Anti-tissue factor humanized antibodies, produced antibody-drug conjugates and uses

Abstract

The present invention provides an anti-tissue factor humanized antibody, an antibody-drug conjugate prepared therefor, and uses thereof, which belong to the field of biopharmaceuticals. The amino acid sequence of the heavy chain of the anti-tissue factor humanized antibody is set forth in SEQ ID NO:3, and the amino acid sequence of the light chain is set forth in SEQ ID NO:4. The anti-tissue factor humanized antibody has high affinity for tissue factor. The present invention also provides an antibody-drug conjugate obtained by coupling the anti-tissue factor humanized antibody with a cytotoxic drug. Based on the characteristics of the anti-tissue factor humanized antibody, which has high affinity for both low- and high-tissue factor tumor cell lines and a short cellular endocytosis time, the antibody-drug conjugate can rapidly reach tumor targets and exert a cytotoxic effect, while also effectively suppressing the growth of tumor cells and subcutaneous tumors in mice. Therefore, the antibody-drug conjugate plays an important role in cancer treatment.

Description

[Technical Field]

[0001] This application claims priority to a Chinese patent application filed with the China Patent Office on March 24, 2023, with application number 202310308580.X and titled "Anti-tissue factor humanized antibody, prepared antibody-drug conjugate and use," the entire contents of which are incorporated herein by reference.

[0002] The present invention belongs to the technical field of biopharmaceuticals, and specifically relates to an anti-tissue factor humanized antibody, the prepared antibody-drug conjugate and its use. [Background technology]

[0003] Antibody-drug conjugates (ADCs) consist of three components: a monoclonal antibody, a cytotoxic drug, and a linker. The advantage of ADCs is that they utilize the targeting properties of antibody drugs to precisely target small molecule cytotoxic drugs to tumor tissue, releasing highly active cytotoxins to kill tumor cells, effectively improving the targeting properties of tumor drugs. At the same time, ADC drugs precisely target tumors without affecting normal, non-cancerous cells, significantly reducing the harmful side effects of tumor chemotherapy. In recent years, ADC drugs have become a research hotspot in the field of precision tumor therapy because they combine the powerful killing effect of traditional small molecule chemotherapy with the tumor-targeting properties of antibody drugs, becoming a popular area of ​​new drug research and development.

[0004] Tissue factor (TF) is a 47-kDa transmembrane protein that initiates the extrinsic coagulation pathway. However, recent scientific studies have demonstrated that tissue factor is abnormally expressed in various tumor cells to varying degrees, and tumor tissue factor expression is positively correlated with tumor malignancy. For example, the positive rates are 100% in pancreatic and cervical cancer, 34%-88% in non-small cell lung cancer, 14%-100% in endometrial cancer, 47%-75% in prostate cancer, 75%-100% in ovarian cancer, 43%-91% in esophageal cancer, and 50%-78% in bladder cancer. Abnormally high tissue factor expression in these tumors leads to increased coagulation activity in tumor tissue and blood vessels, enhanced tumor cell adhesion, and promoted tumor cell migration and escape from blood vessels. Furthermore, in tumor cells, TF expression is enhanced through intracellular signaling, promoting VEGF transcription and tumor angiogenesis. High expression of tissue factor is closely related to tumor growth, angiogenesis, metastasis, and clinical treatment, and the development of tumor treatment drugs targeting tissue factor has gradually attracted attention in the industry.

[0005] However, there are very limited reports on monoclonal antibodies against tissue factor in the prior art, and the affinity of monoclonal antibodies is not ideal, which undoubtedly hinders the development and research of antibody-drug conjugates targeting tissue factor. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, an object of the present invention is to provide a humanized anti-tissue factor antibody that has high affinity for tissue factor.

[0007] Another object of the present invention is to provide an antibody-drug conjugate formed by coupling an anti-tissue factor humanized antibody with a cytotoxic drug, which is characterized by a short cellular endocytosis time and high affinity for tumor cell lines with low tissue factor expression, and which plays an important role in the treatment of tumors and cancers. [Means for solving the problem]

[0008] 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 being represented by SEQ ID NO:3, and the amino acid sequence of the light chain of the anti-tissue factor humanized antibody being represented by SEQ ID NO:4.

[0009] The present invention provides an antibody-drug conjugate obtained by coupling the anti-tissue factor humanized antibody with a cytotoxic drug.

[0010] Preferably, the cytotoxic drug comprises at least one of the dolastatin derivative MMAE, the anti-tubulin inhibitor MMAF, the maytansine derivative DM1, and the DNA topoisomerase I inhibitor DX8951; Preferably, the anti-tissue factor humanized antibody and the cytotoxic drug are linked via a linker, and the linker comprises at least one of 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, MC-SMCC-DM1; The DAR value of the antibody-drug conjugate is 2-8.

[0011] The present invention provides a method for producing the antibody-drug conjugate, mixing an anti-tissue factor humanized antibody, tris(2-chloroethyl)phosphate, and a coupling buffer solution, and performing a reduction reaction to obtain a reduction product; and performing a coupling reaction between the reduction product and a linker-cytotoxic drug solution to obtain an antibody-drug conjugate.

[0012] Preferably, the mass / volume ratio of the anti-tissue factor humanized antibody to tris(2-chloroethyl)phosphate is (1 to 2):(2 to 3).

[0013] The mass ratio of the anti-tissue factor humanized antibody to the linker-cytotoxic drug is 1:(5-9).

[0014] Preferably, the temperature of the reduction reaction is 23 to 27°C, and the time of the reduction reaction is 1 to 2 hours. The temperature of the coupling reaction is 23 to 27° C., and the time of the coupling reaction is 1 to 2 hours.

[0015] The present invention provides a single-chain fusion protein, which is a fusion protein formed from the heavy chain variable region and light chain variable region of the humanized anti-tissue factor antibody and a reporter protein.

[0016] The amino acid sequence of the heavy chain variable region of the humanized anti-tissue factor antibody is shown in SEQ ID NO:47, and the amino acid sequence of the light chain variable region of the humanized anti-tissue factor antibody is shown in SEQ ID NO:48.

[0017] The present invention provides the use of the single-chain fusion protein in detecting the expression of tumor cell surface tissue factor.

[0018] The present invention provides use of the antibody-drug conjugate in the manufacture of a medicament for treating cancer.

[0019] The present invention provides the use of the antibody-drug conjugate in combination with other anti-cancer drugs in the manufacture of a medicament for treating cancer.

[0020] Preferably, the cancer is a cancer in which tissue factor is abnormally expressed, and the cancer in which tissue factor is abnormally expressed includes at least one of ovarian cancer, non-small cell lung cancer, primary adenocarcinoma, intestinal cancer, cervical cancer, prostate cancer, endometrial cancer, pancreatic cancer, esophageal cancer, bladder cancer, gastric cancer, liver cancer, colon cancer, and breast cancer. [Effects of the Invention]

[0021] The present invention provides an anti-tissue factor humanized antibody, the heavy chain amino acid sequence of which is shown in SEQ ID NO:1, and the light chain amino acid sequence of which is shown in SEQ ID NO:2. The present invention performs affinity screening on hybridoma cells prepared from animals immunized with tissue factor, obtaining two hybridoma cell lines with relatively strong affinity. Two anti-tissue factor antibodies secreted from the cells are humanized and recombinantly expressed to obtain humanized antibodies that specifically bind to tissue factor. SPR detection reveals that one humanized antibody protected by the present invention has a higher affinity, while another humanized antibody has a lower affinity. In an example of the present invention, affinity kinetic analysis experiments revealed that the KD of the humanized antibody MAb01 protected by the present invention for tissue factor is 3.57×10 -10 M, whereas the KD of another humanized antibody, MAb02, was 1.002 × 10 -9 In summary, the high-affinity anti-tissue factor humanized antibody has a strong affinity for the antigen tissue factor.

[0022] The present invention provides an antibody-drug conjugate obtained by coupling the anti-tissue factor humanized antibody with a cytotoxic drug. In cell endocytosis experiments, two anti-tissue factor humanized antibodies with different affinities were used to prepare antibody-drug conjugates. The resulting ADC drug was labeled with FITC fluorescence. Tests on high- and low-tissue factor tumor cells demonstrated that the high-affinity anti-tissue factor humanized antibody ADC drug achieved endocytosis in low-tissue factor tumor cells, significantly facilitating preclinical development, clinical transformation, and patient treatment efficacy. At the same time, cellular bioactivity detection experiments demonstrated that the antibody-drug conjugate prepared by the present invention can inhibit the biological activity of tumor cells with both low and high tissue factor expression. Furthermore, animal experiments demonstrated that the antibody-drug conjugate prepared by the present invention can effectively inhibit tumor growth. The antibody-drug conjugate provided by the present invention was found to be able to inhibit not only the growth of tumors with high tissue factor expression but also the growth of tumors with low tissue factor expression, providing a versatile drug for the treatment of various tumors and cancers and having great clinical value. [Brief explanation of the drawings]

[0023] [Figure 1] This shows the results of verification of a recombinant vector expressing a humanized anti-tissue factor antibody. [Figure 2] 1 shows the results of recombinant expression of the heavy and light chains of two anti-tissue factor humanized antibodies. [Figure 3A] Figure 1 shows the results of a tissue affinity test using anti-tissue factor humanized antibody-ADC drugs. Section A shows the results for the anti-tissue factor humanized antibody MAb01-ADC drug, and section B shows the results for the anti-tissue factor humanized antibody MAb02-ADC drug. [Figure 3B] Figure 1 shows the results of a tissue affinity test using anti-tissue factor humanized antibody-ADC drugs. Section A shows the results for the anti-tissue factor humanized antibody MAb01-ADC drug, and section B shows the results for the anti-tissue factor humanized antibody MAb02-ADC drug. [Figure 3C]Figure 1 shows the results of a tissue affinity test using anti-tissue factor humanized antibody-ADC drugs. Section A shows the results for the anti-tissue factor humanized antibody MAb01-ADC drug, and section B shows the results for the anti-tissue factor humanized antibody MAb02-ADC drug. [Figure 4] This shows the results of purification of an anti-tissue factor single-chain antibody-GFP fusion protein. [Figure 5] 1 shows standard curves for tissue factor protein, where (A) is a standard curve for detecting tissue factor protein using GFP-tissue factor single-chain antibody, and (B) is a standard curve for detecting tissue factor protein using FITC-tissue factor antibody. [Figure 6] This is the result of MAb01-ADC coupling. [Figure 7] This is the result of MAb02-ADC coupling. [Figure 8] Figure 1 shows the endocytosis results of anti-tissue factor humanized antibody MAb-ADC drugs in tumor cells. A shows the endocytosis results of MAb02-ADC in SK-OV-3 cells, B shows the endocytosis results of MAb02-ADC in BXPC cells, C shows the endocytosis results of MAb001-ADC in SK-OV-3 cells, D shows the endocytosis results of MAb001-ADC in BXPC cells, and E shows the endocytosis results of MAb001-ADC in A549 cells. [Figure 9] Figure 1 shows the results of the inhibitory effects of the anti-tissue factor humanized antibody MAb01-ADC drug on different types of tumor cells. A shows the inhibitory effect of MAb001-ADC on tumor cells, and B shows the inhibitory effect of MAb002-ADC on tumor cells. [Figure 10] Figure 1 shows the inhibitory effect of the anti-tissue factor humanized antibody MAb01-ADC drug on tumor growth in nude mouse subcutaneous tumor models. (A) BXPC cells, (B) SK-OV-3 cells. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention provides an anti-tissue factor humanized antibody, and the amino acid sequence of the heavy chain (HC) of the anti-tissue factor humanized antibody is SEQ ID NO: 3 (QIQLVQSGPELVKPGASVQVSCKTSGYSFTDYNVYWVRQSPAKGIEWIGYIDPYNGLTIYEQNFRGKGTLSLDHSTSTAYMELNSLRYEDTAVYFCARDVTTALDFWGQGTSVTVSSEFAstkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppk pkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk), encoding 449 amino acids, and the amino acid sequence of the light chain (LC) of the anti-tissue factor humanized antibody is SEQ It is shown in ID NO:4 (DIQMTQSPASISASIGERVTITCLASQTIDTWLAWFLQKPGRSPNLLIYAATNLADGVPYRFSASGSGNDFSLTISSLNPEDVATYYCQQVYSSPFTFGQGNKLEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) and encodes 214 amino acids.

[0025] In the present example, two anti-tissue factor monoclonal antibodies obtained by screening were humanized and recombinantly expressed to obtain two anti-tissue factor humanized antibodies. Affinity analysis showed that the anti-tissue factor humanized antibody MAb01 protected by the present invention has strong affinity and its ability to specifically recognize tissue factor is significantly superior to that of another anti-tissue factor humanized antibody MAb02. The amino acid sequence of the corresponding heavy chain is SEQ ID NO:1 (QIQLVQSGPEVVKPGASVRVSCKGSGYSFTD). YNIY WVRQSPAKGLEWIGYIDPYNGLTIYDQNFRAKATLSVDHSTSNAYMEINSLRYEDTAVYFCARDVTSALEFWGQGTSVTVSSEFAstkgpsvfplapsskstsggtaal gclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtl misrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk), and the amino acid sequence of the corresponding light chain is SEQ ID NO:2(DIQMTQSPASISASVGERVTITCLGSQTIDTYLAWYLQKPGRSPQLLIYAATQLADGVPSRFSASGSGTDFSLTISSLQPEDVATYYCQNVYSSPFTFGQGNKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

[0026] The present invention provides an antibody-drug conjugate obtained by coupling the anti-tissue factor humanized antibody with a cytotoxic drug.

[0027] In the present invention, the cytotoxic drug includes at least one of the dolastatin derivative MMAE, the anti-tubulin inhibitor MMAF, the maytansine derivative DM1, and the DNA topoisomerase I inhibitor DX8951. The anti-tissue factor humanized antibody and the cytotoxic drug are linked via a linker. The linker preferably includes at least one of 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.

[0028] The present invention provides a method for producing the antibody-drug conjugate, mixing an anti-tissue factor humanized antibody, tris(2-chloroethyl)phosphate, and a coupling buffer solution, and performing a reduction reaction to obtain a reduction product; and performing a coupling reaction between the reduction product and a linker-cytotoxic drug solution to obtain an antibody-drug conjugate.

[0029] In the present invention, the mass / volume ratio of the anti-tissue factor humanized antibody to tris(2-chloroethyl)phosphate is preferably (1-2):(2-3), more preferably 1:3, 1:2, or 2:3, and most preferably 1:3. The temperature of the reduction reaction is preferably 23-27°C, more preferably 25°C. The time period of the reduction reaction is preferably 1-2 hours, more preferably 1.5 hours. The DAR value of the antibody-drug conjugate is preferably 2-8, more preferably 3.6-4.4.

[0030] In the present invention, the mass ratio of the anti-tissue factor humanized antibody to the linker-cytotoxic drug is preferably 1:(5 to 9), more preferably 1:5, 1:6, 1:7, 1:8, or 1:9, and more preferably 1:7. The temperature of the coupling reaction is preferably 23 to 27°C, more preferably 25°C. The time period for the coupling reaction is preferably 1 to 2 hours, more preferably 1.5 hours.

[0031] The present invention provides a single-chain fusion protein formed by the heavy chain variable region and light chain variable region of the anti-tissue factor humanized antibody and a reporter protein. The amino acid sequence of the heavy chain variable region of the anti-tissue factor humanized antibody is shown in SEQ ID NO: 47 (QIQLVQSGPELVKPGASVQVSCKTSGYSFTDYNVYWVRQSPAKGIEWIGYIDPYNGLTIYEQNFRGKGTLSLDHSTSTAYMELNSLRYEDTAVYFCARDVTTALDFWGQGTSVTVSS), and the amino acid sequence of the light chain variable region of the anti-tissue factor humanized antibody is shown in SEQ ID NO: 48 (DIQMTQSPASISASIGERVTITCLASQTIDTWLAWFLQKPGRSPNLLIYAATNLADGVPYRFSASGSGNDFSLTISSLNPEDVATYYCQQVYSSPFTFGQGNKLEIR).

[0032] In the present invention, there is no particular limitation on the type of reporter protein, and any reporter protein known in the art, such as green fluorescent protein, may be used. In an embodiment of the present invention, the amino acid sequence of the single-chain fusion protein is shown in SEQ ID NO:5, and the nucleotide sequence of the gene encoding the single-chain fusion protein is shown in SEQ ID NO:6. In the present invention, there is no particular limitation on the method for expressing the fusion protein, and any method known in the art for producing recombinant expressed proteins may be used.

[0033] The present invention provides the use of the single-chain fusion protein in detecting the expression of tumor cell surface tissue factor.

[0034] In the present invention, the method for detecting tumor cell surface tissue factor expression preferably involves adding tumor cells to a tissue factor protein-coated black ELISA detection plate, incubating and washing, then adding the single-chain fusion protein, incubating, washing, adding buffer, and resuspending the cells. Detection is performed under conditions of an excitation wavelength of 493 nm and an emission wavelength of 528 nm, obtaining absorbance values, and calculating the tumor cell surface tissue factor expression level according to the regression equation obtained from the standard curve. The washing solution is preferably PBS solution. The resuspension buffer is preferably PBS solution. The incubation time is preferably 1 to 1.5 hours. The results show that the SK-OV-3 and BXPC-3 cell lines express the highest levels of tissue factor on the surface, while the SW620 and A549 cell lines express the lowest levels. 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.

[0035] In the present invention, the antibody-drug conjugate prepared by the present invention has a high affinity for tissue factor and a short cellular endocytosis time, allowing the conjugated cytotoxin to rapidly reach the tumor cell site and exert its cytotoxic or cytocidal effect. At the same time, the anti-tissue factor humanized antibody has a high affinity for tumor cell lines with low tissue factor expression, ensuring that tumor types with low tissue factor expression are also suitable for antibody-drug conjugate therapy. Experiments have shown that the antibody-drug conjugate effectively inhibits the growth of tumor cells and subcutaneous tumors in mice, thereby playing an important role in the treatment of various cancer diseases. In light of this, the present invention provides the use of the antibody-drug conjugate in the manufacture of a medicament for treating cancer, and also provides the use of the antibody-drug conjugate in combination with another anti-cancer drug in the manufacture of a medicament for cancer treatment. The mass ratio of the antibody-drug conjugate to the other anti-cancer drug is 1:(23-27), more preferably 1:25. The other anti-cancer drug preferably includes gemcitabine.

[0036] In the present invention, the cancer is a cancer in which tissue factor is abnormally expressed, and the cancer in which tissue factor is abnormally expressed preferably includes at least one of ovarian cancer, non-small cell lung cancer, primary adenocarcinoma, colon cancer, cervical cancer, prostate cancer, endometrial cancer, pancreatic cancer, esophageal cancer, bladder cancer, gastric cancer, liver cancer, and breast cancer.

[0037] The following provides a detailed description of the anti-tissue factor humanized antibody provided by the present invention, the prepared antibody-drug conjugate, and its use with reference to examples, but should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1 Sequence modification experiments of anti-tissue factor humanized antibody The tissue factor humanized antibody disclosed in our previously filed patent (Publication No.: CN 107446047 A) was artificially modified, and virtual amino acid mutations of the humanized antibody based on interaction forces were performed using Discovery Studio to improve the binding affinity of the antibody. This modification produced two anti-tissue factor humanized antibody sequences, MAb01 and MAb02, whose modified sequences are shown in Tables 1 and 2.

[0039] [Table 1]

[0040] [Table 2]

[0041] The heavy chain HC of the anti-tissue factor humanized antibody MAb02 encoded 449 amino acids and had the amino acid sequence of SEQ ID NO: 1. The light chain LC encoded 214 amino acids and had the amino acid sequence of SEQ ID NO: 2. The heavy chain HC of the anti-tissue factor humanized antibody MAb01 encoded 449 amino acids and had the amino acid sequence of SEQ ID NO: 3. The light chain gene LC encoded 214 amino acids and had the amino acid sequence of SEQ ID NO: 4.

[0042] Example 2 Expression of anti-tissue factor humanized antibody and screening of stable cell lines This example conforms to the screening method of our patent (application publication number: CN 107446047 A). After artificially synthesizing the genes encoding the humanized antibodies MAb01 and MAb02, the heavy chain gene fragments of MAb01 and MAb02 and the pinsulator4X-MSA vector were double-digested with Sal I and Asc I, respectively. After 1% agarose gel electrophoresis, the insert fragment 1 fragment (1425 bp) and the linearized vector fragment 1 fragment (11938 bp) were recovered from the gel and ligated overnight at 4°C to form the ligation product. After synthesizing the gene sequences of the MAb01 and MAb02 light chain gene fragments, the MAb01 and MAb02 light chain gene fragments and the pCAGGS-IRES-AscI vector were double-digested with NotI and BciI, respectively. Two insert fragments (720 bp) and two linearized vector fragments (6838 bp) were recovered from electrophoresis gels and ligated overnight at 4°C to form the ligation product. The ligation product was transformed into competent E. coli DH5α and single colonies were screened with ampicillin. Single colonies were cultured overnight at 37°C, and the plasmids were extracted from the cells and identified as correct by enzymatic digestion.

[0043] After the recombinant plasmids were identified, pinsulator4X-MAb01 heavy chain-dhfr and pinsulator4X-MAb02 heavy chain-dhfr were double-digested with SalI and MauBI, and the resulting insert 3 (13,349 bp) fragment was isolated by electrophoresis on a 1% agarose gel. pCAGGS-MAb01 light chain-IRES-AscI and pCAGGS-MAb02 light chain-IRES-AscI were double-digested with SalI and AscI, and the resulting insert 3 (4,179 bp) fragment was isolated by electrophoresis. Linearized vector 3 and insert 3 were then ligated to form the pinsulator4X-CAG-MAb01-dhfr and pinsulator4X-CAG-MAb02-dhfr expression vectors, which were then transformed into competent E. coli DH5α and screened for single colonies using ampicillin.

[0044] Single colonies were cultured overnight at 37°C and 180 rpm, and the cells were harvested. Plasmids were extracted and subjected to enzyme digestion and sequence analysis. Figure 1 shows the results of 1% agarose gel electrophoresis of the SwaI-digested expression vectors pinsulator4X-CAG-MAb01-dhfr and pinsulator4X-CAG-MAb02-dhfr. The electrophoresis results were consistent with the theoretical values ​​(2890 bp and 14638 bp, respectively).

[0045] Sequence analysis of the obtained vectors showed that the heavy and light chain genes of the pinsulator4X-CAG-MAb01-dhfr and pinsulator4X-CAG-MAb02-dhfr vectors were completely identical to the sequences of the synthesized genes.

[0046] Example 3 1. Expression of humanized anti-tissue factor antibodies 6 × 10 CHO-dhfr- cells in a 6-well plate 5 The cells were inoculated at a density of 1000 kJ / ml and cultured overnight. Preparation of transfection complexes: Tube A contained 25 μl of Opti-MEM and 4 μl of Lipofectamine 3000, and the mixture was shaken for 1-2 seconds at room temperature. Tube B contained 25 μl of OptiMEM and 2 μg of plasmids (pinsulator4X-CAG-MAb01-dhfr and pinsulator4X-CAG-MAb02-dhfr). The two tubes were mixed homogeneously, left for 5 minutes, and then added to a 6-well culture plate. After 24 hours, the cells were digested with trypsin, placed in a culture dish, and subjected to cell pressure screening with the addition of MTX to a final concentration of 50 μM. After 20 days, single cell clusters were harvested and cultured in a 6-well plate. When the cells reached 90% confluency, they were transferred to a T25 culture flask and expanded. At a cell density of 5 × 10 5 When the cell mass reached 1 / ml, the cells were transferred to an Erlenmeyer flask and cultured at 37°C, 5% CO2, and 130 rpm with shaking. After culturing for 15 days, the cell supernatant was collected.

[0047] 2. Purification of anti-tissue factor humanized antibody (1) Pretreatment of cell solution: The cell supernatant was collected, adjusted to pH 9.0, and then centrifuged at 10,000 rpm for 20 minutes at 4°C. Then, the solution was filtered through a 0.22 μm membrane. (2) Purification of Mabselet protein A: Column A was equilibrated with 5 column volumes of balancing solution (20 mM Tris·HCl, 0.15 M NaCl, pH 9.0). Cell supernatant was passed through column A at a flow rate of 2 ml / min. The absorbance peak was recorded. After loading the sample, the column was washed with 5 column volumes of balancing solution until the baseline was reached. The target protein was eluted with 100 mM glycine·HCl, pH 3.0, and the elution peak was collected. Column A was then washed with balancing solution. The collected solution was adjusted to pH 7.0 with 1 M Tris. The target protein was analyzed by SDS-PAGE electrophoresis. The analytical results are shown in Figure 2.

[0048] Analysis of purification results: The impure cell solution was purified using a protein A column and gel chromatography, resulting in a homogeneous protein. Based on the estimated antibody molecular weight and SDS-PAGE results, the heavy chain band of the humanized anti-tissue factor antibody was located at 50 KD, and the light chain band of the humanized anti-tissue factor antibody was located at 25 KD.

[0049] Example 3 Kinetic analysis of the affinity of anti-tissue factor humanized antibodies to the antigen tissue factor The anti-tissue factor humanized antibody was coupled to the second channel of the CM5 chip surface via the amino group. The first and third channels were used as reference channels, and tissue factor was used as the analyte (mobile phase) flowing across the chip surface. The tissue factor mother solution was diluted to injection concentrations of 18 nM, 9 nM, 4.5 nM, 2.25 nM, and 1.125 nM, injected for 120 seconds, dissociated for 600 seconds, and regenerated with glycine HCl pH 2.0 for 30 seconds. The affinity was determined using the Kinetics / Affinity program provided by the Bicore S200. The affinity of our humanized antibody before modification was 3.371 x 10 -8The measurement method was the same as that used in this experiment, and the tissue factor mother 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.

[0050] The results are shown in Figure 3. The binding constant of the anti-tissue factor humanized antibody MAb01 to tissue factor was KD = 3.57 × 10 -10 M, and the binding constant of the anti-tissue factor humanized antibody MAb02 to tissue factor is KD = 1.002 × 10 -9 Both were higher than the original patented antibody before modification. The anti-tissue factor humanized antibody MAb01 had the strongest affinity for the antigen tissue factor.

[0051] Example 4 Method for detecting expression of tumor cell surface tissue factor 1. Expression and purification of anti-tissue factor single-chain fv antibody-GFP fusion protein (TF-scFv-GFP) 600 When the pH reached 0.6, IPTG was added to a final concentration of 1 mM, and the mixture was cold-induced overnight at 16°C. The cells were collected by centrifugation, disrupted by sonication, and subjected to Ni column affinity chromatography to obtain a TF-scFv-GFP fusion protein with a molecular weight of approximately 57 kDa. The results of the purification of TF-scFv-GFP are shown in Figure 4.

[0052] 2. Detection of cell surface tissue factor protein using anti-tissue factor single-chain antibody-GFP fusion protein A 96-well ELISA black test plate was coated with tissue factor protein (purchased from Taiyuan Boaote Biotechnology Co., Ltd., catalog number: BAT1001) at concentrations of 300 pmol, 150 pmol, 75 pmol, 37.5 pmol, 18.75 pmol, and 9.375 pmol, respectively, and a fixed amount of tumor cells (SK-OV-3 / BXPC-3 cell line, approximately 1 × 10 6 , HeLa cell line approximately 1 x 10 7 , A549 / SW620 cell line approximately 1 x 10 8 ) were collected and immobilized on a 96-well cell culture plate. Anti-tissue factor single-chain antibody-GFP fusion protein and a positive control antibody (Abcam FITC fluorescent anti-tissue factor antibody

[05] (ab275690) were added, incubated for 1 hour, washed once or twice with PBS (pH 7.3), resuspended in PBS buffer, and detected under conditions of an excitation wavelength of 493 nm and an emission wavelength of 528 nm.

[0053] The standard curve constructed is shown in FIG.

[0054] The expression level of tissue factor on tumor cells was detected using the above method. As shown in Table 3, the results of the detection of cell surface tissue factor protein using the anti-tissue factor single-chain antibody-GFP fusion protein were consistent with those using the positive control antibody, demonstrating the feasibility of the detection method used in this example. The results showed that the expression levels of tissue factor on the surface of SK-OV-3 and BXPC-3 cell lines were highest, while the expression levels of tissue factor on the surface of SW620 and A549 cell lines were lowest. 6.02 × 10 per mole. 23 Based on the fact that each type of tumor cell contained 100 molecules, the number of tumor cell surface tissue factor targets was calculated and is shown in Table 4.

[0055] [Table 3]

[0056] [Table 4]

[0057] Example 5 Anti-tissue factor humanized antibody-ADC drug coupling experiment Direct coupling was performed after replacing the antibody (5 mg / mL) with coupling buffer (20 mM His-His.HCl, pH 5.96). As shown in Table 5, antibody, DTPA (diethylenetriaminepentaacetic acid, 10 mM), and TCEP (tris(2-carboxyethyl)phosphine hydrochloride, 10 mM) were added in order. After each addition, the mixture was vortexed immediately and then centrifuged to collect the entire reaction mixture at the bottom of the tube. The mixture was then placed in a thermostatic mixer and subjected to the reduction reaction according to the conditions shown in Table 5. After the reduction reaction was completed, the corresponding amount of DMSO (dimethyl sulfoxide) was added to each reaction mixture in an ice-water bath and vortexed to mix thoroughly. Next, the corresponding amount of linker payload (5 mM) was added, vortexed to mix thoroughly, and centrifuged to collect the entire reaction mixture at the bottom of the tube. The mixture was then placed in a thermostatic mixer and subjected to the coupling reaction according to the conditions shown in Table 6. After the coupling was completed, an appropriate amount of ADC was taken and the DAR value was detected using the HIC-HPLC method (see Table 7), and after the ADC was repeatedly dialyzed in an ultrafiltration centrifuge tube (30 KDa), an appropriate amount was taken for concentration, HIC-HPLC, SEC-HPLC, and free drug residue testing.

[0058] [Table 5]

[0059] [Table 6]

[0060] [Table 7]

[0061] Example 6 Anti-tissue factor humanized antibody MAb01-ADC drug endocytosis experiment Tumor cells with high and low tissue factor expression were cultured and digested with trypsin after reaching a cell density of 90%. 250,000 cells were placed in a confocal dish and allowed to adhere to the wall. After the cells had adhered, the original culture medium was aspirated, and 1 ml of culture medium containing 1 μl of either the fluorescently labeled (FITC) MAb01-ADC drug or MAb02-ADC antibody was added. The cells were observed under a confocal microscope (Zeiss LSM 710, 20x magnification) from the time of antibody addition until 0 minutes later. Fields containing uniform density and single cells were selected and photographed after 6 / 8 cycles, with the magnification set to 20 minutes per cycle.

[0062] As shown in Figure 8, the antibody MAb01-ADC drug initiated endocytosis in 30 minutes and was fully endocytosed within 2 hours, achieving endocytosis in both SK-OV-3 and A549 cell lines. The endocytosis time for the antibody MAb02-ADC-ADC drug was relatively long, achieving endocytosis in the SK-OV-3 cell line but not in the A549 cell line. The high-affinity anti-tissue factor humanized antibody-ADC drug was found to be able to achieve endocytosis in tumor cells with low tissue factor expression.

[0063] Example 7 High-affinity anti-tissue factor humanized antibody-ADC drug bioactivity 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. 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), and HeLa cell line (cervical cancer cell line) were used. IgG-MMAE drug was included as a control. Logarithmic growth phase cells were seeded into a 96-well cell culture plate at a density of 5,000 cells per well, with 100 μl per well. After incubation at 37°C and 5% carbon dioxide for approximately 16 hours, different concentrations of antibody MAb01-ADC drug were added, with three replicate wells set up for each drug concentration. After 3 days of incubation, the culture medium was removed, and 100 μl of CCK-8 reaction solution was added per well. The incubation was continued at 37°C until the expected color intensity was reached. The cell viability (OD ) of each group was measured. 450 ) was measured, and cell viability was calculated according to the following formula I: MAb02-ADC drug was also tested in cell lines capable of achieving 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 administered - OD blank) / (OD control - OD blank) x 100% Formula I

[0064] The above data were analyzed using GraphPad Prism 5 software to determine the IC of TF-ADC against different cell lines. 50 values ​​were calculated.

[0065] The results are shown in Figure 9. The antibody MAb01-ADC drug could effectively inhibit the proliferation of tumor cells with high tissue expression in vitro, and the inhibitory effect was proportional to the number of TF molecules on the cell surface. The antibody MAb02-ADC drug also had a certain inhibitory effect on tumor cell lines that achieved endocytosis.

[0066] IC of various cancer cell types against antibody MAb01-ADC drug 50 The values ​​are shown in Table 8.

[0067] [Table 8]

[0068] Example 8 Experimental study of anti-tissue factor humanized antibody-ADC drug inhibiting subcutaneous tumors in mice A nude mouse subcutaneous tumor model was used to select tumor cells. One tumor cell was implanted per 100 nude mice. When tumor volumes reached 64–100 mm3, six experimental groups were established, each with eight mice. The MAb01-ADC drug was administered at doses of 1 mg / kg, 2 mg / kg, or 4 mg / kg. The ADC 2 mg / kg was combined with gemcitabine 50 mg / kg as a positive control, and PBS as a negative control. Tail vein administration began on day 0. Nude mice were weighed every other day, and tumor diameters were measured with a vernier caliper. At the end of the experiment, blood was collected and serum was preserved. The animals were sacrificed by cervical dislocation, and tumors were excised and weighed. Drug efficacy was assessed based on changes in tumor weight and relative volume. Tumor volume was calculated according to formula II. V=a×b×c Formula II. Among these, a, b, and c represent the length, width, and height of the tumor, respectively.

[0069] As shown in Figure 10, the antibody MAb01-ADC drug was able to significantly inhibit the growth of ovarian cancer and pancreatic cancer tumors in a dose-dependent manner.

[0070] It should be pointed out that the above are only preferred embodiments of the present invention, and those skilled in the art may make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the protection scope of the present invention.

Claims

1. An anti-tissue factor humanized antibody, wherein the amino acid sequence of the heavy chain of the anti-tissue factor humanized antibody is set forth in SEQ ID NO: 3, and the amino acid sequence of the light chain of the anti-tissue factor humanized antibody is set forth in SEQ ID NO:

4.

2. 10. An antibody-drug conjugate, which is obtained by coupling the humanized anti-tissue factor antibody of claim 1 with a cytotoxic drug.

3. mixing an anti-tissue factor humanized antibody, tris(2-chloroethyl)phosphate, and a coupling buffer solution, and performing a reduction reaction to obtain a reduction product; and performing a coupling reaction between the reduction product and a linker-cytotoxic drug solution to obtain an antibody-drug conjugate.

4. A single-chain fusion protein formed from the heavy chain variable region and light chain variable region of the anti-tissue factor humanized antibody of claim 1 and a reporter protein, The amino acid sequence of the heavy chain variable region of the anti-tissue factor humanized antibody is shown in SEQ ID NO: 47, A single-chain fusion protein, wherein the amino acid sequence of the light chain variable region of the humanized anti-tissue factor antibody is set forth in SEQ ID NO:

48.

5. Use of the antibody-drug conjugate of claim 2 in the manufacture of a drug for treating cancer.

6. 10. Use of the antibody-drug conjugate of claim 2 in combination with another anti-cancer drug in the manufacture of a drug for treating cancer.

7. The use according to claim 5 or 6, characterized in that the cancer is a cancer in which tissue factor is abnormally expressed, and the cancer in which tissue factor is abnormally expressed includes at least one of ovarian cancer, non-small cell lung cancer, primary adenocarcinoma, intestinal cancer, cervical cancer, prostate cancer, endometrial cancer, pancreatic cancer, esophageal cancer, bladder cancer, gastric cancer, liver cancer, colon cancer, and breast cancer.

8. An anti-cancer drug, comprising the antibody-drug conjugate of claim 2 and another anti-cancer drug.

9. The anti-cancer drug of claim 8, wherein the other anti-cancer drug comprises gemcitabine.

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