A bispecific antibody drug targeting tf and her2 and a preparation method and application thereof

Abstract

The application provides a bispecific antibody conjugate drug targeting TF and Her2, and a preparation method and application thereof, and belongs to the technical field of biological drug preparation. The bispecific antibody provided by the application can target TF and / or Her2 antigens in tumor cells, has high stability, and has the advantages of easy expression, purification and conjugation. The bispecific antibody can specifically bind to tumor surface antigens and be internalized into tumor cells, and can specifically kill tumor cells. The bispecific antibody conjugate drug prepared based on the bispecific antibody has good tumor inhibition effect in a cell model and an animal model, is non-toxic and harmless to animals, and has excellent potential for treating cancer.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical preparation technology, specifically relating to a bispecific antibody-drug conjugate targeting TF and Her2, its preparation method, and its application. Background Technology

[0002] Bispecific antibodies (BsAbs) can target a variety of tumors compared to traditional monoclonal antibodies. They combine the advantages of bispecific antibodies and antibody-drug conjugates (ADCs), offering more precise targeting: bispecific ADCs contain two different antigen-binding sites, enabling them to simultaneously recognize two different antigens on tumor cells. Compared to traditional monoclonal ADCs, their recognition of tumor cells is more accurate, effectively reducing non-specific binding to normal tissues and lowering off-target toxicity.

[0003] Human epidermal growth factor receptor-2 (HER2) is highly expressed in many tumors such as breast cancer and gastric cancer, but it is not uniformly expressed in all tumor cells, and HER2 may also be expressed at low levels in some normal tissues. The low or heterogeneous expression of HER2 in some tumor cells may limit the killing effect of marketed HER2-ADC drugs on these cells. Tissue factor (TF) is highly expressed in various solid tumors such as pancreatic cancer, cervical cancer, and non-small cell carcinoma, but lowly expressed in normal tissues. Antibody drugs targeting TF antigens can specifically treat solid tumors such as pancreatic cancer, cervical cancer, and non-small cell carcinoma. Currently, there is a lack of antibody drugs that can simultaneously treat multiple tumors. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a bispecific antibody targeting TF and Her2, which has strong binding properties and endocytosis effect against pancreatic cancer, breast cancer, and ovarian cancer, providing an effective tool for the treatment of antibody-drug conjugates.

[0005] This invention provides a bispecific antibody targeting tissue factor and human epidermal growth factor receptor-2, comprising a heavy chain structure targeting tissue factor, a light chain structure targeting tissue factor, a heavy chain structure targeting human epidermal growth factor receptor-2, and a light chain structure targeting human epidermal growth factor receptor-2.

[0006] The amino acid sequence of the heavy chain structure of the targeted tissue factor is shown in SEQ ID NO:2;

[0007] The amino acid sequence of the light chain structure of the target tissue factor is shown in SEQ ID NO:4;

[0008] The amino acid sequence of the heavy chain structure targeting human epidermal growth factor receptor-2 is shown in SEQ ID NO:6;

[0009] The amino acid sequence of the light chain structure targeting human epidermal growth factor receptor-2 is shown in SEQ ID NO:8.

[0010] This invention provides a method for preparing the bispecific antibody, comprising the following steps:

[0011] Recombinant vectors were constructed to express heavy chain structures targeting tissue factors, light chain structures targeting tissue factors, heavy chain structures targeting human epidermal growth factor receptor-2, and light chain structures targeting human epidermal growth factor receptor-2, respectively.

[0012] The four recombinant vectors were co-transfected into mammalian cells for expression and purification to obtain bispecific antibodies.

[0013] Preferably, in the recombinant vector, the nucleotide sequence of the gene encoding the heavy chain structure of the target tissue factor is shown in SEQ ID NO:1;

[0014] The nucleotide sequence of the gene encoding the light chain structure of the target tissue factor is shown in SEQ ID NO:3;

[0015] The nucleotide sequence of the gene encoding the heavy chain structure targeting human epidermal growth factor receptor-2 is shown in SEQ ID NO:5;

[0016] The nucleotide sequence of the gene encoding the light chain structure targeting human epidermal growth factor receptor-2 is shown in SEQ ID NO:7.

[0017] This invention provides the application of the bispecific antibody or the bispecific antibody prepared by the preparation method in the preparation of antitumor antibody-drug conjugates.

[0018] This invention provides the application of the bispecific antibody or the bispecific antibody prepared by the preparation method in the preparation of drugs that reduce resistance to antitumor drugs.

[0019] 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, head and neck cancer, and gastric cancer.

[0020] Preferably, the host source of the tumor includes humans and / or primates.

[0021] This invention provides a bispecific antibody-drug conjugate, comprising an active drug molecule and the bispecific antibody or a bispecific antibody prepared by the preparation method described above;

[0022] The active drug molecule is conjugated to the bispecific antibody.

[0023] Preferably, the active pharmaceutical ingredient includes an antitumor drug.

[0024] Preferably, the antitumor drug includes at least one of the following: monomethylolpropamine E, DNA topoisomerase I inhibitor DXd, 7-ethyl-10-hydroxycamptothecin, and microtubule protein inhibitor DM1.

[0025] This invention provides a bispecific antibody targeting tissue factor and human epidermal growth factor receptor-2 (HFR-2), comprising a heavy chain structure targeting tissue factor, a light chain structure targeting tissue factor, a heavy chain structure targeting HFR-2, and a light chain structure targeting HFR-2, with corresponding amino acid sequences as shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. Experiments show that the bispecific antibody exhibits good antigen-antibody binding and strong affinity for TF and Her2, and has the same affinity as monoclonal antibodies targeting the corresponding targets. Simultaneously, the binding activity and endocytosis effect of the drug conjugate carrying the bispecific antibody were measured with tumor cells expressing TF and / or Her2. The results showed that the bispecific antibody not only binds to the surface of tumor cells, but also has a higher endocytosis effect than the single-target antibody group. Furthermore, cytotoxicity experiments showed that the bispecific antibody exhibited the same activity as the single-target antibody in tumor cells expressing a single target, and showed higher activity than the single-target antibody and superior activity to the control bispecific antibody B83P in tumor cells with low to moderate expression. As can be seen, the bispecific antibody provided by this invention not only exhibits strong specificity in binding to TF and Her2, but also demonstrates superior binding properties compared to single-target antibodies and conventionally designed bispecific antibodies like B83P. This bispecific antibody provides an effective tool for the preparation of antibody-drug conjugates for antitumor therapy. Furthermore, given its dual-target design, it significantly reduces the likelihood of drug resistance and prolongs the duration of treatment for patients. Therefore, this bispecific antibody provides a new approach for preparing drugs with reduced resistance.

[0026] This invention also provides an antibody-drug conjugate (ADC) based on the aforementioned bispecific antibody. The ADC utilizes the binding of the carried bispecific antibody to TF and / or Her2 on the surface of tumor cells, and internalizes into the tumor. Within the cell, lysosomes degrade the ADC, releasing active drug molecules to achieve cytotoxicity and thus kill tumor cells. Both cellular and subcutaneous tumor models have demonstrated that the ADC can specifically kill tumor cells, exhibiting good tumor-suppressing effects in animal models and being non-toxic and harmless to animals, demonstrating excellent potential for cancer treatment. Attached Figure Description

[0027] Figure 1This is a schematic diagram of the structure of a bispecific antibody, where A is the schematic diagram of the structure of the B836 antibody and B is the schematic diagram of the structure of the B83P bispecific antibody.

[0028] Figure 2 The results are for bispecific antibody purification detection, where A is the purification result of B836 antibody, B is the purification result of B83P bispecific antibody, R represents reducing electrophoresis, and NR represents non-reducing electrophoresis.

[0029] Figure 3 The results show the ELISA affinity assays for B836 antibody with target antigens from different species.

[0030] Figure 4 The SPR affinity of B836 antibody with its corresponding monoclonal antibody and target antigen was detected.

[0031] Figure 5 The results show the binding activity of B836 antibody on the surface of Bxpc-3 cells.

[0032] Figure 6 Results of B836 antibody binding activity on the surface of sk-br-3 cells;

[0033] Figure 7 Results of the binding activity of B836 antibody on the surface of SK-OV-3 cells;

[0034] Figure 8 Results of the endocytosis effect of B836 antibody in Bxpc-3 cells;

[0035] Figure 9 Results of the endocytosis effect of B836 antibody in sk-br-3 cells;

[0036] Figure 10 Results of the endocytosis effect of B836 antibody in SK-OV-3 cells;

[0037] Figure 11 The results show the inhibition rate of B836 antibody against Bxpc-3;

[0038] Figure 12 The results show the inhibition rate of B836 antibody against sk-ov-3.

[0039] Figure 13 The results show the inhibition rate of B836 antibody against sk-br-3.

[0040] Figure 14 Results of the B836 antibody assay in a Bxpc-3 mouse subcutaneous tumor model;

[0041] Figure 15 Tumor weight on day 28 after different doses of B836 antibody were administered to a mouse model of tumors;

[0042] Figure 16 Results of the B836 antibody assay in a sk-ov-3 mouse subcutaneous tumor model;

[0043] Figure 17 Tumor weight on day 28 after different doses of B836 antibody were administered to a mouse model of tumors. Detailed Implementation

[0044] This invention provides a bispecific antibody targeting tissue factor and human epidermal growth factor receptor-2, comprising a heavy chain structure targeting tissue factor, a light chain structure targeting tissue factor, a heavy chain structure targeting human epidermal growth factor receptor-2, and a light chain structure targeting human epidermal growth factor receptor-2.

[0045] In this invention, the bispecific antibody comprises two sides: one side consists of a heavy chain structure and a light chain structure targeting tissue factor, and the other side consists of a heavy chain structure and a light chain structure targeting human epidermal growth factor receptor-2 (HGF-2), wherein the heavy chain structure targeting tissue factor and the heavy chain structure targeting HGF-2 are linked by disulfide bonds. The heavy chain structure targeting tissue factor comprises VH-CH1-CH2-CH3, with the amino acid sequence as shown in SEQ ID NO:2. The light chain structure targeting tissue factor is VL-CL, with the amino acid sequence as shown in SEQ ID NO:4. The CrossMab technique is used to exchange the positions of CH1 and CL in the heavy chain structure targeting HGF-2, which helps ensure the correct pairing of the light chain with its respective heavy chain in the bispecific antibody, thereby reducing the problem of light chain mismatch and giving the bispecific antibody better stability and targeting ability in vivo. The heavy chain structure of the target human epidermal growth factor receptor-2 is VH-CL-CH2-CH3, and the corresponding amino acid sequence is shown in SEQ ID NO:6; the light chain structure of the target human epidermal growth factor receptor-2 is CH1-VL1, and the corresponding amino acid sequence is shown in SEQ ID NO:8. The bispecific antibody is named B836.

[0046] This invention provides a method for preparing the bispecific antibody, comprising the following steps:

[0047] Recombinant vectors were constructed to express heavy chain structures targeting tissue factors, light chain structures targeting tissue factors, heavy chain structures targeting human epidermal growth factor receptor-2, and light chain structures targeting human epidermal growth factor receptor-2, respectively.

[0048] The four recombinant vectors were co-transfected into mammalian cells for expression and purification to obtain bispecific antibodies.

[0049] This invention constructs recombinant vectors that respectively express a heavy chain structure targeting tissue factors, a light chain structure targeting tissue factors, a heavy chain structure targeting human epidermal growth factor receptor-2, and a light chain structure targeting human epidermal growth factor receptor-2.

[0050] In this invention, the nucleotide sequence of the gene encoding the heavy chain structure targeting tissue factor in the recombinant vector is preferably as shown in SEQ ID NO:1; the nucleotide sequence of the gene encoding the light chain structure targeting tissue factor is shown in SEQ ID NO:3; the nucleotide sequence of the gene encoding the heavy chain structure targeting human epidermal growth factor receptor-2 is shown in SEQ ID NO:5; and the nucleotide sequence of the gene encoding the light chain structure targeting human epidermal growth factor receptor-2 is shown in SEQ ID NO:7. This invention does not impose any special restrictions on the type of backbone vector of the recombinant vector; any mammalian expression vector type well known in the art can be used, such as the pCDNA3.1 vector. This invention does not impose any special restrictions on the construction method of the recombinant vector; homologous recombination or artificial synthesis methods well known in the art can be used.

[0051] After constructing the recombinant vectors, the present invention co-transfects the four constructed recombinant vectors into mammalian cells for expression, purification, and obtains bispecific antibodies.

[0052] In this invention, there are no particular limitations on the co-transfection method; conventional transfection reagents in the art can be used. During co-transfection, the four recombinant vectors are mixed with the transfection reagent in equal mass ratios. This invention also does not have particular limitations on the type of mammalian cells used; mammalian cells well-known in the art, such as Chinese hamster ovary cells, can be used. The purification is preferably performed using an A-column purification method.

[0053] This invention provides the application of the bispecific antibody or the bispecific antibody prepared by the preparation method in the preparation of antitumor antibody-drug conjugates.

[0054] In this invention, the tumor is a tumor cell expressing TF and / or Her2. The tumor preferably includes at least one of the following: pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, colon cancer, head and neck cancer, non-small cell lung cancer, and gastric cancer. The host source of the tumor preferably includes humans and / or primates.

[0055] This invention provides the application of the bispecific antibody or the bispecific antibody prepared by the preparation method in the preparation of drugs that reduce resistance to antitumor drugs.

[0056] In this invention, the drug prepared from the bispecific antibody can simultaneously bind to two different antigens. Even if tumor cells downregulate or lack expression of one antigen, it can still exert its effect by recognizing the other antigen, thereby more comprehensively covering different subtypes of tumor cells, overcoming the treatment challenges brought about by tumor heterogeneity, and improving treatment efficacy; reducing the risk of drug resistance: the bispecific antibody ADC is designed for two antigens, and compared with drugs targeting a single antigen, the probability of tumor cells developing resistance to both antigens simultaneously is relatively low. Therefore, the bispecific antibody ADC can, to a certain extent, delay the development of drug resistance and provide patients with a more durable treatment effect.

[0057] The present invention provides a bispecific antibody-drug conjugate, comprising an active drug molecule and the bispecific antibody or a bispecific antibody prepared by the preparation method thereof; wherein the active drug molecule is conjugated to the bispecific antibody.

[0058] In this invention, the active pharmaceutical ingredient preferably includes an antitumor drug. The antitumor drug preferably includes at least one of the following: monomethylolpropionate E (MMAE), DNA topoisomerase I inhibitor DXd, 7-ethyl-10-hydroxycamptothecin (SN-38), and microtubule protein inhibitor DM1. In the bispecific antibody-drug conjugate, the bispecific antibody is conjugated to MMAE via a disulfide bond, and the linker can be VC.

[0059] In this invention, the preparation method of the bispecific antibody-drug conjugate preferably includes the following steps: reducing a conjugation buffer containing a bispecific antibody with TCEP, and then conjugating it with an active drug molecule linked to a linker to obtain the bispecific antibody-drug conjugate. The molar ratio of the bispecific antibody to TCEP is 1:2 to 8, and can be 1:4. The temperature of the reduction reaction is preferably 23 to 27°C, and can be 25°C. The time of the reduction reaction is preferably 2.0 to 2.5 h. The molar ratio of the bispecific antibody to the active drug molecule linked to the linker is preferably 1:4 to 8, and can be 1:4. The time of the conjugation reaction is preferably 0.8 to 1.2 h, and can be 1 h. The DAR value of the prepared bispecific antibody-drug conjugate, detected by HIC-HPLC, is 3.9.

[0060] In one embodiment of the present invention, the biological activity of the bispecific antibody-drug conjugate (B836-ADC) against human pancreatic cancer cells (Bxpc-3), human breast cancer cells (sk-br-3), and human ovarian cancer cells (sk-ov-3) was tested. The results showed that B836 had a higher inhibition rate against the three tumor cell lines than the single-target monoclonal antibody T320, Herceptin-MMAE, and the control bispecific antibody B83P, exhibiting a better tumor cell killing effect. In another embodiment of the present invention, experiments were conducted to construct nude mouse subcutaneous tumor models using Bxpc-3 and sk-ov-3 tumor cells to verify the in vivo antitumor activity of B836-ADC. The results showed that B836-ADC could significantly inhibit tumor growth in a dose-dependent manner. In the Bxpc-3 pancreatic cancer animal model, it was superior to the commercially available drugs RC48 and DS8201; in the sk-ov-3 animal model, it was superior to the commercially available drug DS8201 and comparable to RC48.

[0061] The following detailed description, in conjunction with embodiments, illustrates a bispecific antibody-drug conjugate targeting TF and Her2, its preparation method, and its application, but these should not be construed as limiting the scope of protection of this invention.

[0062] Example 1

[0063] Design, expression and purification of bispecific antibody B836

[0064] The B836 antibody targets both tissue factor (TF) and human epidermal growth factor receptor-2 (HER2). To further verify the function of this bispecific antibody, this invention also constructed a bispecific antibody, B83P, as a control. B83P also targets both tissue factor (TF) and human epidermal growth factor receptor-2 (HER2), but the antibody sequences are different.

[0065] Both bispecific antibodies B836 and B83P employ CrossMab technology, which specifically involves exchanging the heavy chain (CH1) and light chain (CL) domains of the HER2-targeting antibody. This ensures the correct pairing of the light chain with its respective heavy chain in the bispecific antibody, thereby reducing the problem of light chain mismatch and giving the bispecific antibody better stability and targeting ability in vivo.

[0066] B836 dual-antibody structure, such as Figure 1 As shown in Figure A, the heavy chain structure of the TF-targeting antibody is VH-CH1-CH2-CH3(B836-). Signal peptides -VH-CH (Human IgG1 Mutation) nucleotide sequence as shown in SEQ ID NO:1 (GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGC) ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC QIQLVQSGGEVKKPGASVRVSCKASGYSFTDYNVYWVRQSPGKGLEWIGYIDPYNGITIYDQNFKGKATLTVDKSTSTAYMELSSLRSEDTAVYFCARDVTTALDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:2);

[0067] The light chain VL-CL (B836- signal peptide -VL-CL (Human Kappa)) nucleotide sequence is as SEQ ID NO:3 (GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAA As shown in TGTGGGAGATAGAGTGACCATTACCTGTCTGGCCAGCCAGACAATTGACACCTGGCTGGCCTGGTACCTGCAGAAACCCGGAAAGTCCCCCCAGCTGCTGATTTACGCCGCCACAAACCTGGCCGACGGCGTGCCTTCTCGATTCTCTGGAAGCGGCTCCGGCACCGATTTCTCCTTCACTATCTCCTCCCTGCAGCCCGAGGACTTCGCAACTTACTACTGCCAGCAGGTGTACTCCTCCCCCTTCACCTTCGGCCAGGGTACTAAGCTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGATTCTAGA), the corresponding amino acid sequence is as DIQMTQSPASLSASVGDRVTITCLASQTIDTWLAWYLQKPGKSPQLLIYAATNLADGVPSRFSGSGSGTDFSFTISSLQPEDFATYYCQQVYSSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:4).

[0068] The heavy chain structure of the anti-HER2 antibody, VH-CL-CH2-CH3 (B836- signal peptideThe nucleotide sequence -VH-CL(Human Kappa)-Fc(Human IgG1 E356D M358LT366W) is shown in SEQ ID. As shown in (SEQ ID NO:6): the nucleotide sequence of the light chain CH1-VL1 (B836-VL-CH1 (Human IgG1)) is as follows: GCGGCCGCAAA -VL-CH1 (Human IgG1)) nucleotide sequence is as SEQ ID NO:7 (GCGGCCGCAAA As shown in SEQ ID NO:8, the corresponding amino acid sequence is as follows: MHSSALLCCLVLLTGVRADIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC(SEQ ID NO:8).

[0069] The heavy chain nucleotide sequence and light chain nucleotide sequence of the TF-targeting antibody and the heavy chain nucleotide sequence and light chain nucleotide sequence of the HER2-targeting antibody were cloned into the pCDNA3.1 vector NotI / XbaI, respectively. The four plasmids are pCDNA3.1-B836-TF-VH-CH1-CH2-CH3, pCDNA3.1-B836-TF-VL-CL, pCDNA3.1-B836-HER2-VH-CL-CH2-CH3, and pCDNA3.1-B836-HER2-CH1-VL1.

[0070] B83P dual-resistance structure, such as Figure 1 As shown in Figure B, the heavy chain structure VH-CH1-CH2-CH3 and the light chain structure of the TF-targeting antibody are the same as those in B836; the heavy chain structure of the HER2-targeting antibody is VH-CL-CH2-CH3 (B83P). The nucleotide sequence -VH-CL(Human Kappa)-Fc(Human IgG1E356D M358L T366W) is shown in SEQ ID NO:9 (GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAG EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR D E L TKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:10), the light chain structure CH1-VL1 of the HER2-targeting antibody (B83P- -VL-CH1 (Human IgG1)) nucleotide sequence is as shown in SEQ ID NO:11 (GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGATATTCAGATGACCCAGTCCCCCTCTTCCCTTTCCGCCAGCGTAGGCGATCGGGTGACCATCACATGCAAGGCTTCTCAAGACGTATCTATCGGCGTGGCATGGTATCAGCAAAAGCCGGGAAAGGCCCCAAAGCTCTTGATCTATTCTGCTAGCTACCGCTATACAGGGGTGCCTAGTAGGTTTTCTGGAAGTGGCTCCGGGACAGACTTCACCTTGACCATCTCTAGCCTGCAGCCTGAGGACTTTGCCACCTACTACTGCCAGCAGTATTACATCTATCCCTACACTTTTGGCCAGGGGACCAAGGTGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTTGATTCTAGA), and the corresponding amino acid sequence is as DIQMTQSPSSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC(SEQ ID NO:12). The heavy chain nucleotide sequence and light chain nucleotide sequence of the TF-targeting antibody and the heavy chain nucleotide sequence and light chain nucleotide sequence of the HER2-targeting antibody were cloned into the pCDNA3.1 vector, respectively, to construct plasmids pCDNA3.1-B836-TF-VH-CH1-CH2-CH3, pCDNA3.1-B83P-TF-CH1-VL1, pCDNA3.1-B83P-HER2-VH-CL-CH2-CH3, and pCDNA3.1-B836-HER2-VL-CL.

[0071] Take CHO cells in good condition with viability above 98% and in the logarithmic growth phase, and dilute them to 3×10⁻⁶. 6 ~4×10 6 Cells / ml; after culturing for two days, dilute the cells to 6×10⁻⁶. 6 For each plasmid, add 25 μg of each plasmid to the transfection buffer at a mass ratio of 1:1:1:1, along with the transfection reagent. Mix thoroughly and incubate at room temperature for 1–5 min. After incubation, transfer the mixture to pre-separated CHO cells, shake well, and incubate at 37°C. After 8–10 days, collect the supernatant for purification of the antibiotics using column A. Equilibrate the column to 3–5 column volumes (equilibration buffer: 50 mM Tris, 150 mM NaCl, pH 9.0). Load the sample onto the column, wash with equilibration buffer, and elute the column with 100 mM acetic acid (pH 3.0) (add an appropriate amount of Tris-HCl before elution). After elution, adjust the pH of the eluent to neutral and replace the eluent with PBS buffer to obtain the antibiotics.

[0072] SDS electrophoresis results are as follows Figure 2 As shown, both recombinant antibodies yielded bands of the target size, and the purity of both recombinant antibodies reached over 95%.

[0073] Example 2

[0074] Affinity test of B836 with antigen

[0075] Two methods were used to detect the affinity of B836 antibody. The first method was ELISA, which tested the affinity of B836 with two targets from different species. The experimental method involved coating TF protein (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) or Her2 protein (human Her2 Cat:10004-H08H, Beijing Yiqiao Shenzhou Technology Co., Ltd.; cynomolgus monkey Her2 Cat:90295-C08H, Beijing Yiqiao Shenzhou Technology Co., Ltd.; mouse Her2:HY-P73929, MCE) onto an ELISA plate at a concentration of 1 μg / ml. The plates were incubated overnight at 4°C and then coated with 3% BSA. Block at 37°C for 2 hours, add different concentrations of B836 bispecific antibody, incubate at 37°C for 1 hour, add HRP-conjugated Affinipure Goat Anti-Human IgG (H+L) diluted 1:5000, incubate at 37°C for 1 hour, add 100 μL of two-component TMB chromogenic solution (Solarbio), incubate for 5 minutes, add 50 μL of 2N sulfuric acid to stop the reaction, and read the OD. 450 value.

[0076] See results Figure 3 The results showed that the B836 antibody could specifically bind to human and monkey tissue factor and Her2 targets, but not to mouse proteins, laying the foundation for the preparation of ADC drugs using B836.

[0077] The second affinity detection method is surface plasmon resonance (SPR) technology to detect the affinity of bispecific antibody B836, monoclonal antibody T320, and Herceptin to two target sites. The antibodies are conjugated to a CM5 chip, and antigens TF and Her2 (maximum concentration 10 μM, loading conditions 30 μl / min, 2 min, dissociation 300 s) are used as analytical streams for chip detection. The data are fitted using the analysis software SPRAnalysis to obtain the affinity data between the corresponding samples.

[0078] See results Figure 4See Table 1. The results show that the affinity KD of B836 antibody to the target antigen tissue factor is 9.07E-11, while the KD of the corresponding monoclonal antibody T320 to tissue factor is 7.86E-11, indicating that their affinities are comparable. The affinity KD of B836 to the target antigen HER2 is 1.31E-08, while the KD of the corresponding monoclonal antibody Herceptin to HER2 is 1.18E-08, indicating that their affinities are comparable. This suggests that the bispecific antibody B836 did not alter its affinity for the target antigen.

[0079] Table 1. SPR test results of the affinity of B836 antibody and control antibody to the two target antigens.

[0080]

[0081]

[0082] Example 3

[0083] B836 antibody binds to the surface of tumor cells and exhibits endocytic activity.

[0084] 1. The key to ADC drug development is that the antibody can bind to the target site on the surface of tumor cells, achieving ADC drug internalization. In this example, three tumor cell lines were selected: Bxpc-3 (human pancreatic cancer cells, CL-0042, Pronosai), sk-br-3 (human breast cancer cells, CL-0211, Pronosai), and sk-ov-3 (human ovarian cancer cells, CL-0215, Pronosai). The three types of tumor cells in the logarithmic growth phase were selected at a cell density of 1×10⁻⁶. 6 Plates were prepared at a density of 1 / ml and incubated with antibody T320, Herceptin, B836, and negative control Anti-HEL Human IgG1 (BioLegend) (starting at 300 nM, serially diluted 3-fold, for a total of 11 concentrations). After incubation for half an hour, fluorescent secondary antibody PE anti-human IgG FcAntibody was added. Flow cytometry was used to detect the effect of different drug concentrations on cell binding ability. Flowjo and GraphPadPrism 6 software were used to fit curves after inputting MFI data.

[0085] See results Figures 5-7 The results showed that in the Bxpc-3 cell line, which highly expressed TF, B836 exhibited binding activity comparable to that of the highly expressed target monoclonal antibody T320. Similarly, in the sk-br-3 cell line, which highly expressed Her2, B836 also showed activity comparable to that of the highly expressed target monoclonal antibody Herceptin. In the sk-ov-3 cell line, which expressed TF but low Her2, B836 showed higher cell surface activity than the corresponding target monoclonal antibody, demonstrating the advantage of a bispecific antibody.

[0086] 2. To assess the endocytic activity of antibodies in tumor cells, cell lines exhibiting binding activity to the tumor cell surface were selected. For tumor cells in the logarithmic growth phase, 0.6 × 10⁻⁶ antibodies were added to a 1.5 ml EP tube. 5 Cells were collected and centrifuged to remove the supernatant. The test antibodies (antibody T320, Herceptin antibody, B836 antibody, and negative control Anti-HEL Human IgG1) 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 antibodies were 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 for resuspending, 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 for resuspending. The cell suspension was then aliquoted into four 1.5 ml tubes. Two EP tubes were labeled with 0 hours and two with 48 hours. The EP tubes labeled with 48 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-chilled 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-chilled complete culture medium and analyzed by flow cytometry. The EP tubes labeled with 48 hours were incubated at 37°C until the end of the incubation. Secondary antibody incubation and washing were performed before analysis.

[0087] The experimental results showed that the B836 antibody had a higher endocytosis efficiency than the corresponding monoclonal antibody in all three tumor cell lines (detection rate was higher). Figures 8-10 ).

[0088] Example 4

[0089] In vitro cellular biological activity of B836-ADC

[0090] 1. Fabrication of B836-ADC

[0091] Bismuth subunit antibody B836 or B83P and Herceptin were coupled to a small molecule toxin via a disulfide bond. The linker was valine-citrulline (VC), and the small molecule toxin was MMAE. B836 or B83P 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 the antibody linker-payload was added, and the mixture was briefly vortexed to mix thoroughly. The entire reaction mixture was collected at the bottom of the tube and then 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.

[0092] The DAR value of B836-ADC is 3.9, that of B83P-ADC is 3.97, that of Herceptin-MMAE is 3.8, and that of T320-MMAE is 3.92. All have a purity of over 95%.

[0093] 2. Cellular activity assay of B836-ADC

[0094] The biological activities of successfully conjugated B836-ADC and B83P-ADC in tumor cells Bxpc-3 (human pancreatic cancer cells), sk-br-3 (human breast cancer cells), and sk-ov-3 (human ovarian cancer cells) were detected. Tumor cells were cultured at a concentration of 1 × 10⁻⁶ cells / year. 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, 0.0009nM. After 5 days of incubation, add CCK-8 for colorimetric analysis. OD [value missing] 450 reading.

[0095] Test results are shown Figures 11-13 See Tables 2-4. The results showed that B836 had a higher inhibition rate against the three cell lines than the single-target monoclonal antibody T320, Herceptin-MMAE, and the dual-target B83P, exhibiting a better tumor cell killing effect.

[0096] Table 2. Effects of different conjugated drugs on IC50 in Bxpc-3 cells. 50 result

[0097] <![CDATA[IC 50 ]]> Herceptin-MMAE 8.434nM DS8201 3.510nM B83P 1.978nM B836 0.0024nM T320 0.0031nM MMAE 1.596nM

[0098] Table 3. Effects of different conjugated drugs on IC50 in SK-OV-3 cells. 50 result

[0099] <![CDATA[IC 50 ]]> Herceptin-MMAE 1.075nM DS8201 3.177nM B83P 0.1244nM B836 0.0089nM T320 0.024nM MMAE 0.3997nM

[0100] Table 4. Effects of different conjugated drugs on IC50 in SK-BR-3 cells. 50 result

[0101] <![CDATA[IC 50 ]]> DS8201 0.03187nM MMAE 0.3507nM B83P 0.051nM B836 0.0226nM T320 0.5727nM Herceptin-MMAE 0.01901nM

[0102] Example 5

[0103] In vivo biological activity of B836-ADC

[0104] 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 5 Amedium + 10% FBS for sk-ov-3), and the cell concentration was adjusted 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 200mm... 3At approximately 10:00 AM, animals were randomly divided into seven groups of eight based on tumor volume. In the Bxpc-3 cell subcutaneous tumor assay in mice, B836 was administered once weekly at doses of 1 mg / kg, 2 mg / kg, and 4 mg / kg, for a total of four treatments. Antitumor drug RC48 was administered once weekly at 2 mg / kg for a total of four treatments; antitumor drug DS8201 was administered once weekly at 10 mg / kg; the positive control was gemcitabine at 60 mg / kg twice weekly for a total of three weeks; and the negative control was PBS. In the SK-OV-3 cell subcutaneous tumor experiment in mice, B836 was administered at doses of 2 mg / kg, 4 mg / kg, and 8 mg / kg, once weekly for a total of four treatments; RC48 was administered at 2 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 a total of four weeks; the negative control was PBS, administered via tail vein starting on Day 0. The weight of nude mice was measured every three days, and the length and short diameter of the tumors were measured with calipers. At the end of the experiment, blood was collected and serum was preserved. Animals were euthanized by neck dislocation, and the tumor was removed and weighed. The efficacy of the drugs was evaluated based on changes in tumor weight and relative volume. The formula for calculating tumor volume is given in Formula I.

[0105] Formula I: V = a × b × c;

[0106] Where a, b, and c represent the length, width, and height of the tumor, respectively, in mm.

[0107] like Figures 14-17 As shown, B836 significantly inhibits tumor growth in a dose-dependent manner. In the Bxpc-3 pancreatic cancer animal model, it is superior to the commercially available drugs RC48 and DS8201. In the sk-ov-3 animal model, it is superior to the commercially available drug DS8201 and has comparable efficacy to RC48.

[0108] 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 and human epidermal growth factor receptor-2, characterized in that, Including the heavy chain structure of targeting tissue factor, the light chain structure of targeting tissue factor, the heavy chain structure of targeting human epidermal growth factor receptor-2, and the light chain structure of targeting human epidermal growth factor receptor-2. The amino acid sequence of the heavy chain structure of the targeted tissue factor is shown in SEQ ID NO:2; The amino acid sequence of the light chain structure of the target tissue factor is shown in SEQ ID NO:4; The amino acid sequence of the heavy chain structure targeting human epidermal growth factor receptor-2 is shown in SEQ ID NO:6; The amino acid sequence of the light chain structure targeting human epidermal growth factor receptor-2 is shown in SEQ ID NO:

8.

2. The method for preparing the bispecific antibody according to claim 1, characterized in that, Includes the following steps: Recombinant vectors were constructed to express heavy chain structures targeting tissue factors, light chain structures targeting tissue factors, heavy chain structures targeting human epidermal growth factor receptor-2, and light chain structures targeting human epidermal growth factor receptor-2, respectively. The four recombinant vectors were co-transfected into mammalian cells for expression and purification to obtain bispecific antibodies.

3. The method for preparing the bispecific antibody according to claim 2, characterized in that, In the recombinant vector, the nucleotide sequence of the gene encoding the heavy chain structure of the target tissue factor is shown in SEQ ID NO:1; The nucleotide sequence of the gene encoding the light chain structure of the target tissue factor is shown in SEQ ID NO:3; The nucleotide sequence of the gene encoding the heavy chain structure targeting human epidermal growth factor receptor-2 is shown in SEQ ID NO:5; The nucleotide sequence of the gene encoding the light chain structure targeting human epidermal growth factor receptor-2 is shown in SEQ ID NO:

7.

4. The use of the bispecific antibody of claim 1 or the bispecific antibody prepared by the preparation method of claim 2 or 3 in the preparation of antitumor antibody-drug conjugates, wherein the tumor is any one of pancreatic cancer, breast cancer, and ovarian cancer.

5. The application according to claim 4, characterized in that, The host sources of the tumors include humans and / or primates.

6. A bispecific antibody-drug conjugate, characterized in that, The active pharmaceutical ingredient comprises the bispecific antibody of claim 1 or the bispecific antibody prepared by the preparation method of claim 2 or 3; the bispecific antibody is coupled to the active pharmaceutical ingredient monomethyl olritatin E via a disulfide bond.

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