A bispecific antibody targeting tissue factor, antibody drug conjugate, and methods of making and using the same

Abstract

This invention provides a bispecific antibody targeting tissue factors, an antibody-drug conjugate (ADC), its preparation method, and its application, belonging to the field of biopharmaceutical preparation technology. The bispecific antibody provided by this invention can efficiently target tissue factors in tumor cells, exhibiting advantages such as strong affinity and high endocytosis efficiency. This invention also provides an ADC, which, under the action of the bispecific antibody, specifically binds to tumor surface antigens and is internalized into tumor cells, achieving precise drug delivery for antitumor drugs. The ADC provided by this invention exhibits good tumor-suppressing effects in both cell and animal models, and is non-toxic and harmless to animals, demonstrating excellent potential for cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical preparation technology, specifically relating to a bispecific antibody targeting tissue factors, an antibody-drug conjugate, its preparation method, and its application. Background Technology

[0002] Tissue factor (TF) is abnormally expressed to varying degrees in various tumor cells, and its expression is positively correlated with tumor malignancy. For example, in pancreatic cancer, cervical cancer, breast cancer, non-small cell lung cancer, endometrial cancer, prostate cancer, ovarian cancer, esophageal cancer, and bladder cancer, abnormally high expression of TF leads to increased coagulation activity in tumor tissue and blood vessels, enhanced tumor cell adhesion, and promotes the metastasis and escape of tumor cells that invade blood vessels. Within tumor cells, TF promotes VEGF transcription through intracellular signal transduction, inducing tumor angiogenesis. High expression of TF is closely related to tumor growth, angiogenesis, metastasis, and clinical treatment, making the development of tumor therapeutics targeting TF a growing focus in the industry.

[0003] Antibody-drug conjugates (ADCs) improve antitumor efficacy and reduce systemic toxicity by targeting and delivering highly potent cytotoxic drugs to tumor cells. However, traditional monoclonal antibody-drug conjugates have limitations, such as limited affinity for target antigens and low internalization efficiency, leading to insufficient drug delivery efficiency and poor efficacy. Summary of the Invention

[0004] The purpose of this invention is to provide a bispecific antibody targeting tissue factor, which has strong binding properties and endocytosis efficiency against pancreatic cancer and ovarian cancer, providing an effective tool for the treatment of antibody-drug conjugates.

[0005] The present invention provides a bispecific antibody targeting tissue factor, comprising a heavy chain structure 1 targeting tissue factor, a light chain structure targeting tissue factor, and a heavy chain structure 2 targeting tissue factor containing a single chain antibody. The amino acid sequence of the heavy chain structure 1 of the target tissue factor is shown in SEQ ID NO:2; The amino acid sequence of the heavy chain structure 2 containing the single-chain antibody that targets tissue factor is shown in SEQ ID NO:4; The amino acid sequence of the light chain structure of the target tissue factor is shown in SEQ ID NO:6.

[0006] This invention provides a gene encoding a bispecific antibody targeting the tissue factor.

[0007] Preferably, the nucleotide sequence of the gene is shown in SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5.

[0008] The present invention provides a recombinant vector comprising the aforementioned gene.

[0009] The present invention provides a recombinant cell comprising the gene or the recombinant vector.

[0010] The present invention provides an antibody-drug conjugate, characterized in that it comprises an antitumor drug and a bispecific antibody targeting a tissue factor conjugated with the antitumor drug, or a bispecific antibody targeting a tissue factor prepared from the gene, the recombinant vector, or the recombinant cell.

[0011] Preferably, the antitumor drug includes at least one of the following: monomethylolpropamine E, monomethylolpropamine F, microtubule inhibitor DM1, microtubule inhibitor DM4, DNA topoisomerase I inhibitor DXd, and 7-ethyl-10-hydroxycamptothecin.

[0012] The present invention provides a method for preparing the antibody-drug conjugate, comprising the following steps: linking a bispecific antibody targeting tissue factor and an antitumor drug via disulfide bonds.

[0013] This invention provides the application of the antibody-drug conjugate or the antibody-drug conjugate prepared by the method described above in the preparation of antitumor drugs.

[0014] Preferably, the tumor includes at least one of the following: pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, non-small cell lung cancer, colon cancer, endometrial cancer, prostate cancer, esophageal cancer, and bladder cancer; The host sources of the tumors include humans and / or primates.

[0015] This invention provides a bispecific antibody B830 targeting tissue factor, comprising a heavy chain structure 1 targeting tissue factor, a light chain structure targeting tissue factor, and a heavy chain structure 2 containing a single-chain antibody targeting tissue factor; the amino acid sequences are shown in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. The bispecific antibody comprises two antibodies targeting tissue factor, exhibiting a Y-shaped structure, a complete structure composed of two different heavy chains and one light chain, thereby reducing light chain mismatch problems and resulting in good stability and targeting ability of the bispecific antibody in vivo. Experiments show that the bispecific antibody has high affinity for tissue factor derived from primates and humans, and exhibits good binding ability and endocytosis efficiency for tumor cells specifically expressing tissue factor. Therefore, the bispecific antibody provides an effective targeting tool for the preparation of antibody-drug conjugates.

[0016] This invention also provides antibody-drug conjugates, including an antitumor drug and a bispecific antibody targeting tissue factor conjugated to the antitumor drug, or a bispecific antibody targeting tissue factor prepared from the gene, the recombinant vector, or the recombinant cells. Experiments show that using B830 as the targeting antibody can significantly improve the entry of antitumor drugs into tumor cells. Compared with single-target antibodies, it can greatly enhance the tumor-killing ability of tissue factor-specific expression. Furthermore, animal tumor model results show that the ADC drug prepared from B830 can significantly inhibit tumor growth in a dose-dependent manner. In the Bxpc-3 pancreatic cancer animal model, it is superior to the commercially available drug DS8201, and in the sk-ov-3 animal model, it is superior to the commercially available drug DS8201. Attached Figure Description

[0017] Figure 1 Schematic diagram of the B830 dual-resistance structure design; Figure 2 Results of SDS-PAGE electrophoresis of B830 bispecific antibody; Figure 3 The affinity of B830 bispecific antibody to TF protein was determined by ELISA. Figure 4 Results of surface plasmon resonance (SPR) assay for the affinity between B830 bispecific antibody and TF protein. Figure 5 Results of detection of B830 bispecific antibody binding on the surface of human ovarian cancer cell line SK-ov-3; Figure 6 Results of endocytosis efficiency of Bxpc-3 bispecific antibody against B830 in human pancreatic cancer cell line; Figure 7 The results show the endocytosis efficiency of the B830 double antibody in cell line sk-ov-3. Figure 8 The results show the survival rate of Bxpc-3 tumor cells at different concentrations of B830 bispecific antibody. Figure 9 The results show the survival rate of SK-OV-3 tumor cells against different concentrations of B830 bispecific antibody. Figure 10 The results show the survival rate of JIMT-1 tumor cells (human breast cancer cells) against different concentrations of B830 bispecific antibody; Figure 11 The results show the survival rate of different concentrations of B830 bispecific antibody against tumor cells SiHa (human cervical squamous cell carcinoma cells); Figure 12 The effect of different drug administration groups on tumor weight in an animal tumor model induced by Bxpc-3 tumor cells; Figure 13The effect of different drug administration groups on tumor volume in an animal tumor model induced by Bxpc-3 tumor cells; Figure 14 The effect of different drug administration groups on tumor weight in an animal tumor model induced by sk-ov-3 in tumor cells; Figure 15 The effect of different drug administration groups on tumor volume in an animal tumor model induced by sk-ov-3 in tumor cells. Detailed Implementation

[0018] This invention provides a bispecific antibody targeting tissue factor, comprising a heavy chain structure 1 targeting tissue factor, a light chain structure targeting tissue factor, and a heavy chain structure 2 targeting tissue factor containing a single-chain antibody; the amino acid sequence of the heavy chain structure 1 targeting tissue factor is shown in SEQ ID NO:2. The amino acid sequence of the heavy chain structure 2 containing the single-chain antibody that targets tissue factor is shown in SEQ ID NO:4; The amino acid sequence of the light chain structure of the target tissue factor is shown in SEQ ID NO:6.

[0019] In this invention, the bispecific antibody has a Y-shaped structure (see...). Figure 1 The heavy chain structure 1 of the targeting tissue factor is coupled to the light chain structure of the targeting tissue factor via disulfide bonds and non-covalent interactions. The heavy chain structure 1 of the targeting tissue factor and the heavy chain structure 2 of the targeting tissue factor containing a single-chain antibody are coupled via disulfide bonds. The structure of the heavy chain structure 1 of the targeting tissue factor is VH1-CH1-CH2-CH3. The structure of the heavy chain structure 2 of the targeting tissue factor containing a single-chain antibody is VH2-VL2-CH2-CH3.

[0020] In this invention, ELISA and surface plasmon resonance (SPR) assays showed that the bispecific antibody B830 exhibits good affinity for human and monkey tissue factor, but does not bind to mouse tissue factor. Furthermore, the affinity KD of B830 antibody for the target antigen tissue factor is 1.32E-10, while the KD of monoclonal antibody T320 for the antigen tissue factor is 7.86E-11. Tumor cell surface binding and endocytosis activity assays indicated that the B830 bispecific antibody can bind to tumor cells (human ovarian cancer cells), demonstrating superior binding activity compared to the monoclonal antibody. Simultaneously, the B830 bispecific antibody showed higher endocytosis efficiency in tumor cells than the corresponding monoclonal antibody, establishing the foundation for B830 bispecific antibody as a targeted drug delivery carrier and providing an effective tool for the delivery of small-molecule toxic drugs in antitumor therapy.

[0021] This invention provides a gene encoding a bispecific antibody targeting the tissue factor.

[0022] This invention does not impose any special limitations on the gene mentioned, as long as it is a gene capable of encoding a bispecific antibody targeting tissue factors, such as a gene codon-optimized according to the codon preference of the expressing host. In embodiments of this invention, to achieve extracellular secretion of the gene expression product, the gene further includes a signal peptide. The preferred nucleotide sequences of the gene containing the signal peptide are shown in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.

[0023] The present invention provides a recombinant vector comprising the aforementioned gene.

[0024] This invention does not impose any special limitation on the type of backbone vector for the recombinant vector; any backbone vector well-known in the art can be used. In embodiments of this invention, the backbone vector is preferably a pCDNA3.1 vector, and the cloning site is preferably... Not I / Xba I. Restriction site. To ensure correct gene editing, it is preferable to clone the three genes into the backbone vector separately, thus constructing three recombinant vectors, namely pCDNA3.1-B830-TF-VH1-CH1-CH2-CH3, pCDNA3.1-B830-TF-VH2-VL2-CH2-CH3, and pCDNA3.1-B830-TF-VL1-CL.

[0025] The present invention provides a recombinant cell comprising the gene or the recombinant vector.

[0026] In this invention, the recombinant cells are preferably CHO cells. The recombinant vector is transferred into the cells via a transfection reagent.

[0027] The present invention provides an antibody-drug conjugate comprising an antitumor drug and a bispecific antibody targeting a tissue factor conjugated to the antitumor drug, or a bispecific antibody targeting a tissue factor prepared from the gene, the recombinant vector, or the recombinant cell.

[0028] This invention does not impose any special limitations on the type of antitumor drug; any small-molecule toxic drugs well-known in the art for pancreatic cancer, breast cancer, cervical cancer, and non-small cell carcinoma may be used. The antitumor drug preferably includes at least one of the following: monomethylolpropionate E, monomethylolpropionate F, microtubule inhibitor DM1, microtubule inhibitor DM4, DNA topoisomerase I inhibitor DXd, and 7-ethyl-10-hydroxycamptothecin.

[0029] In this invention, the antibody-drug conjugate preferably conjugates an oncology drug and a bispecific antibody targeting tissue factors via a linker. This invention does not impose any particular limitation on the type of linker; any linker well-known in the art can be used, such as valine-citrulline (VC).

[0030] The present invention provides a method for preparing the antibody-drug conjugate, comprising the following steps: linking a bispecific antibody targeting tissue factor and an antitumor drug via disulfide bonds.

[0031] This invention provides the application of the antibody-drug conjugate or the antibody-drug conjugate prepared by the method described above in the preparation of antitumor drugs.

[0032] In this invention, the tumor preferably includes at least one of the following: pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, non-small cell lung cancer, endometrial cancer, prostate cancer, esophageal cancer, and bladder cancer; the host source of the tumor preferably includes humans and / or primates.

[0033] Tissue factor is highly expressed in non-small cell lung cancer, endometrial cancer, prostate cancer, esophageal cancer, and bladder cancer. Targeting tissue factor can achieve the treatment of tumors or cancers (Patent CN116789837A: a humanized anti-tissue factor antibody and its preparation and application; Patent CN107446047A: a bifunctional humanized tissue factor antibody and its preparation method).

[0034] In this invention, the antibody-drug conjugate can effectively and precisely target tumor cells at both the cellular and animal levels, achieving binding and endocytosis, exerting the efficacy of anti-tumor drugs, thereby inhibiting tumor growth and achieving tumor killing effects.

[0035] The following detailed description, in conjunction with embodiments, illustrates a bispecific antibody targeting tissue factors, an antibody-drug conjugate, its preparation method, and its application provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1 Design, expression and purification of bispecific antibody B830 The bispecific antibody B830 targets tissue factor (TF) and employs a Y-body structure. The structure of the B830 bispecific antibody is as follows: Figure 1 As shown, a complete structure consisting of two different heavy chains and one light chain is formed, thereby reducing the problem of light chain mismatch and enabling the bispecific antibody to have better stability and targeting ability in vivo.

[0037] Heavy chain structure of TF-targeting antibody 1 VH1-CH1-CH2-CH3 (B830- signal peptide-VH-CH (Human IgG1E356D M358L T366W), its nucleotide sequence is shown in SEQ ID NO:1: GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGC ATGCACAGCTCAGCACTGCTCTGTTGCCTG GTCCTCCTGACTGGGGTGAGGGCC

[0038] The amino acid sequence corresponding to the heavy chain structure 1 VH1-CH1-CH2-CH3 is SEQ ID NO:2: MHSSALLCCLVLL TGVRA QVQLQESGPGLVKPSETLSLTCTVSGFSLTLYGVHWIRQPPGKGLEWLGLTWPGGITDYNSALMSRLTISKDNSKSQVFLKLSSVTAADTAVYYCARDDYGWAMDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0039] The heavy chain structure 2 of the TF-targeting antibody is VH2-VL2-CH2-CH3 (B830- signal peptide -scFv-Fc(Human IgG1Mutation)), whose gene sequence is shown in SEQ ID NO:3: GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGC ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCCAGATCCAGCTGGTGCAGTCCGGAGGAGAGGTGAAAAAGCCCGGCGCCTCCGTGAGGGTGTCCTGCAAGGCCTCCGGCTACTCCTTCACCGACTACAACGTGTACTGGGTGAGGCAGTCCCCCGGCAAGGGCCTGGAGTGGATCGGCTACATCGACCCCTACAACGGCATCACCATCTACGACCAGAACTTCAAGGGCAAGGCCACCCTGACCGTGGACAAGTCCACCTCCACCGCCTACATGGAGCTGTCCTCCCTGAGGTCCGAGGACACCGCCGTGTACTTCTGCGCCAGGGACGTGACCACCGCCCTGGACTTCTGGGGCCAGGGCACCACCGTGACCGTGTCCTCC GGTGGTGGCGGATCAGGTGGGGGAGGCTCTGGTGGAGGCGGTAGT

[0040] The amino acid sequence corresponding to heavy chain structure 2, VH2-VL2-CH2-CH3, is SEQ ID NO:4. MHSSALLCCLVL LTGVRA QIQLVQSGGEVKKPGASVRVSCKASGYSFTDYNVYWVRQSPGKGLEWIGYIDPYNGITIYDQNFKGKATLTVDKSTSTAYMELSSLRSEDTAVYFCARDVTTALDFWGQGTTVTVSSG GGGSGGGGSGGGGSDIQMTQSPASSLSASVGDRVTITCLASQTIDTWLAWYLQKPGKSPQLLIYAATNLADGVPSRFSGSGSGTDFSFTISSLQPEDFATYYCQQVYSSPFTFGQGTKL EIKEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0041] The light chain is VL1-CL (B830- signal peptide -VL-CL (Human Kappa) structure, the nucleotide sequence of which is shown in SEQ ID NO: 5: GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGC ATGCACAGCTCAGCACTGCTCTGTTGCCT GGTCCTCCTGACTGGGGTGAGGGCCGACATCGTGATGACACAGAGCCCTAGCAGCCTGGCCGTGAGCCTGGGCGAGAGAGCCACAATCAACTGTGCCAGCAGCCAGAGCCTGCTGAACAGCAGAAACAGACAGAACTACCTGGCCTGGTACCAGCAGAAGCCTGGCCAGCCTCCTAAGCTGCTGATCTACGCCGCCAGCAGCAGAGGCGCTGGCGTGCCTGACAGATTCAGCGGCAGCGGCAGCGGCACAGACTTCACACTGACAATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGTAAGCAGAGCAGCAACCTGTACACATTCGGCGGCGGCACAAAGCTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGATTCTAGA。

[0042] The amino acid sequence corresponding to the light chain structure VL1-CL is SEQ ID NO:6: MHSSALLCCLVLLTGVRA DIVMTQSPSSLAVSLGERATINCASSQSLLNSRNRQNYLAWYQQKPGQPPKLLIYAASSRGAGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSSNLYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0043] The nucleotide sequences of the above three structures were cloned into the pCDNA3.1 vector, respectively. Not I / Xba I restriction enzyme sites were used to construct three expression plasmids: pCDNA3.1-B830-TF-VH1-CH1-CH2-CH3, pCDNA3.1-B830-TF-VH2-VL2-CH2-CH3, and pCDNA3.1-B830-TF-VL1-CL.

[0044] Take CHO cells that are in good condition, have a viability of over 98%, and are in the logarithmic growth phase, and adjust the cell density to 3 × 10⁻⁶. 6 ~4×10 6 Cells / mL; after culturing for 24 hours, the cells were diluted to 6 × 10⁻⁶. 6 Mix the three plasmids at a mass ratio of 1:1:1, with 30 μg of each plasmid. Add transfection buffer and transfection reagent, mix thoroughly, and incubate at room temperature for 3 minutes. Add the mixture to CHO cells, mix well, and incubate at 37°C for 8-10 days. Collect the supernatant for purification of the double antibody B830.

[0045] Purification was performed using Protein A affinity chromatography. The column was first equilibrated with 3-5 column volumes of 50 mM Tris and 150 mM NaCl (pH 7.4) equilibration buffer. After loading the sample, the column was washed with equilibration buffer, followed by elution with 100 mM glycine-HCl (pH 3.0). During elution, an appropriate amount of Tris-HCl buffer was added to neutralize the eluent. The eluent was collected and the pH was adjusted to neutral. Finally, the eluent was transferred to PBS buffer to obtain purified bispecific antibody B830, which was then detected by SDS-PAGE electrophoresis.

[0046] SDS-PAGE electrophoresis results are as follows: Figure 2 As shown, the recombinant antibody B830 obtained a band of the expected size and a purity of over 95%, indicating that a high-purity, bispecific antibody targeting tissue factor, B830, was successfully obtained.

[0047] Example 2 Affinity assay of the bispecific antibody B830 targeting tissue factor epitopes with the antigen 1. ELISA detection method Affinity testing of B830 with targets from different species was conducted. The experimental method involved coating TF proteins (human TF protein purchased from Shanxi Biological Research Institute Co., Ltd.; cynomolgus monkey TF protein purchased from Sino Biological InC cat:90885-C08H, LC16DE2303; mouse TF protein purchased from Sino Biological InC cat:50413-M08H, LC16JU1712) onto ELISA plates at a concentration of 1 μg / ml. The plates were incubated overnight at 4°C, blocked with 3% BSA solution at 37°C for 2 hours, and then incubated with different concentrations of B830 antibiotics at 37°C for 1 hour. HRP-conjugated human IgG (HRP-conjugated AffinipureGoat Anti-Human IgG (H+L)) diluted 1:5000 was added, and the plates were incubated at 37°C for 1 hour. Finally, 100 μL of two-component TMB chromogenic buffer (Solarbio) was added, and after 5 minutes of development, 50 μL of TMB chromogenic buffer was added. Stop at 2N sulfuric acid solution, read OD. 450 value.

[0048] See results Figure 3 The results showed that the B830 antibody could specifically bind to human and monkey tissue factor targets, but not to mouse proteins, laying the foundation for the preparation of ADC drugs from B830.

[0049] 2. Surface Plasmon Resonance (SPR) technique to detect the affinity of bispecific antibody B830, monoclonal antibody T320, and monoclonal antibody T330 for the target. The B830 antibody was conjugated to the CM5 chip, and the antigen TF (maximum concentration 10µM, loading conditions 30µl / min, 2min, dissociation 300s) was used as the analytical stream for chip detection. The data were fitted using the analysis software SPR Analysis to obtain the affinity data between the corresponding samples.

[0050] See results Figure 4 See Table 1. The results showed that the affinity KD of antibody B830 to the target antigen tissue factor was 1.32E-10, while the corresponding monoclonal antibody T320 had an affinity KD of 7.86E-11, and the corresponding monoclonal antibody T330 had an affinity KD of 1.19E-9. These results indicate that the bispecific antibody B830 maintained excellent affinity comparable to its two parent monoclonal antibodies, with a KD value close to that of the high-affinity monoclonal antibody T320, and no significant loss of function.

[0051] Table 1. SPR test results of the affinity of B830 antibody and control antibody to the target antigen.

[0052] Example 3 B830 antibody binds to the surface of tumor cells and exhibits endocytic activity. 1. The key to ADC drug development is that the antibody can bind to the target site on the surface of tumor cells, enabling the internalization of the ADC drug. Tumor cells SK-OV-3 (human ovarian cancer cells, CL-0215, Pronosai) were selected, and tumor cells in the logarithmic growth phase were taken at a cell density of 1×10⁻⁶. 6 Plates were seeded at a density of / ml and incubated with antibodies T320, T330, Herceptin, B830, and the negative control HYHEL-10 (ProBio) for half an hour. Then, fluorescent secondary antibody PE anti-human IgG Fc Antibody was added. Finally, the effects of different drugs on cell binding ability were detected by flow cytometry. Flowjo and GraphPadPrism 6 software were used to fit curves after inputting MFI data.

[0053] See results Figure 5 The results showed that in the sk-ov-3 cell line, which expressed the target TF and had low Her2 expression, the cell surface activity of B830 was higher than that of the corresponding target monoclonal antibody, demonstrating the advantage of bispecific antibodies.

[0054] 2. The cell lines selected for endocytosis activity of the antibody in tumor cells were Bxpc-3 (human pancreatic cancer cells, CL-0042, Pronosai) and sk-ov-3 (human ovarian cancer cells, CL-0215, Pronosai). Tumor cells in the logarithmic growth phase were taken, and 0.6 × 10⁻⁶ antibodies were added to a 1.5 ml EP tube. 5Cells were collected and centrifuged to remove the supernatant. The test antibodies (antibody T320, Herceptin, B830, and negative control HYHEL-10) were diluted to 0.6 ml with pre-chilled complete medium, resulting in a final concentration of 10 μg / ml. The secondary antibody (goat anti-human AF647) was diluted 1:300 with pre-chilled complete medium. The diluted test antibody was added to the pellet in a 1.5 ml EP tube, resuspended, and mixed. The tube was incubated on ice for 30 minutes. The cell suspension was centrifuged at 300 g for 3 minutes to remove the supernatant. 600 μl of pre-chilled complete medium was added to resuspend the cell suspension, and the tube was centrifuged at 300 g for 3 minutes to remove the supernatant. 0.6 ml of pre-chilled complete medium was added to the cell pellet to resuspend the cell suspension. The cell suspension was then aliquoted into four 1.5 ml tubes. Two EP tubes were labeled with 0 hours and two with 24 hours. The EP tubes labeled with 24 hours were incubated at 37°C for 48 hours, while the tubes labeled with 0 hours were incubated with secondary antibody. The EP tubes were centrifuged at 300g for 3 minutes to remove the supernatant. 100 μl of secondary antibody dilution buffer was added to each tube and mixed well. The EP tubes were incubated on ice for 30 minutes. The cell suspension was centrifuged at 300g for 3 minutes to remove the supernatant. 600 μl of pre-cooled complete culture medium was added to resuspend the cells, and the supernatant was removed by centrifugation. The cells in the 0-hour tubes were resuspended in 200 μl of pre-cooled complete culture medium and analyzed by flow cytometry. The EP tubes labeled with 24 hours were incubated at 37°C until the end of the incubation. After secondary antibody incubation and washing, the cells were analyzed by flow cytometry.

[0055] The experimental results showed that the B830 antibody had a higher endocytosis efficiency in tumor cells than the corresponding monoclonal antibody. Figure 6 , Figure 7 ).

[0056] Example 4 In vitro cellular biological activity of B830-ADC 1. Fabrication of B830-ADC Bismuth subunit antibody B830 or T320 was coupled to a small molecule toxin via a disulfide bond. The linker was valine-citrulline (VC), and the small molecule toxin was MMAE. B830 was replaced in the coupling buffer (20 mM His-His·HCl, pH=5.96). 60 μM antibody and TCEP were mixed at a molar ratio of 1:4 and the reaction was carried out at 25 °C for 2 hours. After the reaction, 6.5 times the molar amount of linker-payload was added, and the mixture was briefly vortexed to mix thoroughly. The entire reaction mixture was then collected at the bottom of the tube and placed in a constant temperature mixer for 1 hour of coupling reaction. After coupling, an appropriate amount of ADC was taken and the DAR value was detected by HIC-HPLC.

[0057] The DAR value of B830-ADC is 2.9, and the DAR value of T320-MMAE is 3.92. Both have a purity of over 90%.

[0058] 2. Cellular activity assay of B830-ADC The biological activity of successfully conjugated B830-ADC in tumor cells Bxpc-3 (human pancreatic cancer cells), sk-ov-3 (human ovarian cancer cells), JIMT-1 (human breast cancer cells), and SiHa (human cervical squamous cell carcinoma cells) was detected. Tumor cells were cultured at a concentration of 1×10⁻⁶. 4 Inoculate 96-well plates with a concentration of [value missing] / ml and incubate overnight at 37°C in a 5% CO2 incubator. Add diluted antibody reagent, with 20 concentration gradients for each sample. The specific sample dilution gradients are as follows: 500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.625nM, 7.8nM, 3.9nM, 1.95nM, 0.975nM, 0.48nM, 0.24nM, 0.12nM, 0.06nM, 0.03nM, 0.015nM, 0.0075nM, 0.00375nM, 0.001875nM, and 0.0009nM. After 5 days of incubation, add CCK-8 for colorimetric analysis. OD [value missing] 450 Readings were taken. The same experiment was also conducted using a T320-MMAE as a control.

[0059] Test results are shown Figures 8-11 See Tables 2-5. The results showed that B830 had a higher inhibition rate against the four cell lines than the single-target monoclonal antibody T320, exhibiting a better tumor cell killing effect.

[0060] Table 2. Effects of different conjugated drugs on IC50 in Bxpc-3 cells. 50 result

[0061] Table 3. Effects of different conjugated drugs on IC50 in SK-OV-3 cells. 50 result

[0062] Table 4. Effects of different conjugate drugs on IC50 in JIMT-1 cells 50 result

[0063] Table 5. Effects of different conjugated drugs on IC50 in SiHa cells. 50 result

[0064] Example 5 In vivo biological activity of B830-ADC A subcutaneous tumor model was constructed in nude mice, with Bxpc-3 and sk-ov-3 cells seeded. Bxpc-3 and sk-ov-3 cells were grown to the logarithmic growth phase and resuspended in basal medium (RM1640 + 10% FBS for Bxpc-3, McCoy's 5A medium + 10% FBS for sk-ov-3), adjusting the cell concentration to 5 × 10⁶ cells / year. 7 / mL. Under aseptic conditions, 0.1 mL of cell suspension was inoculated into the right fat pad of mice at an inoculation concentration of 5 × 10⁹ / mL. 6 / 0.1mL / mouse. When the average tumor volume reaches 200 mm... 3 At approximately 10:00 AM, animals were randomly divided into seven groups of eight based on tumor volume. In the Bxpc-3 cell subcutaneous tumor experiment in mice, B830 was administered at doses of 0.5 mg / kg, 1 mg / kg, and 2 mg / kg, once weekly for a total of four treatments. The antitumor drug DS8201 was administered at 10 mg / kg once weekly; the positive control was gemcitabine at 60 mg / kg twice weekly for three weeks; the negative control was PBS. In the sk-ov-3 cell subcutaneous tumor experiment in mice, B830 was administered at doses of 2 mg / kg, 4 mg / kg, and 8 mg / kg, once weekly for a total of four treatments; DS8201 was administered at 10 mg / kg once weekly; the positive control was paclitaxel at 15 mg / kg twice weekly for four weeks; the negative control was PBS, administered via tail vein starting on Day 0. The weight of nude mice was measured every 3 days, and the long and short diameters of the tumors were measured with calipers. At the end of the experiment, blood was collected and serum was preserved. The animals were euthanized by neck dislocation, and the tumor was removed and weighed. The efficacy of the drug was evaluated based on changes in tumor weight and relative volume. The formula for calculating tumor volume is given in Formula I. V = a × b × c (Formula I) Where a, b, and c represent the length, width, and height of the tumor, respectively, in mm.

[0065] like Figures 12-15 As shown, the B830 ADC drug significantly inhibited tumor growth in a dose-dependent manner. In the Bxpc-3 pancreatic cancer animal model, it was superior to the commercially available drug DS8201, and in the sk-ov-3 animal model, it was also superior to the commercially available drug DS8201.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A bispecific antibody targeting tissue factor, characterized in that, Including a heavy chain structure 1 of targeting tissue factors, a light chain structure of targeting tissue factors, and a heavy chain structure 2 of targeting tissue factors containing single-chain antibodies; The amino acid sequence of the heavy chain structure 1 of the target tissue factor is shown in SEQ ID NO:2; The amino acid sequence of the heavy chain structure 2 containing the single-chain antibody that targets tissue factor is shown in SEQ ID NO:4; The amino acid sequence of the light chain structure of the target tissue factor is shown in SEQ ID NO:

6.

2. A gene encoding a bispecific antibody targeting tissue factor as described in claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:

5.

4. A recombinant vector, characterized in that, Includes the gene described in claim 2 or 3.

5. A recombinant cell, characterized in that, Includes the gene of claim 2 or 3 or the recombinant vector of claim 4.

6. An antibody-drug conjugate, characterized in that, This includes antitumor drugs and bispecific antibodies targeting tissue factors as described in claim 1 conjugated with the antitumor drugs, or bispecific antibodies targeting tissue factors prepared from the gene described in claim 2 or 3, the recombinant vector described in claim 4, or the recombinant cells described in claim 5.

7. The antibody-drug conjugate according to claim 6, characterized in that, The antitumor drug includes at least one of the following: monomethylolpropamine E, monomethylolpropamine F, microtubule inhibitor DM1, microtubule inhibitor DM4, DNA topoisomerase I inhibitor DXd, and 7-ethyl-10-hydroxycamptothecin.

8. A method for preparing the antibody-drug conjugate according to claim 6 or 7, characterized in that, The process includes the following steps: linking a bispecific antibody targeting tissue factor and an anti-tumor drug via disulfide bonds.

9. The use of the antibody-drug conjugate of claim 6 or 7 or the antibody-drug conjugate prepared by the preparation method of claim 8 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The tumors include at least one of the following: pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, non-small cell lung cancer, colon cancer, endometrial cancer, prostate cancer, esophageal cancer, and bladder cancer; The host sources of the tumors include humans and / or primates.

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