Engineered bispecific antibody coupling drug as well as preparation method and application thereof

Abstract

The invention relates to an engineered bispecific antibody coupling medicine as well as a preparation method and application thereof. The ratio of a conjugate to an antibody of the engineered bispecific antibody coupling medicine is 2; the location of the drug loading includes a hinge domain. According to the present invention, the conjugate-antibody ratio of the antibody coupling drug is 2, the drug is connected to the hinge structure domain, and the disulfide bond between the chains of the double antibodies is adopted to carry out coupling without introducing any brand new amino acid sequence, such that the immunogenicity caused by the obtained engineered antibody in vivo is less;

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an engineered bispecific antibody-drug conjugate and a preparation method and application thereof. 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] In order to solve the above technical problems, the present invention provides an engineered bispecific antibody-drug conjugate and a preparation method and application thereof. The conjugate-antibody ratio of the engineered bispecific antibody-drug conjugate is 2, that is, two drug molecules are connected to each antibody, and the drug is loaded at the hinge domain. Since the interchain disulfide bonds of the bispecific antibody itself are used for conjugation without introducing any new amino acid sequences, the resulting engineered antibody will cause less immunogenicity in vivo.

[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-drug conjugate, wherein the conjugate-antibody ratio of the engineered bispecific antibody-drug conjugate is 2;

[0007] The location of drug loading includes the hinge domain.

[0008] In the present invention, the antibody is a bispecific antibody, which can not only improve the selectivity of tumor co-expression targeting, but also significantly increase the internalization of antibody-drug conjugates in tumor cells. Compared with monoclonal antibodies, the bispecific antibodies in the combination can target tumor cells more specifically, overcome drug resistance, and increase drug safety.

[0009] Preferably, the engineered bispecific antibody comprises a first Fab domain, a second Fab domain, a hinge domain and an Fc region.

[0010] Preferably, the second Fab domain comprises a TCR constant domain.

[0011] 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 the "knob-in-hole" technology, the heterodimerization of the bispecific antibody can be ensured.

[0012] Preferably, the Fc domain comprises a KiH mutation.

[0013] Preferably, the drug is linked to two thiol groups on the bispecific antibody.

[0014] Preferably, the two thiol groups are respectively 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.

[0015] Preferably, the two sulfhydryl groups exist in the form of a disulfide bond when not linked.

[0016] 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.

[0017] Preferably, the hinge region contains at least two interchain disulfide bonds.

[0018] Preferably, the KiH mutation site is located in the Fc structural region.

[0019] Preferably, the Fc domain contains a first CH2, a second CH2, a first CH3 and a second CH3.

[0020] Preferably, the first CH3 and the second CH3 in the Fc domain are connected via a KiH mutation.

[0021] Preferably, the drug comprises any one of paclitaxel, methotrexate, 5-fluorouracil, cytarabine, maytansine, monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin D, Dxd, SN38 or MC-VC-PAB-MMAE, or a combination of at least two thereof.

[0022] The Chinese name of the MC-VC-PAB-MMAE is maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E.

[0023] The drug 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 microtubule 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.

[0024] Preferably, the engineered bispecific antibody comprises any one of antibody A or antibody B.

[0025] Preferably, the amino acid sequence of the first heavy chain of antibody A is as shown in SEQ ID NO:1.

[0026] SEQ ID NO: 1:

[0027] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVL EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ IVSAEAWGRASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0028] Preferably, the amino acid sequence of the first light chain of antibody A is as shown in SEQ ID NO:2.

[0029] SEQ ID NO:2:

[0030] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFG QGTKVEIKPDIQNPDCAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPECTFFPS.

[0031] Preferably, the amino acid sequence of the second heavy chain of antibody A is as shown in SEQ ID NO:3.

[0032] SEQ ID NO:3:

[0033] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0034] Preferably, the amino acid sequence of the second light chain of antibody A is as shown in SEQ ID NO:4.

[0035] SEQ ID NO:4:

[0036] DIQMTQSPSSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0037] Preferably, the amino acid sequence of the first heavy chain of antibody B is as shown in SEQ ID NO:5.

[0038] SEQ ID NO:5:

[0039] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVL EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ IVSAEAWGRASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0040] Preferably, the amino acid sequence of the first light chain of antibody B is as shown in SEQ ID NO:6.

[0041] SEQ ID NO:6:

[0042] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFG QGTKVEIKPDIQNPDCAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPECTFFPS.

[0043] Preferably, the amino acid sequence of the second heavy chain of antibody B is as shown in SEQ ID NO:7.

[0044] SEQ ID NO:7:

[0045] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWNTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQ GSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0046] Preferably, the amino acid sequence of the second light chain of antibody B is as shown in SEQ ID NO:8.

[0047] SEQ ID NO:8:

[0048] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0049] In a second aspect, the present invention provides a method for preparing the engineered bispecific antibody-drug conjugate described in the first aspect, the preparation method comprising adding a reducing agent and a salt solution of transition metal ions to the engineered bispecific antibody to form a mixed solution and letting it stand, adding an oxidant to carry out an oxidation reaction, then adding a quenching oxidant to quench the oxidation reaction, then adding a chelating agent and a drug to react, and obtaining the product after purification.

[0050] In the present invention, transition metal ions are selective in disulfide bond reduction and oxidation, and 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 2 is formed. The antibody-drug conjugate prepared by this method has a higher uniformity than that produced by a conventional conjugation process. Since the interchain disulfide bond of the bispecific antibody itself is used for conjugation without introducing any new amino acid sequence, the resulting engineered antibody will cause less immunogenicity in vivo.

[0051] Preferably, the concentration of the engineered bispecific antibody in the mixed solution is 0.03-0.05 mM, for example, it can be 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.

[0052] Preferably, the reducing agent includes any one or a combination of at least two of tris(2-carboxyethyl)phosphine, dithiothreitol, diphenylphosphinoacetic acid, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(diphenylphosphino)propionic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, 1,3,5-triaza-7-phosphatricyclo[3.3.1.13.7]decane and n-butyldi(1-adamantyl)phosphine, sodium borohydride or lithium aluminum hydride.

[0053] Preferably, the concentration of the reducing agent in the mixed solution is 0.3-0.5 mM, for example, it can be 0.3 mM, 0.32 mM, 0.34 mM, 0.36 mM, 0.37 mM, 0.4 mM, 0.42 mM, 0.44 mM, 0.46 mM or 0.5 mM, etc.

[0054] Preferably, the transition ions include Zn 2+ 、Cd 2+ or Hg 2+ Any one or a combination of at least two of the following.

[0055] Preferably, the salt in the salt solution includes any one of ZnCl2, Zn(NO3)2, ZnSO4, Zn(CH3COO)2, ZnI2, ZnBr2 or C2H2O4Zn, or a combination of at least two thereof.

[0056] 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, 0.12 mM, 0.14 mM, 0.16 mM, 0.18 mM or 0.2 mM.

[0057] Preferably, the standing time is 12-18 hours and the temperature is 10-15° C. The 12-18 hours may be, for example, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours or 18 hours, etc. The 10-15° C. may be, for example, 10° C., 11° C., 12° C., 13° C., 14° C. or 15° C., etc.

[0058] Preferably, the oxidant comprises 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.

[0059] Preferably, the concentration of the oxidant is 0.2-0.4 mM, for example, it can be 0.2 mM, 0.22 mM, 0.24 mM, 0.26 mM, 0.28 mM, 0.3 mM, 0.32 mM, 0.34 mM, 0.36 mM, 0.38 mM or 0.4 mM, etc.

[0060] Preferably, the oxidation reaction time is 0.5-2h, and the temperature is 10-15°C. The 0.5-2h may be, for example, 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h or 2h, etc. The 10-15°C may be, for example, 10°C, 11°C, 12°C, 13°C, 14°C or 15°C, etc.

[0061] Preferably, the quenching oxidant comprises any one or a combination of at least two of tris(2-carboxyethyl)phosphine, tris(3-hydroxypropyl)phosphine, dithiothreitol, 2-diphenylphosphinobenzoic acid or triphenylphosphine trimersulfonic acid (DiPPBS3).

[0062] Preferably, the concentration of the quenching oxidant is 0.05-0.065 mM, for example, it can be 0.05 mM, 0.052 mM, 0.054 mM, 0.056 mM, 0.058 mM, 0.06 mM, 0.062 mM, 0.064 mM or 0.065 mM, etc.

[0063] Preferably, the quenching oxidation reaction time is 0.1-1h, and the temperature is 10-15°C. The 0.1-1h may be, for example, 0.1h, 0.2h, 0.3h, 0.4h, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h or 1h, etc. The 10-15°C may be, for example, 10°C, 11°C, 12°C, 13°C, 14°C or 15°C, etc.

[0064] Preferably, the chelating agent includes any one of ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid, nitrilotriacetic acid, N-hydroxyethylethylenediaminetriacetic acid or diethyltriaminepentaacetic acid, or a combination of at least two thereof.

[0065] 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 or 6 mM, etc.

[0066] Preferably, the concentration of the drug in the reaction is 0.4-0.6 mM, for example, it can be 0.4 mM, 0.42 mM, 0.44 mM, 0.46 mM, 0.48 mM, 0.5 mM, 0.52 mM, 0.54 mM, 0.56 mM or 0.6 mM, etc.

[0067] 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.

[0068] Preferably, the purification method comprises any one of filtration, ion exchange, ultrafiltration or column chromatography, or a combination of at least two thereof.

[0069] In a third aspect, the present invention provides a use of the engineered bispecific antibody-drug conjugate according to the first aspect in the preparation of an anticancer drug.

[0070] Compared with the prior art, the present invention has at least the following beneficial effects:

[0071] 1. The present invention provides an engineered bispecific antibody-drug conjugate. Since the antibody is a bispecific antibody, it targets tumor cells more specifically, overcomes drug resistance, and increases drug safety. The antibody-drug conjugate has a conjugate-antibody ratio of 2, and the drug is connected to the hinge domain. Since the bispecific antibody itself is used for conjugation without introducing any new amino acid sequence, the resulting engineered antibody will cause less immunogenicity in vivo.

[0072] 2. The preparation method of the present invention is a metal-assisted coupling technology, which controls disulfide bond reduction and sulfhydryl oxidation through chemical reactions, does not require protein engineering, introduction of enzyme engineering, and modification of payloads. After reacting with a sulfhydryl-reactive load, a bispecific antibody with improved uniformity and a conjugate antibody ratio of 2 can be produced for the bispecific antibody, and the operation is simple and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Schematic diagram of the reaction of Example 1-2.

[0074] Figure 2 This is a characterization diagram of the bispecific antibody-drug conjugate prepared in Example 1.

[0075] Figure 3 This is a characterization diagram of the bispecific antibody-drug conjugate prepared in Example 2. DETAILED DESCRIPTION

[0076] 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.

[0077] The sources of the reagents used in the following examples are:

[0078] TCEP: Sigma-Aldric, C4706-2G;

[0079] ZnCl2: Sigma-Aldrich, STBK6550;

[0080] 5,5'-Dithiobis(2-nitrobenzoic acid): Sigma-Aldrich, D8130-500MG;

[0081] DiPPBS3: WuXi Biologics;

[0082] Dimethylacetamide: Sigma-Aldrich;

[0083] MC-VC-PAB-MMAE: WuXi AppTec;

[0084] Ultrafiltration tube: Thermo Fisher, UFC803024.

[0085] Example 1

[0086] This example prepares antibody A-MC-VC-PAB-MMAE bispecific antibody conjugate drug

[0087] 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.

[0088] (1) The bispecific antibody A was dissolved in a pH 7, 40 mM phosphate buffer to a concentration of 0.038 mM, TCEP and ZnCl2 were added to form a mixed solution, wherein the concentration of TCEP was 0.38 mM and the concentration of ZnCl2 was 0.15 mM. The mixed solution was placed at 12° C. and allowed to stand for 16 h.

[0089] (2) Add 5,5'-dithiobis(2-nitrobenzoic acid) to 0.25 mM and react at 12°C for 1 h. Then add 0.060 mM DiPPBS3 and react at 12°C for 0.5 h to quench the oxidation reaction.

[0090] (3) Add EDTA to a concentration of 5 mM to capture Zn 2+ , MC-VC-PAB-MMAE dissolved in dimethylacetamide was added to a concentration of 0.64 mM, and the reaction was continued at 12°C for 1 h.

[0091] (4) Use an ultrafiltration tube (30K) to replace the phosphate 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.

[0092] Example 2

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

[0094] The bispecific 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.

[0095] (1) Antibody B was dissolved in pH 7, 40 mM phosphate buffer to a concentration of 0.038 mM, TCEP and ZnCl2 were added to form a mixed solution, wherein the concentration of TCEP was 0.38 mM and the concentration of ZnCl2 was 0.15 mM. The mixed solution was placed at 12° C. and allowed to stand for 16 h.

[0096] (2) Add 5,5'-dithiobis(2-nitrobenzoic acid) to 0.32 mM and react at 12°C for 1 h. Then add 0.060 mM DiPPBS3 and react at 12°C for 0.5 h to quench the oxidation reaction.

[0097] (3) Add 5 mM EDTA to capture Zn 2+, a 0.50 mM solution of MC-VC-PAB-MMAE in dimethylacetamide was added simultaneously, and the reaction was continued at 12°C for 1 h.

[0098] (4) Use an ultrafiltration tube (30K) to replace the phosphate 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.

[0099] Test Example 1

[0100] In this test example, the DAR value and drug distribution of the bispecific antibody-drug conjugates prepared in Examples 1 and 2 were determined using hydrophobic interaction chromatography (HIC).

[0101] The bispecific antibody-drug conjugates prepared in Examples 1-3 were characterized and identified, and the specific results are shown in Table 1.

[0102] Table 1

[0103]

[0104] The results show that: Figure 2 and Figure 3 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 80%, which significantly improves the uniformity of the product.

[0105] 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.

[0106] 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-drug conjugate, characterized in that: The engineered bispecific antibody-drug conjugate has a conjugate-antibody ratio of 2; The location of drug loading includes the hinge domain.

2. The engineered bispecific antibody-drug conjugate according to claim 1, characterized in that: The engineered bispecific antibody comprises a first Fab domain, a second Fab domain, a hinge domain and an Fc structural region; Preferably, the second Fab domain comprises a TCR constant domain; Preferably, the Fc domain comprises a KiH mutation; Preferably, the drug is linked to two thiol groups on the bispecific antibody; Preferably, the two thiol groups are respectively 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; Preferably, the two sulfhydryl groups exist in the form of a disulfide bond when not linked.

3. The engineered bispecific antibody-drug conjugate according to claim 2, 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 by a KiH mutation; Preferably, the drug comprises any one of paclitaxel, methotrexate, 5-fluorouracil, cytarabine, maytansine, monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin D, Dxd, SN38 or MC-VC-PAB-MMAE, or a combination of at least two thereof.

4. The engineered bispecific antibody-drug conjugate according to any one of claims 1 to 3, characterized in that: The engineered bispecific antibody comprises any one of antibody A or antibody B.

5. The engineered bispecific antibody-drug conjugate according to claim 4, 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.

6. A method for preparing an engineered bispecific antibody-drug conjugate according to any one of claims 1 to 5, characterized in that: The preparation method comprises adding a reducing agent and a salt solution of transition metal ions to the engineered bispecific antibody to form a mixed solution and letting it stand, adding an oxidant to carry out an oxidation reaction, then adding a quenching oxidant to carry out a quenching oxidation reaction, then adding a chelating agent and a drug to react, and purifying to obtain the product.

7. The preparation method according to claim 6, characterized in that: The concentration of the engineered bispecific antibody in the mixed solution is 0.03-0.05 mM; Preferably, the reducing agent comprises any one or a combination of at least two of tris(2-carboxyethyl)phosphine, dithiothreitol, diphenylphosphinoacetic acid, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3-(diphenylphosphino)propylamine, 3-(diphenylphosphino)propionic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, 1,3,5-triaza-7-phosphatricyclo[3.3.1.13.7]decane and n-butyldi(1-adamantyl)phosphine, sodium borohydride or lithium aluminum hydride; Preferably, the concentration of the reducing agent in the mixed solution is 0.3-0.5 mM; Preferably, the transition 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 C2H2O4Zn; Preferably, the concentration of the salt solution in the mixed solution is 0.1-0.2 mM; Preferably, the standing time is 12-18 hours and the temperature is 10-15°C.

8. The preparation method according to claim 6 or 7, 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 concentration of the oxidant is 0.2-0.4 mM; Preferably, the oxidation reaction time is 0.5-2h and the temperature is 10-15°C; Preferably, the quenching oxidant comprises any one or a combination of at least two of tris(2-carboxyethyl)phosphine, tris(3-hydroxypropyl)phosphine, dithiothreitol, 2-diphenylphosphinobenzoic acid or triphenylphosphine trimersulfonic acid; Preferably, the concentration of the quenching oxidant is 0.05-0.065 mM; Preferably, the quenching oxidation reaction takes place for 0.1-1 h at a temperature of 10-15 °C.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The chelating agent includes any one of ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid, nitrilotriacetic acid, N-hydroxyethylethylenediaminetriacetic acid or diethyltriaminepentaacetic acid or a combination of at least two thereof; Preferably, the concentration of the chelating agent in the reaction is 4-6 mM; Preferably, the concentration of the drug in the reaction is 0.4-0.6 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 the engineered bispecific antibody-drug conjugate according to any one of claims 1 to 5 in the preparation of anticancer drugs.

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