 mM, 0.44 mM, 0.45 mM, 0.46 mM, 0.08 mM, 0.5 mM, 0.52 mM, 0.54 mM, 0.56 mM, 0.58 mM or 0.6 mM, etc.

[0068] Preferably, the transition metal ions include Zn 2+ 、Cd 2+ or Hg 2+ Any one or a combination of at least two of the following.

[0069] Preferably, the salt in the salt solution comprises ZnCl 2 、Zn(NO 3 ) 2 、ZnSO 4 、Zn(CH 3 COO 2 、ZnI 2 、ZnBr 2 Or any one of zinc formate or a combination of at least two thereof.

[0070] Preferably, the concentration of the salt solution in the mixed solution is 0.1-0.2 mM, for example, it can be 0.1 mM, 0.11 mM, 0.12 mM, 0.13 mM, 0.14 mM, 0.15 mM, 0.16 mM, 0.17 mM, 0.18 mM, 0.19 mM or 0.2 mM, etc.

[0071] Preferably, the standing temperature is 10-15°C and the time is 12-18h. The 10-15°C may be, for example, 10°C, 11°C, 12°C, 13°C, 14°C or 15°C, etc. The 12-18h may be, for example, 12h, 13h, 14h, 15h, 16h, 17h or 18h, etc.

[0072] Preferably, the chelating agent comprises ethylenediaminetetraacetic acid and / or ethylene glycol tetraacetic acid.

[0073] Preferably, the concentration of the chelating agent in the reaction is 4-6 mM, for example, 4 mM, 4.2 mM, 4.4 mM, 4.6 mM, 4.8 mM, 5 mM, 5.2 mM, 5.4 mM, 5.6 mM, 5.8 mM and 6 mM.

[0074] Preferably, the concentration of the drug in the reaction is 0.5-0.8 mM, for example, it can be 0.5 mM, 0.52 mM, 0.54 mM, 0.56 mM, 0.58 mM, 0.6 mM, 0.62 mM, 0.64 mM, 0.66 mM, 0.68 mM, 0.7 mM, 0.72 mM, 0.74 mM, 0.76 mM, 0.78 mM or 0.8 mM, etc.

[0075] Preferably, the reaction temperature is 10-15°C, and the reaction time is 0.5-2h. The 10-15°C may be, for example, 10°C, 11°C, 12°C, 13°C, 14°C or 15°C, etc. The 0.5-2h may be, for example, 0.5h, 0.6h, 0.8h, 1.0h, 1.2h, 1.4h, 1.6h, 1.8h or 2.0h, etc.

[0076] Preferably, the purification method comprises any one of filtration, ion exchange, ultrafiltration or column chromatography, or a combination of at least two thereof.

[0077] In a fourth aspect, the present invention provides an application of an engineered bispecific antibody for preparing a bispecific antibody-drug conjugate according to the first aspect or an antibody-drug conjugate with a conjugate-antibody ratio of 4 according to the second aspect in preparing an anticancer drug.

[0078] Compared with the prior art, the present invention has at least the following beneficial effects:

[0079] 1. The engineered bispecific antibody constructed in the present invention replaces the CH1 / CL constant domain in the antibody Fab domain with the TCR constant domain, thereby ensuring homologous HC-LC pairing, and through the "knob-in-hole" technology, the heterodimerization of the bispecific antibody can be ensured.

[0080] 2. In the present invention, drugs are loaded on the basis of engineered bispecific antibodies, that is, 4 drug molecules are connected to each antibody. The bispecific antibodies can target tumor cells more specifically, promote the coordinated endocytosis of two targets through cross-linking, and improve the efficiency of toxins entering tumor cells. At the same time, it can further inhibit tumor cell growth signals by reducing the expression of receptor proteins on the cell membrane, thereby achieving better therapeutic effects.

[0081] 3. The present invention adopts metal-assisted coupling technology, and by controlling the reduction of disulfide bonds, the sulfhydryl group is selectively coupled to the drug load, and four molecules of drug load are coupled to two pairs of open disulfide bonds of the antibody, which significantly improves the homogeneity of the bispecific antibody-drug conjugate. The process is simple, and the uniformity of the obtained antibody-drug conjugate is significantly improved compared with the conventional method. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 Schematic diagram of the reaction of Examples 1-3.

[0083] Figure 2 This is a characterization diagram of the bispecific antibody-drug conjugate prepared in Example 1.

[0084] Figure 3 This is a characterization diagram of the bispecific antibody-drug conjugate prepared in Example 2.

[0085] Figure 4 This is a characterization diagram of the bispecific antibody-drug conjugate prepared in Example 3. DETAILED DESCRIPTION

[0086] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0087] The sources of the reagents used in the following examples are:

[0088] TCEP: Sigma-Aldric, C4706-2G;

[0089] ZnCl 2 : Sigma-Aldrich, STBK6550;

[0090] Dimethylacetamide: Sigma-Aldrich;

[0091] Ultrafiltration tube: Thermo Fisher, UFC803024.

[0092] Example 1

[0093] This example prepares antibody A-MC-VC-PAB-MMAE bispecific antibody conjugate drug

[0094] The bispecific antibody Antibody A constructed in this example comprises the following four chains: the amino acid sequence of the first heavy chain is SEQ ID NO: 1, the amino acid sequence of the first light chain is SEQ ID NO: 2, the amino acid sequence of the second heavy chain is SEQ ID NO: 3, and the amino acid sequence of the second light chain is SEQ ID NO: 4.

[0095] (1) Dissolve the bispecific antibody Antibody A in pH 7, 40 mM phosphate buffer to a concentration of 0.037 mM, add TCEP and ZnCl 2 A mixed solution was formed in which the concentration of TCEP was 0.45 mM and ZnCl 2 The concentration was 0.15 mM, and the mixed solution was placed at 12°C for 16 h.

[0096] (2) Add EDTA to a concentration of 5 mM, then add MC-VC-PAB-MMAE dissolved in dimethylacetamide to a concentration of 0.64 mM, and react at 12°C for 1 h.

[0097] (3) Use an ultrafiltration tube (30K) to replace the product buffer with a pH 5.5, 20 mM histidine buffer (4.21 mM histidine, 15.79 mM histidine hydrochloride) to remove excess small molecule impurities in the reaction system. The specific reaction process is as follows: Figure 1 shown.

[0098] Example 2

[0099] This example prepares the antibody B-MC-VC-PAB-MMAE bispecific antibody conjugate drug

[0100] The bispecific antibody antibody B constructed in this example includes the following four chains, the amino acid sequence of the first heavy chain is SEQ ID NO: 5, the amino acid sequence of the first light chain is SEQ ID NO: 6, the amino acid sequence of the second heavy chain is SEQ ID NO: 7, and the amino acid sequence of the second light chain is SEQ ID NO: 8.

[0101] (1) Dissolve the bispecific antibody Antibody B in pH 7, 40 mM phosphate buffer to a concentration of 0.038 mM, add TCEP and ZnCl 2 A mixed solution was formed in which the concentration of TCEP was 0.38 mM and ZnCl 2 The concentration was 0.15 mM, and the mixed solution was placed at 12°C for 16 h.

[0102] (2) Add EDTA to a concentration of 5 mM, then add MC-VC-PAB-MMAE dissolved in dimethylacetamide to a concentration of 0.64 mM, and react at 12°C for 1 h.

[0103] (3) Use an ultrafiltration tube (30K) to replace the product buffer with a pH 5.5, 20 mM histidine buffer (4.21 mM histidine, 15.79 mM histidine hydrochloride) to remove excess small molecule impurities in the reaction system. The specific reaction process is as follows: Figure 1 shown.

[0104] Example 3

[0105] This example prepares the antibody C-MC-VC-PAB-MMAE bispecific antibody conjugate drug

[0106] The bispecific antibody antibody C constructed in this example includes the following four chains, the amino acid sequence of the first heavy chain is SEQ ID NO: 9, the amino acid sequence of the first light chain is SEQ ID NO: 10, the amino acid sequence of the second heavy chain is SEQ ID NO: 11, and the amino acid sequence of the second light chain is SEQ ID NO: 12.

[0107] (1) Dissolve the bispecific antibody Antibody C in pH 7, 40 mM phosphate buffer to a concentration of 0.038 mM, add TCEP and ZnCl 2 A mixed solution was formed in which the concentration of TCEP was 0.38 mM and ZnCl 2 The concentration was 0.15 mM, and the mixed solution was placed at 12°C for 16 h.

[0108] (2) Add EDTA to a concentration of 5 mM, then add MC-VC-PAB-MMAE dissolved in dimethylacetamide to a concentration of 0.64 mM, and react at 12°C for 1 h.

[0109] (3) Use an ultrafiltration tube (30K) to replace the product buffer with a pH 5.5, 20 mM histidine buffer (4.21 mM histidine, 15.79 mM histidine hydrochloride) to remove excess small molecule impurities in the reaction system. The specific reaction process is as follows: Figure 1 shown.

[0110] Test Example 1

[0111] In this test example, the DAR value and drug distribution of the bispecific antibody-drug conjugates prepared in Examples 1-3 were determined using hydrophobic interaction chromatography (HIC).

[0112] The bispecific antibody-drug conjugates prepared in Examples 1-3 were characterized and identified, and the specific results are shown in Table 1.

[0113] Table 1

[0114] sample D0％ D1％ D2％ D3％ D4％ D5％ D6％ D7％ D8％ Drug to Antibody Ratio Example 1 0.1 / 7.5 / 75.4 / 17.0 / / 4.2 Example 2 0.1 / 4.8 / 74.8 / 20.3 / / 4.3 Example 3 / / 7.3 0.1 67.2 2.4 22.6 / 0.4 4.4

[0115] The results show that: Figure 2 , Figure 3 and Figure 4 As shown, through characterization, it can be seen that the antibody-drug conjugate with an antibody ratio of 4 in the bispecific antibody-drug conjugate exceeds 67%, which significantly improves the uniformity of the product.

[0116] In summary, the present invention adopts metal-assisted coupling technology to control the reduction of disulfide bonds through chemical reactions, without the need for protein engineering, introduction of enzyme engineering and modification of payloads. After reacting with a thiol-reactive payload, a bispecific antibody-drug conjugate is obtained, which significantly improves the uniformity of the product.

[0117] 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. An engineered bispecific antibody for preparing a bispecific antibody-drug conjugate, characterized in that: The engineered bispecific antibody comprises a first Fab domain, a second Fab domain, a hinge domain and an Fc structural region; The second Fab domain comprises a TCR constant domain; The Fc domain includes a KiH mutation.

2. The engineered bispecific antibody for preparing a bispecific antibody-drug conjugate according to claim 1, characterized in that: The hinge region contains at least two interchain disulfide bonds; Preferably, the KiH mutation site is located in the Fc structural region; Preferably, the Fc domain contains a first CH2, a second CH2, a first CH3 and a second CH3; Preferably, the first CH3 and the second CH3 in the Fc domain are connected via a KiH mutation.

3. The engineered bispecific antibody for preparing a bispecific antibody-drug conjugate according to claim 1 or 2, characterized in that: The engineered bispecific antibody comprises any one of antibody A, antibody B or antibody C.

4. The engineered bispecific antibody for preparing a bispecific antibody-drug conjugate according to claim 3, characterized in that: The amino acid sequence of the first heavy chain of the antibody A is shown in SEQ ID NO: 1; Preferably, the amino acid sequence of the first light chain of antibody A is as shown in SEQ ID NO: 2; Preferably, the amino acid sequence of the second heavy chain of the antibody A is as shown in SEQ ID NO: 3; Preferably, the amino acid sequence of the second light chain of antibody A is as shown in SEQ ID NO: 4; Preferably, the amino acid sequence of the first heavy chain of antibody B is as shown in SEQ ID NO: 5; Preferably, the amino acid sequence of the first light chain of antibody B is as shown in SEQ ID NO: 6; Preferably, the amino acid sequence of the second heavy chain of antibody B is as shown in SEQ ID NO: 7; Preferably, the amino acid sequence of the second light chain of antibody B is as shown in SEQ ID NO: 8; Preferably, the amino acid sequence of the first heavy chain of the antibody C is as shown in SEQ ID NO: 9; Preferably, the amino acid sequence of the first light chain of the antibody C is as shown in SEQ ID NO: 10; Preferably, the amino acid sequence of the second heavy chain of the antibody C is as shown in SEQ ID NO: 11; Preferably, the amino acid sequence of the second light chain of antibody C is as shown in SEQ ID NO:

12.

5. An antibody-drug conjugate with a conjugate-antibody ratio of 4, characterized in that: The antibody-drug conjugate is an engineered bispecific antibody-loaded drug for preparing a bispecific antibody-drug conjugate according to any one of claims 1 to 4.

6. The antibody-drug conjugate with a conjugate-antibody ratio of 4 according to claim 5, characterized in that: The drug comprises any one of paclitaxel, methotrexate, 5-fluorouracil, cytarabine, DM1, DM2, DM3, DM4, monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin D, Dxd, SN38 or MC-VC-PAB-MMAE, or a combination of at least two thereof; Preferably, the location of the drug loading includes the hinge domain and / or the first Fab domain; Preferably, the drug is linked to four thiol groups on the bispecific antibody; Preferably, the four thiol groups are respectively located at the junction of the first Fab domain and the Fc domain, at the junction of the second Fab domain and the Fc domain, at the heavy chain constant region of the first Fab domain, and at the light chain constant region of the first Fab domain; Preferably, the four sulfhydryl groups exist in the form of disulfide bonds when not linked.

7. A method for preparing an antibody-drug conjugate with a conjugate-antibody ratio of 4 according to claim 5 or 6, characterized in that: The preparation method comprises the steps of adding a reducing agent and a salt solution of transition metal ions to an engineered bispecific antibody to form a mixed solution, allowing the solution to stand, adding a chelating agent and a drug to react, and purifying the solution to obtain the bispecific antibody.

8. The preparation method according to claim 7, characterized in that: The concentration of the engineered bispecific antibody in the mixed solution is 0.02-0.05 mM; Preferably, the reducing agent comprises tris(2-carboxyethyl)phosphine and / or dithiothreitol; Preferably, the concentration of the reducing agent in the mixed solution is 0.3-0.6 mM; Preferably, the transition metal ions include Zn 2+ 、Cd 2+ or Hg 2+ Any one or a combination of at least two of the following: Preferably, the salt in the salt solution includes any one or a combination of at least two of ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2 or zinc formate; Preferably, the concentration of the salt solution in the mixed solution is 0.1-0.2 mM; Preferably, the standing temperature is 10-15°C and the standing time is 12-18h.

9. The preparation method according to claim 7 or 8, characterized in that: The chelating agent includes ethylenediaminetetraacetic acid and / or ethylene glycol tetraacetic acid; Preferably, the concentration of the chelating agent in the reaction is 4-6 mM; Preferably, the concentration of the drug in the reaction is 0.5-0.8 mM; Preferably, the reaction temperature is 10-15°C and the reaction time is 0.5-2h; Preferably, the purification method comprises any one of filtration, ion exchange, ultrafiltration or column chromatography, or a combination of at least two thereof.

10. Use of an engineered bispecific antibody for preparing a bispecific antibody-drug conjugate according to any one of claims 1 to 4 or an antibody-drug conjugate with a conjugate-antibody ratio of 4 according to claim 5 or 6 in preparing an anticancer drug.

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