Humanized Anti-Tissue Factor Antibody, Antibody-Drug Conjugate Prepared and Used

Abstract

The present invention relates to the technical field of biopharmaceuticals, and provides an anti-tissue factor humanized antibody, a prepared antibody-drug conjugate, and a use. An amino acid sequence of a heavy chain of the anti-tissue factor humanized antibody is as shown in SEQ ID NO: 3, and an amino acid sequence of a light chain of the anti-tissue factor humanized antibody is as shown in SEQ ID NO: 4. The anti-tissue factor humanized antibody has high affinity to tissue factors. The present invention further provides an antibody-drug conjugate, which is obtained by coupling the anti-tissue factor humanized antibody to a cytotoxic drug. On the basis of the characteristics that the anti-tissue factor humanized antibody has high affinity and short cell endocytosis time for tumor cell strains with low-expression and high-expression tissue factors, the antibody-drug conjugate can quickly reach a tumor target to achieve cytotoxicity, and can effectively inhibit the growth of tumor cells and subcutaneous tumor growth of mice. Therefore, the antibody-drug conjugate plays an important role in cancer treatment.

Description

Anti-tissue factor humanized antibody and prepared antibody-coupled drug and application thereof This application claims the priority of the Chinese patent application filed with the China Patent Office on March 24, 2023, with application number 202310308580.X and invention name “An anti-tissue factor humanized antibody and prepared antibody-drug conjugate and application”, the entire contents of which are incorporated by reference into this application. Technical Field The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to an anti-tissue factor humanized antibody and a prepared antibody-coupled drug and application thereof. Background Art Antibody-drug conjugates (ADCs) consist of three parts: monoclonal antibodies, cytotoxic drugs, and linkers. The advantage of ADC drugs is that they use the targeting properties of antibody drugs to accurately direct small molecule cytotoxic drugs to tumor tissues and release highly active cytotoxins to kill tumor cells, effectively improving the targeting properties of tumor drugs. At the same time, ADC drugs accurately identify targets without affecting normal non-cancerous cells, greatly reducing the toxic side effects of tumor chemotherapy. In recent years, ADC drugs have been a research hotspot in the field of precision tumor treatment, and have gradually become a hot track for new drug development, precisely because they have both the powerful killing effect of traditional small molecule chemotherapy and the tumor targeting properties of antibody drugs. Tissue Factor (TF) is a transmembrane protein with a molecular weight of 47 kDa, which initiates the extrinsic coagulation pathway. However, a series of scientific studies in recent years have reported that tissue factor is abnormally expressed in various tumor cells to varying degrees, and the expression of tumor tissue factor is positively correlated with the malignancy of the tumor, such as 100% positive rate in pancreatic cancer and cervical cancer, 34% to 88% positive rate in non-small cell lung cancer, 14% to 100% positive rate in endometrial cancer, 47% to 75% positive rate in prostate cancer, 75% to 100% positive rate in ovarian cancer, 43% to 91% positive rate in esophageal cancer, and 50% to 78% positive rate in bladder cancer. The abnormally high expression of tissue factor in these tumors leads to increased coagulation activity in tumor tissues and blood vessels, enhanced adhesion of tumor cells, and promoted metastasis and escape of tumor cells that invaded blood vessels; in tumor cells, VEGF transcription is promoted through intracellular signal transduction, inducing tumor angiogenesis. The high expression of tissue factor is closely related to tumor growth, angiogenesis, metastasis and clinical treatment. The development of tumor therapeutic drugs targeting tissue factor has gradually become a hot topic in the industry. However, the reports on monoclonal antibodies against tissue factor in the prior art are very limited, and the affinity of the monoclonal antibodies is not ideal, which undoubtedly hinders the development and research of antibody-drug conjugates based on tissue factor as a target. Summary of the invention In view of this, the object of the present invention is to provide an anti-tissue factor humanized antibody having high affinity to tissue factor. The present invention also aims to provide an antibody-drug conjugate, which is formed by coupling an anti-tissue factor humanized antibody and a cytotoxic drug, has the characteristics of short cell endocytosis time and high affinity for tumor cell lines with low tissue factor expression, thereby playing an important role in the treatment of tumors or cancers. The present invention provides an anti-tissue factor humanized antibody, wherein the amino acid sequence of the heavy chain of the anti-tissue factor humanized antibody is shown in SEQ ID NO:3; and the amino acid sequence of the light chain of the anti-tissue factor humanized antibody is shown in SEQ ID NO:4. The present invention provides an antibody-drug conjugate, which is obtained by conjugating the anti-tissue factor humanized antibody and a cytotoxic drug. Preferably, the cytotoxic drug comprises at least one of the following: MMAE, a dolastatin derivative, MMAF, an anti-tubulin inhibitor, DM1, a maytansine derivative, and DX8951, a DNA topoisomerase I inhibitor; Preferably, the anti-tissue factor humanized antibody and the cytotoxic drug are connected via a linker; the linker comprises at least one of the following: a GGFG tetrapeptide linker, a valine-citrulline dipeptide linker, and a SMCC thioether bond linker; The linker-cytotoxic drugs include MC-VC-PAB-MMAE, MC-VC-PAB-MMAF, MC-VC-PAB-DM1, MC-GGFG-DX8951, and MC-SMCC-DM1; The DAR value of the antibody-drug conjugate is 2-8. The present invention provides a method for preparing the antibody-drug conjugate, comprising the following steps: The anti-tissue factor humanized antibody, tris(2-chloroethyl)phosphate and coupling buffer are mixed to perform a reduction reaction to obtain a reduction product; The reduction product is subjected to coupling reaction with a linker-cytotoxic drug solution to obtain an antibody-coupled drug. Preferably, the mass-to-volume ratio of the anti-tissue factor humanized antibody and tris(2-chloroethyl)phosphate is (1-2):(2-3). The mass ratio of the anti-tissue factor humanized antibody to the linker-cytotoxic drug is 1:(5-9). Preferably, the temperature of the reduction reaction is 23-27° C., and the time of the reduction reaction is 1-2 h; The temperature of the coupling reaction is 23-27° C.; the time of the coupling reaction is 1-2 hours. The present invention provides a single-chain fusion protein, which is a fusion protein formed by the heavy chain variable region, light chain variable region and reporter protein of the anti-tissue factor humanized antibody; The amino acid sequence of the heavy chain variable region of the anti-tissue factor humanized antibody is as shown in SEQ ID NO: 47; The amino acid sequence of the light chain variable region of the anti-tissue factor humanized antibody is as shown in SEQ ID NO:48. The present invention provides an application of the single-chain fusion protein in detecting the expression of tissue factor on the surface of tumor cells. The present invention provides the use of the antibody-drug conjugate in preparing a drug for treating cancer. The present invention provides the use of the antibody-drug conjugate combined with other anticancer drugs in the preparation of drugs for treating cancer. Preferably, the cancer is a cancer in which tissue factor is abnormally expressed; the cancer in which tissue factor is abnormally expressed includes at least one of the following: ovarian cancer, non-small cell lung cancer, adenocarcinoma in situ, colon cancer, cervical cancer, prostate cancer, endometrial cancer, pancreatic cancer, esophageal cancer, bladder cancer, gastric cancer, liver cancer and breast cancer. The present invention provides an anti-tissue factor humanized antibody, the amino acid sequence of the heavy chain of the anti-tissue factor humanized antibody is shown in SEQ ID NO:1; the amino acid sequence of the light chain of the anti-tissue factor humanized antibody is shown in SEQ ID NO:2. The present invention performs affinity screening on hybridoma cells prepared from animals immunized with tissue factor, and obtains two hybridoma cell lines with relatively strong affinity. The two anti-tissue factor antibodies secreted by the hybridoma cells are humanized and recombinantly expressed to obtain humanized antibodies that specifically bind to tissue factor. The SPR method is used to detect that the humanized antibody protected by the present invention has a higher affinity, while the other humanized antibody has a lower affinity. In the embodiment of the present invention, the affinity kinetic analysis experiment shows that the KD of the humanized antibody MAb01 protected by the present invention and tissue factor is 3.57×10 -10 M, while the KD of another humanized antibody MAb02 is 1.002×10 -9 M. In summary, the high-affinity anti-tissue factor humanized antibody has a strong affinity for the antigen tissue factor. The present invention provides an antibody-drug conjugate, which is obtained by coupling the anti-tissue factor humanized antibody and a cytotoxic drug. Cell endocytosis experiments show that two anti-tissue factor humanized antibodies with different affinities are used to prepare antibody-drug conjugates respectively, and the obtained ADC drugs are FITC fluorescently labeled and detected in tumor cells with high and low expression of tissue factor. The results show that the ADC drug of the high-affinity anti-tissue factor humanized antibody is internalized in tumor cells with low expression of tissue factor, which can greatly promote preclinical development, clinical transformation and patient treatment effects. At the same time, the cell-level biological activity detection experiment shows that the antibody-drug conjugate prepared by the present invention can inhibit the biological activity of tumor cells with low and high expression of tissue factor. In addition, animal experiments show that the antibody-drug conjugate prepared by the present invention can effectively inhibit the growth of tumors. It can be seen that the antibody-drug conjugate provided by the present invention can not only inhibit the growth of tumors with high expression of tissue factor, but also effectively inhibit the growth of tumors with low expression of tissue factor, providing a universal drug for the treatment of various tumors or cancers, and has great clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS FIG1 is a validation result of a recombinant vector expressing an anti-tissue factor humanized antibody; FIG2 shows the results of recombinant expression of the heavy and light chains of two anti-tissue factor humanized antibodies; Figure 3 shows the affinity test results of anti-tissue factor humanized antibody-ADC drugs with tissues, wherein A is the result of anti-tissue factor humanized antibody MAb01-ADC drug; B is the result of anti-tissue factor humanized antibody MAb02-ADC drug; FIG4 is the purification result of anti-tissue factor single-chain antibody-GFP fusion protein; Figure 5 is a standard curve graph of tissue factor protein, wherein A is a standard curve graph of GFP-tissue factor single chain antibody detecting tissue factor protein, and B is a standard curve graph of FITC-tissue factor antibody detecting tissue factor protein; FIG6 shows the MAb01-ADC coupling results; FIG7 shows the MAb02-ADC coupling results; Figure 8 shows the results of endocytosis of anti-tissue factor humanized antibody MAb-ADC drugs in tumor cells, wherein A is the endocytosis result of MAb02-ADC in SK-OV-3 cells; B is the endocytosis result of MAb02-ADC in BXPC cells; C is the endocytosis result of MAb001-ADC in SK-OV-3 cells; D is the endocytosis result of MAb001-ADC in BXPC cells; E is the endocytosis result of MAb001-ADC in A549 cells; Figure 9 shows the results of the inhibitory effect of anti-tissue factor humanized antibody MAb01-ADC on different types of tumor cells; A shows the inhibitory effect of MAb001-ADC on tumor cells; B shows the inhibitory effect of MAb002-ADC on tumor cells; FIG. 10 shows the results of the inhibitory effect of the anti-tissue factor humanized antibody MAb01-ADC drug on tumor growth in a nude mouse subcutaneous tumor model, wherein A represents BXPC cells and B represents SK-OV-3 cells. DETAILED DESCRIPTION The present invention provides an anti-tissue factor humanized antibody, wherein the amino acid sequence of the heavy chain (HC) of the anti-tissue factor humanized antibody is as shown in SEQ ID NO: 3 (QIQLVQSGPELVKPGASVQVSCKTSGYSFTDYNVYWVRQSPAKGIEWIGYIDPYNGLTIYEQNFRGKGTLSLDHSTSTAYMELNSLRYEDTAVYFCARDVTTALDFWGQGTSVTVS SEFASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), encoding 449 amino acids; the amino acid sequence of the light chain (LC) of the anti-tissue factor humanized antibody is shown in SEQ ID NO:4(DIQMTQSPASISASIGERVTITCLASQTIDTWLAWFLQKPGRSPNLLIYAATNLADGVPYRFSASGSGNDFSLTISSLNPEDVATYYCQQVYSSPFTFGQGNKLEIRRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC), encoding 214 amino acids. In the embodiment of the present invention, the two anti-tissue factor monoclonal antibodies obtained by screening were humanized and recombinantly expressed to obtain two anti-tissue factor humanized antibodies. The affinity analysis results showed that the anti-tissue factor humanized antibody MAb01 protected by the present invention had a strong affinity and its ability to specifically recognize tissue factor was significantly better than that of another anti-tissue factor humanized antibody MAb02. The corresponding heavy chain amino acid sequence was SEQ ID NO: 1(QIQLVQSGPEVVKPGASVRVSCKGSGYSFTDYNIYWVRQSPAKGLEWIGYIDPYNGLTIYDQNFRAKATLSVDHSTSNAYMEINSLRYEDTAVYFCARDVTSALEFWGQGTSVTVSSEFASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP (CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), the corresponding light chain amino acid sequence is SEQ ID NO:2(DIQMTQSPASISASVGERVTITCLGSQTIDTYLAWYLQKPGRSPQLLIYAATQLADGVPSRFSASGSGTDFSLTISSLQPEDVATYYCQNVYSSPFTFGQGNKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC). The present invention provides an antibody-drug conjugate, which is obtained by conjugating the anti-tissue factor humanized antibody and a cytotoxic drug. In the present invention, the cytotoxic drug includes at least one of the following: MMAE, a dolastatin derivative, MMAF, an anti-tubulin inhibitor, DM1, a maytansine derivative, and DX8951, a DNA topoisomerase I inhibitor. The anti-tissue factor humanized antibody and the cytotoxic drug are connected by a linker. The linker preferably includes at least one of the following: a GGFG tetrapeptide linker, a valine-citrulline dipeptide linker, and an SMCC thioether bond linker. The linker-cytotoxic drug includes MC-VC-PAB-MMAE, MC-VC-PAB-MMAF, MC-VC-PAB-DM1, MC-GGFG-DX8951, and MC-SMCC-DM1. The present invention provides a method for preparing the antibody-drug conjugate, comprising the following steps: The anti-tissue factor humanized antibody, tris(2-chloroethyl)phosphate and coupling buffer are mixed to perform a reduction reaction to obtain a reduction product; The reduction product is subjected to coupling reaction with a linker-cytotoxic drug solution to obtain an antibody-coupled drug. In the present invention, the mass volume ratio of the anti-tissue factor humanized antibody and tris(2-chloroethyl)phosphate is preferably (1-2):(2-3), more preferably 1:3; 1:2; 2:3, and most preferably 1:3. The temperature of the reduction reaction is preferably 23-27°C, more preferably 25°C. The time of the reduction reaction is preferably 1-2h, more preferably 1.5h. The DAR value of the antibody-drug conjugate is preferably 2-8, more preferably 3.6-4.4. In the present invention, the mass ratio of the anti-tissue factor humanized antibody to the linker-cytotoxic drug is preferably 1: (5-9), more preferably 1: 5, 1: 6, 1: 7, 1: 8, 1: 9, and more preferably 1: 7. The temperature of the coupling reaction is preferably 23-27° C., more preferably 25° C. The time of the coupling reaction is preferably 1-2 h, more preferably 1.5 h. The present invention provides a single-chain fusion protein, which is a fusion protein formed by the heavy chain variable region, the light chain variable region and the reporter protein of the anti-tissue factor humanized antibody; the amino acid sequence of the heavy chain variable region of the anti-tissue factor humanized antibody is as shown in SEQ ID NO:47 (QIQLVQSGPELVKPGASVQVSCKTSGYSFTDYNVYWVRQSPAKGIEWIGYIDPYNGLTIYEQNFRGKGTLSLDHSTSTAYMELNSLRYEDTAVYFCARDVTTALDFWGQGTSVTVSS); the amino acid sequence of the light chain variable region of the anti-tissue factor humanized antibody is as shown in SEQ ID NO:48 (DIQMTQSPASISASIGERVTITCLASQTIDTWLAWFLQKPGRSPNLLIYAATNLADGVPYRFSASGSGNDFSLTISSLNPEDVATYYCQQVYSSPFTFGQGNKLEIR). The present invention has no particular limitation on the type of reporter protein, and any reporter protein known in the art may be used, for example Green fluorescent protein. In the embodiment of the present invention, the amino acid sequence of the single-chain fusion protein is shown in SEQ ID NO: 5; the nucleotide sequence of the gene encoding the single-chain fusion protein is shown in SEQ ID NO: 6. The present invention has no special restrictions on the expression method of the fusion protein, and the preparation method of the recombinant expression protein well known in the art can be used. The present invention provides an application of the single-chain fusion protein in detecting the expression of tissue factor on the surface of tumor cells. In the present invention, the method for detecting the expression of tissue factor on the surface of tumor cells preferably includes adding tumor cells to an ELISA black detection plate coated with tissue factor protein for incubation, washing, and then adding a single-chain fusion protein for incubation. After washing, adding a buffer solution for resuspending, and detecting on a machine under the conditions of an excitation wavelength of 493nm and an emission wavelength of 528nm to obtain an absorbance value, and calculating the expression level of tissue factor on the surface of tumor cells according to the regression equation obtained by the standard curve. The washing solution is preferably a PBS solution. The resuspending buffer is preferably a PBS solution. The incubation time is preferably 1 to 1.5h. The results show that the expression of tissue factor on the surface of SK-OV-3 and BXPC-3 cell lines is the highest, and the expression of tissue factor on the surface of SW620 and A549 cell lines is low. The method provided by the present invention can calculate the number of tissue factor targets on the surface of each type of tumor cell, thereby providing guidance for the administration of antibody-drug conjugates. In the present invention, the antibody-drug conjugate prepared by the present invention is based on the fact that the anti-tissue factor humanized antibody has a high affinity for tissue factor and a short cell endocytosis time, which ensures that the coupled cytotoxin quickly reaches the tumor cell site and exerts a cytotoxic or cell-killing effect; at the same time, in view of the fact that the anti-tissue factor humanized antibody has a high affinity for low-expressing tissue factor tumor cell lines, it is ensured that tumor types with low tissue factor expression are also suitable for the treatment of antibody-drug conjugates. Experiments have shown that the antibody-drug conjugate can effectively inhibit the growth of tumor cells and the growth of subcutaneous tumors in mice, and can thus play an important role in the treatment of various cancer diseases. In view of this, the present invention provides the use of the antibody-drug conjugate in the preparation of drugs for treating cancer. The use of the antibody-drug conjugate in combination with other anticancer drugs in the preparation of drugs for treating cancer. The mass ratio of the antibody-drug conjugate to other anticancer drugs is 1: (23-27), more preferably 1: 25. The other anticancer drugs preferably include gemcitabine. In the present invention, the cancer is a cancer with abnormal expression of tissue factor. The cancer with abnormal expression of tissue factor preferably includes at least one of the following: ovarian cancer, non-small cell lung cancer, adenocarcinoma in situ, colon cancer, cervical cancer, prostate cancer, endometrial cancer, pancreatic cancer, esophageal cancer, bladder cancer, gastric cancer, liver cancer and breast cancer. The anti-tissue factor humanized antibody, the prepared antibody-drug conjugate and the application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention. Example 1 Sequence modification experiment of anti-tissue factor humanized antibody The humanized tissue factor antibody disclosed in the patent previously applied by the company (Announcement No.: CN 107446047A) was artificially modified, and the virtual amino acid mutation of the humanized antibody based on the interaction force was performed using Discovery Studio to improve the binding affinity of the antibody. This modification obtained two anti-tissue factor humanized antibody sequences, MAb01 and MAb02. The modified sequences are shown in Tables 1 and 2. Table 1 Heavy chain variable region Table 2 Light chain variable region The heavy chain HC of the anti-tissue factor humanized antibody MAb02 encodes 449 amino acids, and its amino acid sequence is SEQ ID NO: 1; the light chain LC encodes 214 amino acids, and its amino acid sequence is SEQ ID NO: 2. The heavy chain HC of the anti-tissue factor humanized antibody MAb01 encodes 449 amino acids, and its amino acid sequence is SEQ ID NO: 3. The light chain gene LC encodes 214 amino acids, and its amino acid sequence is SEQ ID NO: 4. Example 2 Expression of humanized anti-tissue factor antibody and screening of stable cell lines This example is consistent with the screening method in the patent of our company (application announcement number: CN 107446047A). After the genes encoding humanized antibodies MAb01 and MAb02 are artificially synthesized, the heavy chain gene fragments of MAb01 and MAb02 and the pinsulator4X-MSA vector are double-digested by SalI and AscI, respectively. After 1% agarose gel electrophoresis, the insert fragment 1 fragment (1425bp) and the linearized vector 1 fragment (11938bp) are recovered by gel electrophoresis, and the two are connected overnight at 4°C to form a connection product. At the same time, after the gene sequences of the light chain gene fragments of MAb01 and MAb02 are synthesized, the light chain gene fragments of MAb01 and MAb02 and the pCAGGS-IRES-AscI vector are double-digested by NotI and Bci I, respectively, the insert fragment 2 fragment (720bp) and the linearized vector 2 fragment (6838bp) are recovered by electrophoresis gel, and the two are connected overnight at 4°C to form a connection product. The ligation product was transformed into competent E. coli DH5α, and single colonies were selected with ampicillin. Single colonies were cultured at 37°C overnight, and plasmids were extracted from the bacteria and confirmed by enzyme digestion. After the above recombinant plasmids were correctly identified, the pinsulator4X-MAb01 heavy chain-dhfr and pinsulator4X-MAb02 heavy chain-dhfr were double-digested with SalI and MauB I, and the inserted fragment 3 (13349 bp) fragment of the digestion result was recovered by 1% agarose gel electrophoresis; the pCAGGS-MAb01 light chain-IRES-AscI and pCAGGS-MAb02 light chain-IRES-AscI were double-digested with SalI and AscI, and the inserted fragment 3 (4179) fragment was recovered by electrophoresis. Then the linearized vector 3 and the inserted fragment 3 were connected to form the pinsulator4X-CAG-MAb01-dhfr expression vector and the pinsulator4X-CAG-MAb02-dhfr expression vector, which were transferred into the competent E.coliDH5α, and the single colony was selected by ampicillin. At 37°C and 180rpm, the single colony was cultured overnight, the bacteria were collected, the plasmid was extracted, and the enzyme digestion identification and sequencing analysis were performed. The results of 1% agarose gel electrophoresis of the expression vectors pinsulator4X-CAG-MAb01-dhfr and pinsulator4X-CAG-MAb02-dhfr digested with SwaI are shown in FIG1 . The electrophoresis results are consistent with the theoretical values ​​(2890 bp, 14638 bp). The sequencing results of the obtained vectors showed that the heavy chain gene and light chain gene of the pinsulator4X-CAG-MAb01-dhfr and pinsulator4X-CAG-MAb02-dhfr vectors were completely consistent with the sequences of gene synthesis. Example 3 1. Expression of humanized anti-tissue factor antibody CHO-dhfr- cells were collected at 6×10 5 The cells were inoculated into a 6-well plate at a density of 1 μg / mL and cultured overnight. Preparation of transfection complex: Add 25 μl Opti-MEM and 4 μl Lipofectamine3000 to tube A and shake at room temperature for 1 to 2 seconds. Add 25 μl Opti MEM and 2 μg plasmid (pinsulator4X-CAG-MAb01-dhfr, pinsulator4X-CAG-MAb02-dhfr) to tube B. Mix the above two tubes, let stand for 5 minutes, and then add to a 6-well culture plate. After 24 hours, trypsinize the cells into the culture dish, add MTX with a final concentration of 50 μm for cell pressure screening, and after 20 days, pick single cell clusters and culture them in 6-well plates. When the cell confluence reaches 90%, transfer to T25 The cell density reached 5×10 per ml. 5When the cells reached a certain amount, they were transferred into a conical flask and cultured at 37°C, 5% CO2, and 130 rpm with shaking. After 15 days of culture, the cell supernatant was collected. 2. Purification of humanized anti-tissue factor antibody (1) Cell fluid pretreatment: Collect the cell supernatant, adjust the pH to 9.0, centrifuge at 10,000 rpm for 20 min at 4°C, and filter through a 0.22 μm membrane. (2) Purification of Mabselet protein A: 5 column volumes of equilibration solution (20mM Tris·HCl 0.15M NaCl pH 9.0) were used to equilibrate column A. The cell supernatant was passed through column A at a flow rate of 2ml / min. The absorption peak was recorded. After loading, 5 column volumes of equilibration solution were used to top wash until the baseline was reached. The target protein was eluted with 100mM pH3.0 glycine·HCl eluent. The elution peak was collected and column A was washed with equilibration solution. The pH value of the collected solution was adjusted to 7.0 with 1M Tris. The target protein was subjected to SDS-PAGE electrophoresis. The analysis results are shown in Figure 2. Analysis of purification results: After the cell fluid with impurities was purified by protein A column and gel chromatography, a uniform protein was obtained. According to the molecular weight of the antibody and the SDS-PAGE results, the 50KD position was the heavy chain band of the anti-tissue factor humanized antibody, and the 25KD position was the light chain band of the anti-tissue factor humanized antibody. Example 3 Kinetic Analysis of Affinity between Humanized Antibody against Tissue Factor and Antigen Tissue Factor The anti-tissue factor humanized antibody was coupled to the second channel of the CM5 chip surface through amino coupling, and the 1st and 3rd channels were used as reference channels. The tissue factor was used as the analyte (mobile phase) to flow through the chip surface. The tissue factor mother solution was diluted to 18nM, 9nM, 4.5nM, 2.25nM, and 1.125nM as the injection concentration. The injection time was 120s, the dissociation time was 600s, and the glycine hydrochloride pH 2.0 was regenerated for 30s. The affinity was measured by running the Kinetics / Affinity program of Bicore S200. The affinity of the humanized antibody before the modification was 3.371×10 -8 The determination method was consistent with that used in this experiment. The tissue factor stock solution was diluted to 12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM, respectively. The detection results are shown in Figure 3. The results are shown in Figure 3. The binding constant KD of anti-tissue factor humanized antibody MAb01 and tissue factor is 3.57×10 -10 M, binding constant of anti-tissue factor humanized antibody MAb02 and tissue factor KD = 1.002 × 10 -9 M. All of them are higher than the original patented antibodies of our company before transformation. The humanized anti-tissue factor antibody MAb01 has the strongest affinity with the antigen tissue factor. Example 4 Method for detecting the expression of tissue factor on the surface of tumor cells 1. Expression and purification of anti-tissue factor single-chain antibody-GFP fusion protein (TF-scFv-GFP) The heavy chain variable region and light chain variable region (scFv) of the anti-tissue factor humanized antibody MAb01 were fused with green fluorescent protein (GFP) to express, and the anti-tissue factor single-chain antibody-GFP fusion protein (amino acid sequence of SEQ ID NO: 5, QIQLVQSGPELVKPGASVQVSCKTSGYSFTDYNVYWVRQSPAKGIEWIGYIDPYNGLTIYEQNFRGKGTLSLDHSTSTAYMELNSLRYEDTAVYFCARDVTTALDFWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPASISASIGERVTITCLASQTIDTWLAWFLQKPGRSPNLLIYAATNLADGVPYRFSASGSGNDFSLTISSLNPEDVATYYCQQVYSSPFTFGQGNKLEIRG GAGGGMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGD TLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK).The coding gene (SEQ ID NO: 6, CAAATCCAGCTGGTTCAGAGCGGTCCAGAACTGGTTAAACCGGGCGCTTCTGTACAGGTTTCTTGCAAAACCTCCGGTTACTCCTTCACCGACTACAATGTGTACTGGGTTCGCCAGTCTCCTGCTAAAGGCATCGAGTGGATCGGTTACATCGACCCGTACAACGGCCTGACTATTTACGAACAGAATTTTCGTGGCAAAGGCACCCTGTCTCTGGATCATTCTACCAGCACCGCTTATATGGAACTGAATAGCCTGCGTTACGAAGATACCGCGGTTTATTTCTGTGCTCGTGATGTAACTACTGCCCTGGACTTTTGGGGCCAGGGTACGTCTGTAACCGTAAGCTCTGGTGGTGGCGGTTCTGGCGGTGGTGGTTCTGGTGGTGGTGGTAGCGATATCCAGATGACCCAGTCTCCGGCTTCCATTAGCGCCTCCATCGGTGAGCGTGTCACCATCACTTGCCTGGCCAGCCAGACCATCGATACTTGGCTGGCATGGTTCCTGCAGAAACCGGGTCGTAGCCCAAATCTGCTGATCTACGCTGCAACGAACCTGGCGGACGGCGTTCCGTACCGTTTTTCCGCGTCCGGCTCCGGTAACGACTTCAGCCTGACCATCTCTTCTCTGAACCCTGAAGATGTCGCAACGTACTACTGCCAGCAGGTATACAGCAGCCCGTTCACCTTCGGTCAGGGCAACAAACTGGAGATCCGCGGTGGCGCCGGTGGCGGTATGTCTAAAGGTGAAGAGCTGTTTACTGGTGTTGTTCCGATCCTGGTGGAGCTGGACGGTGATGTTAACGGCCATAAATTCAGCGTGT。 ) was cloned into the NcoI / XhoI multiple cloning site of the expression vector pET-28a. After the recombinant vector was successfully constructed, it was transformed into the competent expression strain BL21(DE3), cultured at 37°C overnight, and inoculated into a new culture medium the next day. The OD 600 When the concentration was 0.6, IPTG was added to a final concentration of 1 mM, and the cells were induced at 16°C overnight. The cells were collected by centrifugation, and the cells were broken by ultrasound. Ni column affinity chromatography was performed to obtain TF-scFv-GFP fusion protein with a molecular weight of about 57 kDa. The purification results of TF-scFv-GFP are shown in Figure 4. 2. Detection of cell surface tissue factor protein using anti-tissue factor single-chain antibody-GFP fusion protein The tissue factor protein (purchased from Taiyuan Bio-Tech Biotechnology Co., Ltd., catalog number: BAT1001) was coated in a 96-well ELISA black test plate at a concentration of 300 pmol, 150 pmol, 75 pmol, 37.5 pmol, 18.75 pmol, and 9.375 pmol of tissue factor protein, respectively. A certain amount of tumor cells (about 1×10 SK-OV-3 / BXPC-3 cell line) were taken. 6 Cells, Hela cell line about 1×10 7 Cells, A549 / SW620 cell line about 1×10 8 Cells) were fixed to a 96-well cell culture plate, and anti-tissue factor single-chain antibody-GFP fusion protein and positive control antibody (abcam FITC fluorescent Anti-Tissue Factor antibody

[0005] (ab275690), incubate for 1 hour, wash 1-2 times with PBS (pH 7.3), resuspend in PBS buffer, and detect on the microscope under the conditions of excitation wavelength of 493nm and emission wavelength of 528nm. The prepared standard curve is shown in Figure 5. The expression of tissue factor on the surface of tumor cells was detected according to the above method. The results are shown in Table 3. The results of anti-tissue factor single-chain antibody-GFP fusion protein detection of cell surface tissue factor protein are consistent with the positive control antibody detection, indicating that the detection method in this embodiment is feasible. The results show that the expression of tissue factor on the surface of SK-OV-3 and BXPC-3 cell lines is the highest, and the expression of tissue factor on the surface of SW620 and A549 cell lines is low. According to 1 mol, there are 6.02×10 23 The number of tissue factor targets on the surface of each type of tumor cells was calculated, as shown in Table 4. Table 3 Expression of tissue factor on tumor cell surface Table 4 Number of targets on tumor cell surface Example 5 Anti-tissue factor humanized antibody-ADC drug conjugate experiment After the antibody (5 mg / mL) was replaced with coupling buffer (20 mM His-His﹒HCl, pH=5.96), coupling was performed directly. Antibodies, DTPA (diethylenetriaminepentaacetic acid, 10 mM) and TCEP (tris(2-carboxyethyl)phosphine hydrochloride, 10 mM) were added in sequence according to Table 5. After each component was added, it was immediately vortexed briefly to fully mix. Finally, the reaction system was collected at the bottom of the tube by brief centrifugation, and then placed in a constant temperature mixer to perform a reduction reaction according to the conditions shown in Table 5. After the reduction reaction was completed, a corresponding amount of DMSO (dimethyl sulfoxide) was added to each reaction system in an ice water bath, and briefly vortexed to fully mix. Then, a corresponding amount of Linker-payload (5 mM) was added, briefly vortexed to fully mix, and then centrifuged to collect the reaction system at the bottom of the tube. The reaction system was placed in a constant temperature mixer and the coupling reaction was performed according to the conditions shown in Table 6. After the coupling was completed, an appropriate amount of ADC was taken to detect the DAR value using the HIC-HPLC method (as shown in Table 7). After the ADC was repeatedly dialyzed several times using an ultrafiltration centrifuge tube (30 KDa), an appropriate amount was taken for concentration, HIC-HPLC and SEC-HPLC, and free drug residue detection. Table 5 Reduction reaction parameters Table 6 Coupling reaction parameters Table 7 DAR reduction-coupling experiment results Example 6 Anti-tissue factor humanized antibody MAb01-ADC drug endocytosis experiment The tissue factor high-expressing and low-expressing tumor cells were cultured and digested with trypsin after the cell density reached 90%. 250,000 cells were plated on a confocal dish and cultured until the cells adhered to the wall. After the cells adhered to the wall, the original culture medium was aspirated and 1 ml of culture medium containing 1 μl of antibody MAb01-ADC drug or antibody MAb02-ADC with fluorescent label (FITC) was added respectively. From the beginning of adding the antibody, the cells were observed under a confocal microscope (Zeiss LSM 710, 20 times microscope), with a time of 0 min, and a uniform density and a single cell field of view were selected. The time was set to 20 min per cycle. After a total of 6 / 8 cycles, the cells were photographed. The experimental results are shown in Figure 8. The antibody MAb01-ADC drug begins to be internalized in half an hour and is completed within 2 hours. Both the SK-OV-3 cell line and the A549 cell line can achieve internalization. The antibody MAb02-ADC-ADC drug takes a long time to be internalized. The SK-OV-3 cell line can achieve internalization, but the A549 cell line cannot achieve internalization. It can be seen that the high-affinity anti-tissue factor humanized antibody-ADC drug can be internalized in tumor cells with low tissue factor expression. Example 7 High affinity anti-tissue factor humanized antibody-ADC drug biological activity detection The cell lines used in this example were purchased from the cell bank of the Chinese Academy of Sciences and cultured according to the corresponding instructions, including SK-vo-3 (human ovarian cancer cell line), A549 (human non-small cell lung cancer), BXPC (pancreatic cancer cell line), SW620 (colon cancer cell line) Hela cell line (cervical cancer cell line), IgG-MMAE drug as a control, the cells in the logarithmic growth phase were inoculated in a 96-well cell culture plate at a density of 5000 cells per well, 100 μl per well, 37 degrees, 5% carbon dioxide culture for about 16 hours, and then different concentrations of antibody MAb01-ADC drugs were added, and 3 replicates were set for each drug concentration. After 3 days of action, the culture medium was poured off, and CCK-8 reaction solution was added, 100 μl per well, and reacted at 37 degrees to the expected color depth, and the cell viability (OD 450 ), and the cell survival rate was calculated according to the following formula I; MAb02-ADC drugs were also tested on cell lines that can achieve endocytosis (SK-vo-3 (human ovarian cancer cell line), BXPC (pancreatic cancer cell line), and Hela cell line (cervical cancer cell line). Survival rate (%) = (OD administration - OD blank) / (OD control - OD blank) × 100% Formula I The above data were analyzed by GraphPad Prism 5 software, and the IC of TF-ADC on different cell lines was calculated. 50 value. The results are shown in Figure 9. In vitro, the antibody MAb01-ADC drug can effectively inhibit the growth of tumor cells with high tissue expression, and its inhibitory effect is proportional to the number of TF molecules on the cell surface. The antibody MAb02-ADC drug also has a certain inhibitory effect on tumor cell lines that achieve endocytosis. IC of different cancer cell types to antibody MAb01-ADC drug 50 The results are shown in Table 8. Table 8 IC values ​​of different types of cancer cells for antibody MAb01-ADC drug 50 value Example 8 Anti-tissue factor humanized antibody-ADC drug inhibits subcutaneous tumor in mice The nude mouse subcutaneous tumor model was used, and tumor cells were selected; one tumor cell was implanted into every 100 nude mice; when the tumor volume reached 64-100 cubic millimeters, 6 experimental groups were determined, with 8 mice in each group, and the antibody MAb01-ADC drug was used to treat the tumor at a dose of 1 mg / kg, 2 mg / kg, and 4 mg / kg, respectively. ADC 2 mg / kg was combined with gemcitabine 50 mg / kg for treatment, the positive control was gemcitabine, the dosage was 50 mg / kg, and the negative control was PBS. The drug was administered through the tail vein starting from Day 0. The body weight of the nude mice was measured every other day, and the long and short diameter data of the tumor were measured with a vernier caliper. At the end of the experiment, blood was collected to store serum, the animals were killed by cervical dislocation, the tumor mass was removed and weighed, and the drug efficacy was evaluated based on the changes in tumor weight and relative volume. The tumor volume was calculated according to formula II. V = a × b × c Formula II. Among them, a, b, and c represent the length, width, and height of the tumor, respectively. As shown in Figure 10, the antibody MAb01-ADC drug can significantly inhibit the growth of ovarian cancer and pancreatic cancer tumors in a dose-dependent manner. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anti-tissue factor humanized antibody, characterized in that: The amino acid sequence of the heavy chain of the anti-tissue factor humanized antibody is shown in SEQ ID NO:3; the amino acid sequence of the light chain of the anti-tissue factor humanized antibody is shown in SEQ ID NO:

4.

2. An antibody-drug conjugate, characterized in that: The anti-tissue factor humanized antibody according to claim 1 is coupled with a cytotoxic drug.

3. The antibody-drug conjugate according to claim 2, characterized in that: The cytotoxic drug comprises at least one of the following: MMAE, a derivative of dolastatin, MMAF, an anti-tubulin inhibitor, DM1, a maytansine derivative, and DX8951, a DNA topoisomerase I inhibitor.

4. The antibody-drug conjugate according to claim 2, characterized in that: The anti-tissue factor humanized antibody and the cytotoxic drug are connected via a linker; The linker comprises at least one of the following: a GGFG tetrapeptide linker, a valine-citrulline dipeptide linker, and a SMCC thioether bond linker; The linker-cytotoxic drugs include MC-VC-PAB-MMAE, MC-VC-PAB-MMAF, MC-VC-PAB-DM1, MC-GGFG-DX8951, and MC-SMCC-DM1; The DAR value of the antibody-drug conjugate is 2-8.

5. A method for preparing the antibody-drug conjugate according to any one of claims 2 to 4, characterized in that: The following steps are involved: The anti-tissue factor humanized antibody, tris(2-chloroethyl)phosphate and coupling buffer are mixed to perform a reduction reaction to obtain a reduction product; The reduction product is subjected to coupling reaction with a linker-cytotoxic drug solution to obtain an antibody-coupled drug.

6. The preparation method according to claim 5, characterized in that: The mass volume ratio of the anti-tissue factor humanized antibody and tris(2-chloroethyl)phosphate is (1-2):(2-3); The mass ratio of the anti-tissue factor humanized antibody to the linker-cytotoxic drug is 1:(5-9).

7. The preparation method according to claim 5, characterized in that: The temperature of the reduction reaction is 23-27° C., and the time of the reduction reaction is 1-2 hours.

8. A single-chain fusion protein, characterized in that It is a fusion protein formed by the heavy chain variable region, light chain variable region and reporter protein of the anti-tissue factor humanized antibody according to claim 1; The amino acid sequence of the heavy chain variable region of the anti-tissue factor humanized antibody is as shown in SEQ ID NO: 47; The amino acid sequence of the light chain variable region of the anti-tissue factor humanized antibody is as shown in SEQ ID NO:

48.

9. Use of the single-chain fusion protein according to claim 8 in detecting the expression of tissue factor on the surface of tumor cells.

10. Use of the antibody-drug conjugate according to any one of claims 2 to 4 in the preparation of a drug for treating cancer.

11. Use of the antibody-drug conjugate according to any one of claims 2 to 4 in the treatment of cancer.

12. Use of the antibody-drug conjugate according to any one of claims 2 to 4 in combination with other anticancer drugs in the preparation of a drug for treating cancer.

13. Use of the antibody-drug conjugate according to any one of claims 2 to 4 in combination with other anticancer drugs in the treatment of cancer.

14. The use according to any one of claims 10 to 13, characterized in that: The cancer is a cancer in which tissue factor is abnormally expressed; the cancer in which tissue factor is abnormally expressed includes at least one of the following: ovarian cancer, non-small cell lung cancer, adenocarcinoma in situ, intestinal cancer, cervical cancer, prostate cancer, endometrial cancer, pancreatic cancer, esophageal cancer, bladder cancer, gastric cancer, liver cancer, colon cancer and breast cancer.

15. An anticancer drug, characterized in that: The invention comprises the antibody-drug conjugate according to any one of claims 2 to 4 and other anticancer drugs.

16. The anticancer drug according to claim 13, characterized in that: The other anticancer drugs include gemcitabine.