JP7020656B2 - Antibodies targeting tissue factor, their preparation methods and their use - Google Patents

Description

The present invention relates to antibodies, particularly antibodies that target human tissue factor (TF), methods of preparation and use thereof.

Tissue factor (TF) is a 47 kDa transmembran glycoprotein. Under normal physiological conditions, TF expression is primarily confined to cells in the subepithelial layer; when blood vessels are damaged in vivo, TF is exposed to the bloodstream and binds to Factor VII and is active. By soliciting, an extrinsic coagulation reaction is initiated.

Studies have found that TF is abnormally activated and expressed in many tumor tissues and plays an important role in tumor development and progression. Most patients are associated with spontaneous thrombosis, such as deep vein thrombosis (DVT), disseminated intravascular coagulation (DIC) and pulmonary embolism (PE), especially in the advanced stages of cancer (Thrombosis research, 2013, 131: S59-S62; Journal of Thrombosis and Haemostasis, 2011, 9 (s1): 306-315); Abnormal expression of TF in tumor cells is a major cause of these symptoms. Analysis of clinical samples of many tumors shows that TF expression levels have a direct impact on exacerbation indicators, such as tumor metastasis and the development of thrombosis in patients, for example, the rate of abnormal TF expression in breast cancer is 85. It shows that it was 8%, 88.5% for pancreatic cancer, 83.6% for lung cancer, 91.3% for esophageal cancer, and so on (Blood, 2012, 119: 924-932).

In addition to initiating an extrinsic coagulation reaction, the TF / FVIIa complex can directly bind to and induce activation of the transmembran G protein-coupled receptor, the protease-activated receptor 2 (PAR2). can. PAR2 is an important signaling pathway that regulates the inflammatory response. Although there are few studies on PAR2 in the field of tumors, it is believed that PAR2 can influence a series of tumor function signals in cells. In summary, TF-PAR2 induces gene expression of key growth factors, immunomodulators, and chemokines (eg, VEGF, CSF1 / 2, IL8, CXCL1, etc.), promotes angiogenesis, and MAPK / ERK. It provides the proper nutrients, energy and suitable environment for tumor growth by phosphorylation. In addition, TF can also enhance tumor cell migration and attachment by interacting with Rac1 and β1 family-related integrins, thereby enhancing the ability of tumor cells to metastasize to hematogenously (generally). Journal of Thrombosis research, 2012, 130: S84-S87; Journal of Thrombosis and Haemostasis, 2013, 11: 285-293; International Journal of Cancer, 2014, doi: 10.1002 / ijc.28959; Blood, 2012, 119: 924- 932).

On the other hand, TF-induced hypercoagulation directly promotes tumor cell survival and hematogenous metastasis (Blood, 2008, 111: 190-9; Cancer Res., 2015, 75 (1 Suppl): Abstract nr B19). That is, TF / FVIIa-initiated coagulation results in the formation of trombin and the deposition of fibrin, which not only allows tumor cells to escape immune attack, but also enhances the interaction between tumor cells and endothelial cells. It helps the spread and infiltration of tumor cells and promotes the development of hematogenous metastases. This is also an important reason why cancer is currently difficult to treat.

Studies have shown that TF also plays a role in thrombotic diseases. In addition to its role in tumor development and progression, TF or MPTF (microparticle tissue factor) -initiated coagulation is also an important reason for inducing venous thromboembolism (VTE), and further in its blood. Content is directly proportional to the severity of VTE. Currently, there are many studies showing that TF can be used as an important marker for clinical diagnosis and status assessment of VTE patients and as a potential target for the treatment of VTE (Thrombosis research, 2010, 125). : 511-512; Lupus., 2010, 19: 370-378; Annual review of physiology, 2011, 73: 515-525). The role of TF in arterial thrombosis is also not negligible. Many clinical data indicate that TF plays an important role in the development and progression of atherosclerosis. In 2009, Steppich BA, Braun SL et al. Conducted a study of 174 patients with unstable thrombosis (uAP) and 112 patients with acute myocardial infarction (AMI), and the results were in plasma. It has been shown that the activity of TF has a direct effect on the mortality of patients with cardiovascular disease, and that TF can be used as a marker for the diagnosis and prognosis of cardiovascular patients (Thrombosis research, 2012, 129). : 279-284; Thromb J., 2009, 7 (11): 1-9); In 2014, Jiang P, Xue D et al. Photochemically induced thrombosis model and FeCl ₃ induced thrombosis. We conducted a study of the disease model and compared it with the intrinsic pathway of blood coagulation to show that the TF-initiated extrinsic pathway of blood coagulation played an important role in the onset and progression of patients with arterial thrombosis, and experiments. The results further demonstrated that the TF-initiated extrinsic pathway of blood coagulation could be used as a target for the treatment of arterial thrombotic patients (Thrombosis research, 2014, 133 (4): 657-666).

TF also plays a role in inflammatory and metabolic disorders. Studies have shown that the onset of inflammatory patients is associated with abnormal angiogenesis and coagulation. Studies conducted by Maria I Bokarewa et al. Have shown that various inflammatory stimuli promote the expression of TF on the surface of endogenous and mononuclear cells, and those experiments show that overexpression of TF It has also been shown to be a major factor in inducing and promoting inflammation (Arthritis Res 2002, 4: 190-195).

In addition, studies have also shown that TF plays a significant regulatory role in the treatment of obesity and diabetes. For example, studies conducted by Leylla Badeanlou et al. Significantly inhibited the development of diet-induced obesity and adipose tissue inflammation by blocking the TF-PAR2 signaling pathway by TF-targeting specific antibodies or knockout of TF. It has been shown that the therapeutic effect of insulin on diabetes can be significantly improved (Nature medicine, 2011, 17 (11): 1490-1497).

Therefore, given the role and function of TF in various related diseases, the development of specific therapeutic antibodies targeting TF is triggered by TF in various patients, such as cancer, thrombosis and inflammation. It is extremely useful for the diagnosis, treatment and prevention of pathological features caused by abnormal coagulation and the like.

An object of the present invention is to specifically target human TF, have an activity of inhibiting tumor growth and metastasis, and have an anticoagulant activity, an activity of inhibiting the production of activated coagulation factor X (FXa), and the like. It is to provide a TF antibody having.

According to the first aspect, the invention includes three complementarity determining regions (CDRs):
CDR1, having the sequence set forth in SEQ ID NO: 1,
CDR2 having the sequence set forth in SEQ ID NO: 2 and CDR3 having the sequence set forth in SEQ ID NO: 3;
Here, any amino acid sequence in said amino acid sequence further comprises a derivative sequence that results from any addition, deletion, modification and / or substitution of at least one amino acid and retains TF-binding affinity. It provides a heavy chain variable region.

According to the second aspect, the invention provides a heavy chain of an antibody having a heavy chain variable region of the first aspect.

According to another preferred aspect, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 7.

According to the third aspect, the present invention is described as follows:
CDR1'with the sequence set forth in SEQ ID NO: 4
CDR2'with the sequence set forth in SEQ ID NO: 5.
CDR3'with the sequence set forth in SEQ ID NO: 6
Contains complementarity determining regions (CDRs) selected from the group consisting of
Provided is a light chain variable region of an antibody, wherein any amino acid sequence in the amino acid sequence results from the addition, deletion, modification and / or substitution of at least one amino acid and comprises a derivative sequence having a TF-binding affinity. do.

According to the fourth aspect, the invention provides a light chain of an antibody having a light chain variable region of the third aspect.

According to another preferred aspect, the light chain variable region has an amino acid sequence as set forth in SEQ ID NO: 8.

According to the fifth aspect, the present invention is described as follows:
(1) Heavy chain variable region on the first side; and / or (2) Light chain variable region on the third side,
Provided an antibody having, or said antibody has a heavy chain of a second aspect; and / or a light chain of a fourth aspect.

According to another preferred example, the antibody is selected from animal-derived antibodies, chimeric antibodies, humanized antibodies or combinations thereof.

According to another preferred example, the number of amino acids added, deleted, modified and / or substituted does not exceed 90% of the total number of amino acids in the initial amino acid sequence.

According to another preferred example, the number of amino acids added, deleted, modified and / or substituted is 1-7.

According to another preferred example, the sequence resulting from the addition, deletion, modification and / or substitution of at least one amino acid is an amino acid sequence having at least 80% homology.

According to another preferred example, the sequence resulting from the addition, deletion, modification and / or substitution of at least one amino acid exhibits activity that inhibits TF-related signaling pathways, anticoagulant activity, and activity that inhibits FXa production. Have.

According to another preferred example, the antibody is selected from the group consisting of TF-mAb-SC1, TF-mAb-Ch, TF-mAb-H39 and TF-mAb-H44.

According to a sixth aspect, the invention is an antibody of the invention for the manufacture of (a) diagnostic agents; and / or (b) pharmaceuticals for the prevention and / or treatment of TF-related diseases. Provide use.

According to another preferred example, the TF-related disease is selected from the group consisting of tumor development, growth and / or metastasis; thrombosis-related disease; inflammation; and metabolism-related disease.

According to another preferred example, the tumor is a tumor with high TF expression.

According to another preferred example, "high TF expression" of expression is when TF transcript and / or protein level L1 in tumor tissue is compared to transcript and / or protein level LO in normal tissue. It means that L1 / L0 ≧ 2, preferably ≧ 3.

According to another preferred example, the tumor is selected from the group consisting of triple negative breast cancer, pancreatic cancer, lung cancer and malignant glioma.

According to another preferred example, the drug is an antibody-drug conjugate.

According to the seventh aspect, the present invention relates to (i) a heavy chain variable region on the first side surface, a heavy chain on the second side surface, a light chain variable region on the third side surface, and a light chain on the fourth side surface. Or an antibody of the fifth aspect; and (ii) optionally provide a recombinant protein having a tag sequence that aids expression and / or purification.

According to the eighth aspect, the present invention relates to (1) a heavy chain variable region on the first side surface, a heavy chain on the second side surface, a light chain variable region on the third side surface, and a light chain on the fourth side surface. , Or the antibody of the fifth aspect; or (2) the recombinant protein of the seventh aspect,
Provided are polynucleotides encoding polypeptides selected from.

According to the ninth aspect, the present invention provides a vector containing the polynucleotide of the eighth aspect.

According to the tenth aspect, the invention provides a genetically engineered host cell comprising a ninth aspect vector or having an eighth aspect polynucleotide integrated into the genome.

According to the eleventh aspect, the present invention is:
(A first side heavy chain variable region, second side heavy chain, third side light chain variable region, fourth side light chain, or fifth side antibody, or a combination thereof. The antibody moiety selected from; and (b) the junction moiety conjugated to the antibody moiety, wherein the junction moiety is selected from detectable markers, toxins, cytokines, radionuclides, enzymes, or combinations thereof. Provides antibody-drug conjugates comprising.

According to another preferred example, the antibody moiety is attached to the junction moiety via a chemical bond or linker.

According to another preferred example, the antibody moiety in the antibody-drug conjugate is selected from the group consisting of TF-mAb-SC1, TF-mAb-Ch, or TF-mAb-H29 to TF-mAb-H48; More preferably, the antibody moiety is selected from the group consisting of TF-mAb-H39 and TF-mAb-H44.

According to a twelfth aspect, the invention provides an immunocyte expressing an antibody of the fifth aspect of the invention or expressing an antibody of the fifth aspect of the invention exposed outside the cell membrane. ..

According to another preferred example, the immune cells include NK cells, T cells.

According to another preferred example, the immune cell is a human immune cell.

According to another preferred example, the antibody is a single chain antibody.

According to the thirteenth aspect, the present invention is:
(I) Heavy chain variable region on the first side surface, heavy chain on the second side surface, light chain variable region on the third side surface, light chain on the fourth side surface, antibody on the fifth side surface, seventh side surface. A pharmaceutical composition comprising a recombinant protein of the eleventh aspect, an antibody-drug conjugate of the eleventh aspect, an active ingredient selected from an immune cell of the twelfth aspect, or a combination thereof; and (ii) a pharmaceutically acceptable carrier. Provide things.

According to the fourteenth aspect, the present invention relates to a heavy chain variable region on the first side surface, a heavy chain on the second side surface, a light chain variable region on the third side surface, a light chain on the fourth side surface, a fifth. Drugs, agents, test panels or kits selected from the antibody of the aspect of, the recombinant protein of the seventh aspect, the antibody of the eleventh aspect-drug conjugate, the immune cell of the twelfth aspect, or a combination thereof. Provides the use of active ingredients for the production of.

According to another preferred example, the agent, test panel or kit
Used to (1) detect TF proteins in samples; and / or (2) detect endogenous TF proteins in tumor cells; and / or (3) detect tumor cells expressing TF proteins. And the medicine is used to treat or prevent diseases such as TF protein expressing tumors, thrombotic diseases, obesity and diabetes.

According to the fifteenth aspect, the present invention has the following steps:
It involves (a) contacting the antibody of the invention with the sample in vitro; and (b) determining whether an antigen-antibody complex is formed, wherein the formation of the complex is in the sample. Provided are methods (including diagnostically or non-diagnostically determining) for determining TF protein in a sample in vitro, indicating the presence of TF protein.

According to the sixteenth aspect, the present invention provides a test panel comprising a substrate (support plate) and a test piece, wherein the test piece comprises an antibody on the fifth side or an immunoconjugate on the eleventh side. ..

According to the seventeenth aspect, the present invention is:
(1) a first container containing the antibody of the present invention; and / or (2) a kit containing a second container containing a second antibody against the antibody of the present invention, or a kit containing a test panel of the sixteenth aspect. I will provide a.

According to the eighteenth aspect, the present invention is:
(A) Cultivate the host cells of the tenth aspect under conditions appropriate for expression; and (b) isolate the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody of the fifth aspect. Alternatively, a method for preparing a recombinant polypeptide, which is a recombinant protein of the seventh aspect, is provided.

According to the nineteenth aspect, the invention uses an antibody of the fifth aspect, an antibody-drug conjugate of said antibody, or CAR-T cells expressing said antibody (eg, administered to a subject in need thereof). ) Provide a method for treating tumors, thrombotic diseases, inflammatory diseases and / or metabolic diseases including.

According to another preferred example, metabolic disorders include obesity or diabetes.

According to a twentieth aspect, the invention relates to 0.005 to 0.10 nM, preferably 0.005 to 0.05 nM, more preferably 0.01 to 0.03 nM or 0. Provided are antibody-TF antibodies characterized by having an ED ₅₀ of 01-0.02 nM.

According to another preferred example, the antibody does not bind to wild-type mouse TF protein.

According to another preferred example, the antibody is described below;
It has one or more characteristics selected from the group consisting of (a) inhibition of tumor cell migration or metastasis; and (b) inhibition of tumor growth.

According to another preferred example, the antibody is TF-mAb-SC1, TF-mAb-Ch, or TF-mAb-H29-TF-mAb-H48; preferably the antibody is TF-mAb-SC1,. It is selected from the group consisting of TF-mAb-Ch, TF-mAb-H39, and TF-mAb-H44.

According to the 21st aspect, the present invention has the following steps:
A step of expressing a human-mouse chimeric antibody by cloning the nucleotide sequence of the murine antibody variable region of the present invention into an expression vector containing a human antibody invariant region and then transfecting animal cells;
A human comprising a step of expressing a humanized antibody by cloning an antibody sequence of an antibody variable region containing a human FR region of the present invention into an expression vector containing a human antibody invariant region and then transfecting animal cells. Provided is a method for preparing a transfection or chimeric antibody.

According to another preferred example, the antibody is a partially or fully humanized monoclonal antibody.

According to the 22nd aspect, the present invention comprises the step of administering the antibody of the present invention, an antibody-drug conjugate of the antibody, or CAR-T cells expressing the antibody to the required subject. It provides a characteristic method for inhibiting the metastasis of tumor cells.

Within the scope of the invention, the technical features of the invention described above and the technical features described below (eg, in the examples) can be combined with each other to form a novel or preferred technical solution. Should be understood. To save space, these combinations are not discussed further here.

FIG. 1 shows the binding activity of a series of original anti-human TF monoclonal antibodies (original hybridomas) to human TF-positive (MDA-MB-231 and BxPC-3) and murine TF-positive (B16-F10) cells, where TF. -MAb-SC1 showed the best binding activity at a concentration of 10 μg / ml.

FIG. 2 shows the binding affinity of TF-mAb-SC1 for the extracellular domain protein of TF as determined by ELISA.

FIG. 3 shows the inhibitory effect of a series of original monoclonal antibodies on the TF-PAR2 intracellular signaling pathway in BxPC-3, as determined by well-turned blots.

FIG. 4 shows restriction enzyme digestion of a human-mouse chimeric antibody (TF-mAb-Ch) expression plasmid by single enzyme digestion and dual enzyme digestion, where FIG. 4A shows single enzyme digestion and dual enzyme digestion. The identification result of the expression plasmid of the heavy chain variable region by digestion is shown, and FIG. 4B shows the identification result of the light chain variable region expression plasmid by single enzyme digestion and double enzyme digestion. FIG. 4 shows restriction enzyme digestion of a human-mouse chimeric antibody (TF-mAb-Ch) expression plasmid by single enzyme digestion and dual enzyme digestion, where FIG. 4A shows single enzyme digestion and dual enzyme digestion. The identification result of the expression plasmid of the heavy chain variable region by digestion is shown, and FIG. 4B shows the identification result of the light chain variable region expression plasmid by single enzyme digestion and double enzyme digestion.

FIG. 5 shows the binding affinity of TF-mAb-SC1 to the cancer cell surface TF, where FIG. 5A shows the result of the binding affinity to BxPC-3 cells, and FIG. 5B shows the MDA-MB-. The results of the binding affinity to 231 cells are shown, FIG. 5C shows the result of the binding affinity to U87MG cells, and FIG. 5D shows the result of the binding affinity to H1975 cells. FIG. 5 shows the binding affinity of TF-mAb-SC1 to the cancer cell surface TF, where FIG. 5A shows the result of the binding affinity to BxPC-3 cells, and FIG. 5B shows the MDA-MB-. The results of the binding affinity to 231 cells are shown, FIG. 5C shows the result of the binding affinity to U87MG cells, and FIG. 5D shows the result of the binding affinity to H1975 cells. FIG. 5 shows the binding affinity of TF-mAb-SC1 to the cancer cell surface TF, where FIG. 5A shows the result of the binding affinity to BxPC-3 cells, and FIG. 5B shows the MDA-MB-. The results of the binding affinity to 231 cells are shown, FIG. 5C shows the result of the binding affinity to U87MG cells, and FIG. 5D shows the result of the binding affinity to H1975 cells. FIG. 5 shows the binding affinity of TF-mAb-SC1 to the cancer cell surface TF, where FIG. 5A shows the result of the binding affinity to BxPC-3 cells, and FIG. 5B shows the MDA-MB-. The results of the binding affinity to 231 cells are shown, FIG. 5C shows the result of the binding affinity to U87MG cells, and FIG. 5D shows the result of the binding affinity to H1975 cells.

FIG. 6 shows the effect of TF-mAb-SC1 on the intracellular signaling pathway of FVIIa-activated TF-PAR2 in BxPC-3 cells.

FIG. 7 shows the activity of TF-mAb-SC1 to inhibit coagulation initiated by TF from BxPC-3 cells (FIG. 7A) and MDA-MB-231 cells (FIG. 7B). FIG. 7 shows the activity of TF-mAb-SC1 to inhibit coagulation initiated by TF from BxPC-3 cells (FIG. 7A) and MDA-MB-231 cells (FIG. 7B).

FIG. 8 shows the results of the activity of TF-mAb-Sc1 to inhibit TF-induced FXa production from BxPC-3 cells (FIG. 8A) and MDA-MB-231 cells (FIG. 8B). FIG. 8 shows the results of the activity of TF-mAb-Sc1 to inhibit TF-induced FXa production from BxPC-3 cells (FIG. 8A) and MDA-MB-231 cells (FIG. 8B).

FIG. 9 shows the activity of TF-mAb-SC1 to inhibit subcutaneous BxPC-3 xenograft tumor growth in nude mice (arrows indicate the time of initiation of administration).

FIG. 10 shows the results of the activity of TF-mAb-SC1 to inhibit subcutaneous U87MG xenograft tumor growth in nude mice (arrows indicate the time of initiation of administration).

FIG. 11 shows the results of the activity of TF-mAb-SC1 for field inhibition of HCC1806 xenograft tumor growth (arrows indicate time of initiation of administration).

FIG. 12 shows the activity of TF-mAb-SC1 to inhibit the accumulation of BxPC-3 tumor matrix collagen, and the results of statistical analysis using Image-pro plus.

FIG. 13 shows the activity of TF-mAb-SC1 and its statistical results for reducing the luminal region of BxPC-3 tumor blood vessels.

FIG. 14 shows the determination of the migration capacity of MDA-MB-231 cells (FIG. 14A) and BxPC-3 cells (FIG. 14B) under conditions where the TF gene is knocked out. FIG. 14 shows the determination of the migration capacity of MDA-MB-231 cells (FIG. 14A) and BxPC-3 cells (FIG. 14B) under conditions where the TF gene is knocked out.

FIG. 15 shows the determination of the activity of TF-mAb-SC1 to inhibit the mobility of MDA-MB-231 cells (FIG. 15A) and BxPC-3 cells (FIG. 15B). FIG. 15 shows the determination of the activity of TF-mAb-SC1 to inhibit the mobility of MDA-MB-231 cells (FIG. 15A) and BxPC-3 cells (FIG. 15B).

FIG. 16 shows the determination of the hematogenous migration capacity of MDA-MB-231-luc cells in mice after the TF gene was knocked out, and the statistical results of its fluorescence intensity.

FIG. 17 shows the determination of the activity of TF-mAb-SC1 for inhibiting the hematogenous migration ability of MDA-MB-231-luc cells in mice, and the statistical results of its fluorescence intensity.

FIG. 18A shows representative images and statistical analysis of the formation of MDA-MB-231 tumor metastases on the lungs of mice, and FIG. 18B shows the results of statistical analysis of lung weight in each group. Is shown. FIG. 18A shows representative images and statistical analysis of the formation of MDA-MB-231 tumor metastases on the lungs of mice, and FIG. 18B shows the results of statistical analysis of lung weight in each group. Is shown.

FIG. 19 shows the results of internalization of TF-mAb-SC1 into lysosomes by MDA-MB-231 cells observed under a laser confocal microscope.

FIG. 20 shows the binding affinity of the chimeric antibody TF-mAb-Ch to the extracellular domain protein of TF, as determined by ELISA.

FIG. 21A shows the binding affinity of TF-mAb-Ch to the TF-positive tumor cell MDA-MB-231, and FIG. 21B shows the result of the binding affinity of TF-mAb-Ch to the TF-positive tumor cell HCC1806. show. FIG. 21A shows the binding affinity of TF-mAb-Ch to the TF-positive tumor cell MDA-MB-231, and FIG. 21B shows the result of the binding affinity of TF-mAb-Ch to the TF-positive tumor cell HCC1806. show.

FIG. 22 shows the activity of TF-mAb-Ch to inhibit the TF-PAR2 intracellular signaling pathway in BxPC-3 cells.

FIG. 23 shows the results of internalization of TF-mAb-Ch into lysosomes by MDA-MB-231 cells.

FIG. 24A shows the binding affinity of the humanized antibody TF-mAb-H39 to the extracellular domain protein of TF as determined by ELISA, and FIG. 24B shows the binding affinity of the humanized antibody TF-mAb-H39 to the extracellular domain protein of TF as determined by ELISA. The binding affinity of the humanized antibody TF-mAb-H44 of the above is shown. FIG. 24A shows the binding affinity of the humanized antibody TF-mAb-H39 to the extracellular domain protein of TF as determined by ELISA, and FIG. 24B shows the binding affinity of the humanized antibody TF-mAb-H39 to the extracellular domain protein of TF as determined by ELISA. The binding affinity of the humanized antibody TF-mAb-H44 of the above is shown.

FIG. 25 shows the results of the effects of humanized antibodies on the TF-PAR2 intracellular signaling pathway in BxPC-3 cells.

FIG. 26 shows the results of the activity of various humanized antibodies to inhibit HCC1806 xenograft tumor growth.

FIG. 27 shows the dose-response activity of the chimeric antibody TF-mAb-Ch and the humanized antibody TF-mAb-H39 to inhibit HCC1806 xenograft tumor growth.

FIG. 28 shows a comparison of TF protein expression results in highly invasive and highly metastatic basal-like breast cancer (particularly triple-negative breast cancer) cell lines and lumen subtype breast cancer cell lines as determined by Western blots. ..

FIG. 29 shows highly invasive and highly metastatic basal-like breast cancer (particularly triple-negative breast cancer) cell lines analyzed according to the database E-TABM-157 (European Molecular Biology Laboratory-European Bioinformatics Institute, EMBL-EBI), and The results of comparison of the expression level of TF mRNA in the lumen subtype breast cancer cell line are shown.

FIG. 30 shows TF protein levels in different pancreatic cancer cell lines as determined by Western blots.

FIG. 31 shows aberrant activation of TF mRNA in pancreatic cancer tissue compared to adjacent normal tissue analyzed according to database GSE15471 (Gene Expression Omnibus, GEO).

By conducting extensive and detailed studies and numerous screenings, we have surprisingly obtained an anti-TF monoclonal antibody (TF-mAb-SC1), and the experimental results show that the monoclonal antibody against the TF protein is. Indicates that it is an IgG2b antibody. The antibody can bind to the TF antigen with high specificity and high affinity (EC ₅₀ is about 0.019 nM as determined by ELISA), and the antibody has significant antitumor activity. , And has no obvious toxic side effects to the mammal itself. In addition, chimeric antibodies, humanized antibodies and their corresponding ADCs, obtained based on TF-mAb-SC1, also have outstanding properties. The present invention has been achieved on the basis of them.

Antibodies As used herein, the term "antibody" or "immunoglobulin" consists of two identical light chains (L) and two identical heavy chains (H). It is a heterotetramer glycoprotein of about 150,000 Da with the same structural properties. Each light chain is attached to a heavy chain via a shared disulfide bond, and different immunoglobulin isotypes have different numbers of disulfide bonds between the heavy chains. Regularly spaced intrachain disulfide bonds are also present in each heavy chain and each light chain. Each heavy chain has a variable region (VH) at one end, followed by many invariant regions. Each light chain has a variable region (VL) at one end and an invariant region at the other end; a light chain invariant region corresponding to the first invariant region of the heavy chain, and a light chain variable region corresponding to the heavy chain invariant region. Have. Certain amino acid residues form an interface between the variable region of the light chain and the variable region of the heavy chain.

As used herein, the term "variable" means that the antibodies differ from each other with respect to the sequence in a particular portion of the variable region, which is the binding of various particular antibodies to those particular antigens. And it means that it is in charge of specificity. However, the variability is not evenly distributed over the variable region of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The storage portion of the variable region is called the framework region (FR). The naturally occurring heavy and light chain variable regions generally exist in a β-sheet structure linked by three CDRs forming a binding loop, and in some cases a partial β-sheet structure. Includes four FR regions capable of forming. The CDRs in each strand are closely bound to each other via the FR region and together with the CDRs of the other strands form the antigen binding site of the antibody (Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The invariant regions are not directly involved in the binding of the antibody to the antigen, however, they exhibit different effects and functions, eg, are involved in the antibody-dependent cytotoxicity of the antibody.

The "light chain" of a vertebrate antibody (immunoglobulin) is classified into one of two distinctly different classes (called κ and λ), depending on the amino acid sequence of its invariant region. obtain. Immune glybrins can be classified into different classes depending on the amino acid sequence of their heavy chain invariant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subclassed into the following subclasses (isotypes): IgG1, IgG2, IgG3, IgG4: , IgA, and IgA2. The heavy chain invariant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit mechanism and three-dimensional arrangement of different classes of immunoglobulins are well known to those of skill in the art.

In general, the antigen binding properties of an antibody are three specific regions located in the heavy and light chain variable regions, called complementarity determining regions (CDRs), which divide the variable region into four framework regions (FRs). Can be explained by; the amino acid sequences of the four FRs are relatively conservative and are not directly involved in the binding reaction. The CDRs form a ring structure and approach each other within the three-dimensional structure via the β-sheet formed by the FR of the ring, and the CDRs on the heavy chain and the CDRs on the corresponding light chain are antibodies. Consists of the antigen binding site of. By comparing the amino acid sequences of antibodies of the same type, it can be determined which amino acids form FR or CDR.

The present invention includes not only intact antibodies, but also fragments of antibodies with immunological activity, or fusion proteins formed by antibodies and other sequences. Accordingly, the invention also includes antibody fragments, derivatives and analogs.

In the present invention, antibodies include mouse, chimeric, humanized or fully human antibodies prepared by techniques well known to those of skill in the art. Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human moieties, are obtained by standard DNA recombinant techniques, all of which are useful antibodies. A chimeric antibody is a chimeric antibody in which different parts have molecules derived from different animal species, such as a variable region from a monoclonal antibody derived from a mouse, and an invariant region from a human immunoglobulin (eg, US Pat. No. 4,816,6). See 567 and 4,816,397; they are incorporated herein by reference in their entirety). Humanized antibodies refer to antibody molecules derived from non-human species having one or more complementarity determining regions (CDRs) derived from non-human species, and framework regions derived from human immunoglobulin molecules. See 5,585,089; which is incorporated herein by reference in its entirety). Their chimeric and humanized antibodies can be prepared by recombinant DNA techniques well known in the art.

In the present invention, the antibody can be unispecific, bispecific, trispecific, or multispecific.

In the present invention, the antibody of the invention further comprises a conservative variant thereof, which is up to 10, preferably up to 8, more preferably up to 5 and most preferably up to the amino acid sequence of the antibody of the invention. References are made to polypeptides formed by substitution of three amino acids with similar amino acids. Those conservative variant polypeptides are preferably prepared by amino acid substitution according to Table A.

Anti-TF Antibodies The present invention provides antibodies with high specificity and high affinity for TF, including heavy and light chains, where the heavy chain is the amino acid sequence of the heavy chain variable region (VH). And the light chain comprises the amino acid sequence of the light chain variable region (VL).

Preferably, the CDRs for the amino acid sequence of the heavy chain variable region (VH) and the amino acid sequence of the light chain variable region (VL) are selected from:
a1) SEQ ID NO: 1;
a2) SEQ ID NO: 2;
a3) SEQ ID NO: 3;
a4) SEQ ID NO: 4;
a5) SEQ ID NO: 5;
a6) SEQ ID NO: 6;
a7) A sequence having a TF-binding affinity resulting from the addition, deletion, modification and / or substitution of at least one amino acid of any of the amino acid sequences of the amino acid sequence.

According to another preferred example, the sequence resulting from the addition, deletion, modification and / or substitution of at least one amino acid is preferably at least 80%, preferably at least 85%, more preferably at least 90%, most preferably. An amino acid sequence having at least 95% homology.

Preferably, the antibody has an activity of inhibiting the TF-related signaling pathway; has an anticoagulant activity; has an activity of inhibiting FXa production; or has a combination thereof.

The antibodies of the invention can be double-stranded or single-chain antibodies and can be selected from the group consisting of animal-derived antibodies, chimeric antibodies, and humanized antibodies; more preferably humanized antibodies and human-animal chimeras. It can be selected from the group consisting of antibodies; and can be fully human antibodies.

The antibodies of the invention can be single chain antibodies and / or antibody fragments such as Fab, Fab', (Fab') ₂ , or other antibody derivatives known in the art, and IgA, IgD, IgE. , IgG and IgM antibodies or subtypes of antibodies, one or more.

In the present invention, the animal is preferably a mammal, for example a mouse.

The antibodies of the invention can be chimeric antibodies, humanized antibodies, CDR transplants and / or modified antibodies that target human TFs.

According to a preferred example of the invention, it results from the addition, deletion, modification and / or substitution of any one or more sequences of SEQ ID NOs: 1-3, or at least one amino acid, and TF-binding affinity. Those sequences having are located in the CDR of the heavy chain variable region (VH).

According to a preferred example of the invention, it results from the addition, deletion, modification and / or substitution of any one or more sequences of SEQ ID NOs: 4-6, or at least one amino acid, and TF-binding affinity. Those sequences having are located in the CDR of the light chain variable region (VL).

According to a more preferred example of the invention, VH CDR1, CDR2, CDR3 may be from any one or more sequences of SEQ ID NOs: 1-3, or from the addition, deletion, modification and / or substitution of at least one amino acid. Generated and selected independently of those sequences having TF-binding affinity; VL CDR1, CDR2, CDR3 are any one or more sequences of SEQ ID NOs: 4-6, or the addition of at least one amino acid. , Deletions, modifications and / or substitutions, and independently selected from those sequences having TF-binding affinity.

In the present invention, the number of amino acids added, deleted, modified and / or substituted does not exceed, preferably more than 40%, more preferably 35% of the total number of amino acids in the initial amino acid sequence. It is more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and even more preferably 15 to 20%.

In the present invention, the number of amino acids added, deleted, modified and / or substituted is more preferably 1 to 7, more preferably 1 to 5, more preferably 1 to 3, and even more preferably. It is 1-2.

According to another preferred example, the antibody targeting TF is TF-mAb-SC1 (original name is TF-mAb).

According to another preferred example, the amino acid sequence of the heavy chain variable region (VH) of antibody TF-mAb-SC1 is the amino acid sequence as set forth in SEQ ID NO: 7.

According to another preferred example, the amino acid sequence of the light chain variable region (V-κ) of antibody TF-mAb-SC1 is the amino acid sequence as set forth in SEQ ID NO: 8.

Preparation of Antibodies Sequences of DNA molecules for the antibodies or fragments thereof of the present invention are obtained by conventional techniques such as PCR amplification or genomic library screening. In addition, the sequences encoding the light and heavy chains are fused together to form a single chain antibody.

Once the related sequence is obtained, a large amount of the related sequence can be obtained by using the recombination method. This is usually done by cloning the sequence into a vector, transforming the cell with the vector, and then separating the relevant sequence from the propagated host cell by conventional methods.

In addition, related sequences are artificially synthesized, especially if the fragment length is short. Usually, several small fragments are first synthesized and then ligated together to give a fragment with a long sequence.

It is currently possible to obtain a DNA sequence encoding an antibody (or a fragment thereof, or a derivative thereof) of the present invention by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention further relates to a vector comprising said appropriate DNA sequence and appropriate promoter or control sequence. Those vectors can be used to transform suitable host cells to allow them to express the protein.

The host cell can be a prokaryotic cell such as a bacterial cell; or a lower eukaryotic cell such as a yeast cell; or a higher eukaryotic cell such as a mammalian cell. Preferred animal cells include, but are not limited to, CHO-S, HEK-293 cells.

Generally, the resulting host cells are cultured under conditions suitable for the expression of the antibodies of the invention. The antibodies of the invention are then subjected to conventional immunoglobulin purification steps, such as conventional separation and purification means well known to those of skill in the art, such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ionization. Purified by using exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.

The resulting monoclonal antibody can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assay (eg, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis (Anal. Biochem., 107: 220 (1980)).

The antibodies of the invention are expressed in cells or on cell membranes or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods according to its physical, chemical or other properties. Those methods are well known to those of skill in the art. Examples of these methods include, but are not limited to, conventional regeneration treatment, treatment with a protein precipitant (salting out method), centrifugation, disruption of penetrating bacteria, ultrasonic treatment, ultracentrifuge. Centrifugal separation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), various other liquid chromatography techniques and combinations of these methods.

Antibody-drug conjugate (ADC)
The invention also provides an antibody-drug conjugate (ADC) based on the antibody of the invention.

Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, where the antibody is conjugated to the effector molecule, and chemical junctions are preferred. Preferably, the effector molecule is a therapeutically active drug. In addition, the effector molecule can be one or more toxic proteins, chemotherapeutic drugs or radionuclides.

The antibodies and effector molecules of the invention can be coupled by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. Non-selective coupling agents refer to compounds that result in a link between an effector molecule and an antibody via a covalent bond, such as glutaraldehyde. The coupling agent utilizing a carboxyl group is one of a cis-aconytic acid anhydride coupling agent (for example, cis-aconytic acid anhydride) and an acylhydrazone coupling agent (coupling site is acylhydrazone). Or it can be more than one.

Specific residues on the antibody (eg, Cys or Lys, etc.) are contrast agents (eg, chromophores and phosphors), diagnostic agents (eg, MRI contrast agents and radioisotopes), stabilizers (eg, eg, MRI contrast agents and radioisotopes). Used to link various functional groups, including poly (ethylene glycol)) and therapeutic agents. The antibody can be conjugated to the functional agent to form an antibody-functional agent conjugate. Functional agents (eg, drugs, detection reagents, stabilizers) are attached to the antibody (by covalent bond). The functional agent is directly or indirectly linked to the antibody via the linker.

The antibody can be attached to the drug to form an antibody-drug conjugate (ADC). Typically, the ADC contains a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker. Typically, the degradable linker is readily degraded in an intracellular environment, for example, the linker is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include enzymatically degraded linkers containing peptidyl-containing linkers that can be degraded intracellularly by proteases (eg, lysosomal proteases or endosomal proteases), sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidase. Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (eg, linkers that are hydrolyzed at pH below 5.5, such as hydrazone linkers) and linkers that are degraded under reducing conditions (eg, disulfide bond linkers). include. Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by the protease.

Prior to ligation to the antibody, the linker has a reactive group capable of reacting with a particular amino acid residue, and ligation is achieved by that reactive group. Thiol-specific reactive groups are preferred, for example, maleimide compounds, halogenated (eg, iodo, bromo, or chloro substituted) amides; halogenated (eg, iodo, bromo, or chloro substituted) esters; halogens. Halogenated (eg, iodo, bromo, or chloro substituted) halogenated benzyl; vinyl sulfone; pyridyl disulfide; mercury derivatives such as 3,6-di- (mercury methyl) dioxane (counterion CH ₃ COO- ^{, Cl-} Or NO _3- ⁾ ; and contains polymethylenedimethylsulfide thiosulfonate. The linker comprises, for example, maleimide linked to the antibody via thiosuccinimide.

The drug can be any cytotoxic, cell proliferation inhibitory or immunosuppressive drug. According to one embodiment, the antibody is linked to the drug via a linker and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino group, a carboxyl group, a thiol group, a hydroxyl group, or a ketone group that can form a bond with the linker. When the drug is linked directly to the linker, the drug has a reactive group prior to being linked to the antibody.

Useful drugs include: anti-tubulin drugs, DNA accessory groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, metabolic antagonists, chemotherapeutic sensitizers, topoisomerase inhibitors, vinca Alkaloid etc. Examples of particularly useful cytotoxic drugs include: DNA accessory groove binding agents, DNA alkylating agents, and tubulin inhibitors; typical cytotoxic drugs include, for example: auristatin, camptothecin, docamycin. / Duocarmycin, etopocid, mateanthin and matetansinoids (eg DM1 and DM4), taxan, benzodiazepines or benzodiazepine-containing drugs (eg, pyrrolo [1,4] benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines). , As well as binca alkaloids.

In the present invention, the drug-linker can be used to form the ADC in a simple process. According to other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, the cysteine residue is reacted with the reactive moiety of the linker in the first step, and then the functional groups on the linker are reacted with the drug in the subsequent steps to form the ADC.

In general, the functional groups on the linker are selected to specifically react with the appropriate reactive groups on the drug moiety. As a non-limiting example, a hideout-based moiety can be used to specifically react with a reactive alkynyl group on a drug moiety. The drug is covalently attached to the linker by the 1,3-dipole cycloaddition between the hideout group and the alkynyl group. Other useful functional groups include, for example: ketones and aldehydes (suitable for reactions with hydrazides and alkoxyamines), phosphines (suitable for reactions with azides); isocyanates and isothiocyanates (amines). And suitable for reaction with alcohols); and activated esters such as N-hydroxysuccinimide esters (suitable for reactions with amines and alcohols). Those and other conjugation methods, such as ^{Bioconjugation} Technology (2nd Edition (Elsevier)), are well known to those of skill in the art. Those skilled in the art will appreciate that if the reactive functional group of the complementary pair is selected for the selective reaction between the drug moiety and the linker, each member of the complementary pair is used for the linker, and also the drug. Understand that it can be used for.

The invention further provides a method of preparing an ADC, further comprising binding the antibody to a drug-linker compound under conditions sufficient to form an antibody-drug conjugate (ADC).

According to certain embodiments, the method of the invention comprises binding an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. According to those embodiments, the method of the invention comprises binding the antibody-linker conjugate to the drug moiety under conditions sufficient to co-bond the drug moiety to the antibody via the linker.

［式中、Ａｂは抗体であり；
ＬＵはリンカーであり、
Ｄは薬物であり；そして
下付文字ｐは、１～８から選択された値である］を有する。 According to some embodiments, the antibody-drug conjugate (ADC) has the following formula:

[In the formula, Ab is an antibody;
LU is a linker,
D is a drug; and the subscript p is a value selected from 1-8].

Detection Applications and Kits The antibodies of the invention or ADCs thereof can be used, for example, in detection applications for use in the detection of samples to provide diagnostic information.

In the present invention, the sample used includes a cell, a tissue sample and a biopsy sample. The term "biopsy" as used in the present invention should include all types of biopsy known to those of skill in the art. Biopsy specimens used in the present invention include, for example, excision samples of tumors and tissue samples prepared by endoscopic methods or puncture or needle puncture biopsy of organs.

The samples used in the present invention include fixed or conserved cell or tissue samples.

The invention further provides a kit simply comprising an antibody (or fragment thereof) of the invention; according to a preferred example of the invention, the kit further comprises a container, instructions, buffer, etc. According to a preferred example, the antibodies of the invention can be immobilized on a test panel.

Uses The present invention further provides the use of the antibodies of the present invention for the manufacture of diagnostic agents or the manufacture of pharmaceuticals for the prevention and / or treatment of TF-related diseases. TF-related diseases include tumor formation, tumor growth and / or metastasis, thrombosis-related diseases, inflammation, metabolism-related diseases and the like.

The use of the antibodies, ADCs or CAR-Ts of the invention includes, but is not limited to:
(I) Diagnosis, prevention and / or treatment of tumor formation, tumor growth and / or metastasis, especially tumors with high TF expression, where tumors include (but are not limited to): cancer (eg, eg). , Triple negative breast cancer), pancreatic cancer, lung cancer, malignant glioma, gastric cancer, liver cancer, esophageal cancer, kidney cancer, colon cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, leukemia, Bone marrow cancer, angiosarcoma, etc .; in particular, triple negative cancer, pancreatic cancer, malignant glioma and lung cancer; more preferably triple negative breast cancer and / or pancreatic cancer;
(Ii) Diagnosis, prevention and / or treatment of thrombosis-related disorders, wherein thrombosis-related disorders include (but are not limited to): atherosclerosis, acute coronary syndrome, acute myocardium. Infarction, stroke, hypertension, deep venous thrombosis, pulmonary embolism, renal embolism and arterial surgery, thrombosis due to coronary artery bypass surgery, etc.;
(Iii) Diagnosis, prevention and / or treatment of inflammation, where inflammation includes, but is not limited to: rheumatoid arthritis, osteoarthritis of the arthritis, tonic spondylitis, gout, Little Syndrome. , Psoriasis arthritis, infectious arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, glomerular nephritis, systemic erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, and idiopathic Pulmonary fibrosis;
(Iv) Diagnosis, prevention and / or treatment of metabolism-related diseases, wherein the metabolism-related diseases include (but are not limited to): diabetes, dietary obesity, fat inflammation and the like.

Pharmaceutical Compositions The present invention further provides compositions. According to a preferred example, the composition is a pharmaceutical composition comprising an antibody or an active fragment thereof, a fusion protein or ADC, or a corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, the substances can be formulated in non-toxic, inert and pharmaceutically acceptable aqueous carrier media, where the pH is generally about 5-8, preferably about 6-. It is 8, but its pH value can be changed depending on the nature of the substance to be blended and the condition to be treated. The pharmaceutical composition to be formulated can be administered by conventional routes such as intratumoral, intraperitoneal, intravenous, or topical administration, but not limited to them.

Antibodies of the invention are also expressed in cells from nucleotide sequences and can be used in cell therapy, eg, antibodies are used in chimeric antigen receptor T cell immunotherapy (CAR-T), and the like.

The pharmaceutical compositions of the present invention can be used directly to bind TF protein molecules and, therefore, to prevent and treat diseases such as tumors. In addition, concomitant therapies can also be used at the same time.

The pharmaceutical composition of the present invention is a safe and effective amount (eg, 0.001 to 99% by weight, preferably 0.01 to 90% by weight, preferably 0.1 to 80% by weight) of the monoclonal antibody of the present invention. (Or a conjugate thereof) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol and combinations thereof. The pharmaceutical product should correspond to the administration method. The pharmaceutical composition of the present invention can be prepared in the form of an injection by a conventional method using physiological saline or an aqueous solution containing glucose and another adjuvant. Pharmaceutical compositions such as injections and solutions should be prepared under sterile conditions. The dose of the active ingredient is a therapeutically effective amount, eg, from about 1 μg / kg body weight / day to about 5 mg / kg body weight / day. In addition, the polypeptides of the invention can also be used in combination with additional therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight and, in most cases, at least about 10 μg / kg body weight. It is about 50 mg or less / kg body weight. Preferably, the dose is from about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the particular dose should depend on factors that are within the skill of a skilled physician, such as the route of administration and the physical condition of the patient.

The main advantages of the present invention include:
a) The antibodies of the invention have outstanding biological activity and properties and have high affinity (EC ₅₀ is 0.01-003 nM as determined by ELISA); in addition, the cell surface. It has a good binding affinity for TF and can be used as a TF-target antibody;
(B) The humanized antibody of the present invention not only has an activity comparable to that of an immune antibody, but also has low immunogenicity;
(C) Both the antibodies and ADCs of the invention have significant antitumor activity and have no obvious toxic side effects to the mammalian itself, and (d) the antibodies and ADCs of the invention. Not only has a significant therapeutic effect in tumor models, it can also be applied to other high TF expression related diseases such as thrombotic and metabolic diseases.

The invention is further described with reference to the specific examples that follow. It should be understood that the following examples are used merely to illustrate the invention, but do not limit the scope of the invention. Experimental methods in subsequent examples have not been shown with specific conditions, but are usually described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). It is carried out according to the conditions to be applied or recommended by the manufacturer. Unless otherwise stated, percentages and parts mean weight percentages and parts by weight. The cell line is a conventional product that is commercially available or purchased from the ATCC, and all plasmids are commercially available products.

Example 1: Expression and preparation of a monoclonal antibody targeting human TF Step 1: Preparation of hybridoma cells:
First, 8-week-old female Balb / c mice are immunized with the extracellular domain of human TF protein (UniProtKB / Swiss-Prot: P13726.1, amino acids from positions 34-251), where TF cells. The extracellular domain was used in an amount of 100 μg / mouse to prepare immunized splenocytes; mouse myeloma cells (SP2 / 0) and feeder cells were timely prepared for fusion purposes.

After preparing the above three cells, fusion of splenocytes and SP2 / 0 cells was mediated by PEG, PEG was removed, and the resulting cells were resuspended in HAT complete medium containing feeder cells. Then, it was cultured in a 96-well plate. Positive wells were screened by ELISA. Finally, cells in positive wells were subjected to clonal culture by limiting dilution, and cells with high titers and good morphology and grown in a monoclonal manner were screened by ELISA or immunofluorescence techniques. Cells grown in a monoclonal manner were further entrusted to subcloning screening for 3 consecutive screenings until the positive cloning rate was 100%.

Step 2: Preparation of ascites of a murine monoclonal antibody targeting human TF:
The hybridoma cells screened in step 1 were entrusted to amplification culture. After increased adaptability, pristane (0.5 ml / mouse) was injected intraperitoneally into the mouse to provide a favorable environment for hybridoma cell proliferation. After 7-10 days, 10 × 10 ⁶ hybridoma cells were injected intraperitoneally in each mouse. Mice were observed daily for ascites and mental state production from day 7 onwards, and ascites was collected, centrifuged, cells removed, and cryopreserved at -80 ° C for later purification.

Step 3: Purification of murine monoclonal antibody targeting human TF:
The ascites cryopreserved in step 2 was thawed on ice and filtered through a 0.45 μm filter and then dialyzed against PBS overnight at 4 ° C. Finally, the antibodies were purified by FPLC technique, ultrafiltered, concentrated to the desired concentration, repackaged and cryopreserved at −80 ° C. for further use.

Step 4: Determination of biological activity and target specificity of murine monoclonal antibody targeting human TF:
After preliminary screening, about 30 hybridomas were selected for secondary limiting dilution cloning, and then 6 antibodies were selected for large-scale expression and purification. Each antibody was determined by flow cytometry at a concentration of 10 μg / ml for its affinity for human breast cancer cells MDA-MB-231, human pancreatic cancer cells BxPC-3 and murine melanoma cells B16-F10.

The results shown in FIG. 1 show that the antibody tested is specific for human TF (MDA-MB-231 and BxPC-3 cells) without specific binding to murine TF (B16-F10 cells). TF-mAb-SC1 was shown to have a higher affinity for human TF than the other 5 antibodies.

The antigen (extracellular domain protein of TF) was diluted to 0.5 μg / ml with a coaching solution and used to coat the ELISA plate overnight at 100 μl / well and 4 ° C. Excess antigen was removed linearly. Plates were blocked with 2% BSA at room temperature for 2 hours, and then individual 3-fold serially diluted monoclonal antigens were added at 100 μl / well and incubated at room temperature for 2 hours. Unbound antibody was removed by washing, and appropriate concentrations of horseradish peroxidase-labeled anti-mouse secondary antibody were added at 100 μl / well and incubated for 1 hour at room temperature. The unbound secondary antibody was removed by washing, a TMB color-developing solution was added, and the color was developed to an appropriate color depth. 2M H ₂ SO ₄ was added at 50 μl / well to stop the color reaction, then the absorbance at 450 nm was determined and the data were analyzed. As shown in FIG. 2, TF-mAb-SC1 had a strong affinity for the extracellular domain protein of TF and had an EC50 of about 0.019 _nM .

On the other hand, 3 × 10 ⁵ pancreatic cancer cells BxPC-3 were spread on a 12-well plate. After 12 hours, cells were washed 3 times with sterilized PBS and then starved for 4 hours at 37 ° C. in a 5% CO ₂ incubator after addition of serum-free medium. Each monoclonal antibody was entrusted to 3-fold gradient dilution and incubated with BxPC-3 in an incubator for 1 hour, followed by activation of the PAR2 intracellular signaling pathway by 25 nM FVIIa in BxPC-3. After 15 minutes of interaction at 37 ° C., cells were washed once with pre-cooled PBS, intracellular proteins were collected on ice, and TF-mAb-SC1 for downstream MAPK / ERK phosphorylation levels. The effect of was identified by Western blot, where only FVIIa stimulation was performed, and cells not incubated with the monoclonal antibody were used as positive controls. If only the solvent was obtained and no antibody was added, it was designated as Veh. The effects of the six antibodies on the TF-PAR2 intracellular signaling pathway in BxPC-3 were determined by Western blot.

The results shown in FIG. 3 suggested that only TF-mAb-SC1 significantly inhibited downstream MAPK / ERK phosphorylation levels with a certain degree of volume dependence.

TF-mAb-SC1 showed very high specificity, very high affinity, and a significant inhibitory effect on MAPK / ERK phosphorylation levels and was selected for sequencing and subsequent studies.

Conventional sequencing and analysis according to Kabat's database yielded the following sequence information:
The amino acid sequence of the CDRs of the heavy chain variable region was as follows:
SEQ ID NO: 1: SYWMN;
SEQ ID NO: 2: MIYPADSETRLNQKFKD;
SEQ ID NO: 3: EDYGSDY.

The complete VH amino acid sequence was as shown in SEQ ID NO: 7.
QVQLQQPGAELVRPGASVKLSCKASGYSFISYWMNWVKQRPGQGLEWIGMIYPADSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCAREDYGSSDYWGQGTTLTVSS (SEQ ID NO: 7).

The amino acid sequence of the CDR of the light chain variable region was as follows:
SEQ ID NO: 4: SASSSVSYMN;
SEQ ID NO: 5: GISNLAS;
SEQ ID NO: 6: QQKSSFPWT.

The complete VL amino acid sequence was as shown in SEQ ID NO: 8.
EILLTQSPAIIAAASPGEKVTTCSASSVSYMNWYLQKPGSSSPKIWIYGISYGISNLASGVPARFSGSGSGTSFSFTTINSMETEDVATYYCQQKSFPWTFGGGTGTGTKLEIK (SEQ ID NO: 8).

Example 2: Preparation of human-mouse chimeric antibody A human-mouse antibody was constructed based on the obtained highly active and specific murine TF-mAb-SC1.

Analysis according to the relevant database showed that the amino acid sequence of the CDRs of the heavy chain variable region was:
SEQ ID NO: 18: MIYPXDESTRLNXKFKD (X is any one selected from the group consisting of A, D, E, Q and Y)
SEQ ID NO: 19: GYSFXSYWMN (X is any one selected from the group consisting of A, I, Y, Q and W)
SEQ ID NO: 20: AREDYGXSDY (X is any one selected from the group consisting of S, P, G, D, M and N).

Analysis according to the relevant database showed that the amino acid sequence of the CDR of the light chain variable region was:
SEQ ID NO: 21: QQXSSFXWT (X is any one selected from the group consisting of S, P, K, G, and H).
SEQ ID NO: 22: SASSXVSYMN (X is any one selected from the group consisting of A, P, D, and S).
SEQ ID NO: 23: GXSNLAS (X is any one selected from the group consisting of P, D, I, and S).

Primers were designed to introduce EcoRI and NheI into the heavy chain variable region and AgeI and BsiWI restriction endonuclease sites into the light chain variable region, and then the antibody heavy chain and light chain obtained above. Variable region sequences were separately cloned into vectors containing the heavy chain invariant region and the κ chain invariant region of human IgG1. After confirmation by identification (FIG. 4A shows the identification results of the enzymatic digestion of the heavy chain, and FIG. 4B shows the identification results of the enzymatic digestion of the light chain, where Sample 1 shows the corresponding heavy chain / light chain. A blank vector, sample 2 is a vector in which the heavy / light chain variable region is cloned, samples 3 and 4 are samples after a single enzymatic digestion, and sample 5 is a double. The chimeric antibody constructed (which was the sample after enzymatic digestion) was expressed and purified and obtained by using a transfection technique and a mammalian expression system (CHO-S or HEK-293 cells). The human-mouse chimeric antibody was named TF-mAb-Ch.

Example 3: Determination of TF-mAb-SC1 binding affinity for TF-positive tumor cells In this experiment, triple negative breast cancer cells MDA-MB-231, pancreatic cancer, which express TF highly on the cell surface. Cell BxPC-3, malignant glioma cell U87MG and non-small cell lung cancer cell H1975 were used as target cells; and 100 μl TF-mAb diluted by 3-fold serial dilutions from 333.33 nM to 0.15 nM. -SC1 is used as the primary antibody and mixed homogeneously with 3 x 10 ⁵ MDA-MB-231 or BxPC-3 suspended in 100 μl RPMI-1640 serum-free medium, respectively; or 66.67 nM. 100 μl of TF-mAb-SC1 diluted to ~ 0.03 nM by 3-fold serial dilution was used as the primary antibody and homogeneous with 3 × 10 ⁵ U87MG suspended in 100 μl MEM serum-free medium. Or mix 100 μl of 33.3 nM and 3.33 nM TF-mAb-SC1 as the primary antibody with 3 × 10 ⁵ H1975 suspended in 100 μl RPMI-1640 serum-free medium. , And then incubated at 4 ° C. for 1 hour. The cells were washed twice with PBS, the terminally bound primary antibody was removed, and the target cells were incubated with 200 μl of PE-labeled secondary antibody (2 μg / ml) at 4 ° C. for 30 minutes. The cells were washed twice with PBS, the terminally bound secondary antibody was removed, and finally the cells were resuspended in 400 μl PBS. TF-mAb-SC1 was determined by flow cytometry for its binding affinity for the corresponding cell surface TF.

TF-mAb-SC1 shown in FIG. 5 has good binding affinities for BxPC-3, MDA-MB-231 and U87MG cells, 2.6 nM (FIG. 5A) and 2.5 nM (FIG. 5B, respectively). ) And 1.6 _nM (FIG. 5C); and FIG. 5D shows that TF-mAb-SC1 also had good binding affinity for H1975 cells.

This suggested that the monoclonal antibody in Example 1 could be used as a target for human TF.

Example 4: Effect of TF-mAb-SC1 on TF-PAR2 intracellular signaling pathways 3 × 10 ⁵ pancreatic cancer cells (BxPC-3) were plated on a 12-well plate. After 12 hours, cells were washed with sterilized PBS and then starved for 4 hours at 37 ° C. in a 5% CO ₂ incubator after addition of serum-free medium. Next, TF-mAb-SC1 is diluted 100 nM to 1.2 nM by 3-fold serial dilution and incubated with BxPC-3 cells in an incubator, followed by 25 nM FVIIa in BxPC-3 cells intracellularly in PAR2 cells. Activated the signaling pathway. After 15 minutes of interaction at 37 ° C., cells were washed once with precooled PBS and cell proteins were collected on ice. The effect of TF-mAb-SC1 on downstream MAPK / ERK phosphorylation levels was identified by Western blots, where only FVIIa stimulation without TF-mAb-SC1 treatment was used as a control.

The results as shown in FIG. 6 showed that TF-mAb-SC1 strongly and dose-dependently inhibited the phosphorylation level of downstream MAPK / ERK signaling.

Example 5: Determination of anticoagulant activity of TF-mAb-SC1 100 nM of TF-mAb-SC1 is serially diluted to 1.5625 nM (final volume of 50 μl) and contains 50 μl Hanks equilibrium containing 5 mM CaCl ₂ . Incubated at room temperature for 15 minutes with 3 × 10 ⁴ MDA-MB-231 and BxPC-3 cells suspended in salt solution (HBSS). Next, 50 μl of human citrate plasma was added and the resulting mixture was mixed rapidly and homogeneously. Absorbance at 405 nM was measured every 15 seconds within the next 2 hours to calculate the anticoagulant effect on TF-initiated coagulation on the cell surface.

In FIG. 7, the coordinates represent the time to achieve the maximum coagulation rate, the abscissa represents the concentration of TF-mAb-SC1, and BxPC-3 (FIG. 7A) and MDA-MB-231 (FIG. 7B). TF on the cell surface was used as a source of TF. Experimental results showed that TF-mAb-SC1 had significant anticoagulant activity at concentrations below 12.5 nM.

Example 6: Determination of TF-mAb-SC1 activity to inhibit FXa production 100 nM of TF-mAb-SC1 is serially diluted to 1.5625 nM (50 μl final volume) and 50 μl of HBSS (3 nM). Incubated separately with 1.5 × 10 ⁴ MDA-MB-231 and BxPC-3 cells suspended in (including FVIIa) under shaking at room temperature. Next, 50 μl of FX (at a final concentration of 50 nM) was added to initiate the reaction, and after 5 minutes, 1 M of EDTA (25 μl) was added to terminate the reaction. Then 3 mM S2765 (25 μl) was added and the resulting mixture was mixed rapidly and homogeneously. The kinetic response curve was measured every 15 seconds for the next 60 minutes to determine the activity of inhibiting FXa production.

As shown in FIG. 8, TF-mAb-SC1 showed good activity to inhibit FXa and had _IC50s of 9.0 nM (FIG. 8A) and 6.4 nM (FIG. 8B).

Example 7: Evaluation of TF-mAb-SC1 for its activity of inhibiting tumor growth in vivo Nude mice were randomly divided into two groups, each group having 10 animals. First, tumor cells (1 × 10 ⁷ BxPC-3, or 5 × 10 ⁶ U87MG, or 2.5 × 10 ⁶ HCC1806) were mixed with 100 μg of TF-mAb-SC1 at room temperature. After a 30-minute incubation at room temperature, the back or breast fat pad of a 6-week-old immunodeficient female mouse (Ball / c nude mouse) was inoculated with the resulting mixture to exert an inhibitory effect on the growth of subcutaneous tumors of BxPC-3. Observed. Another group of mice inoculated with normal mouse IgG (shown as IgG in the figure) was used as a control. Nude mice were periodically measured for their body weight and tumor size, tumor growth curves were blotted, and activity was assessed.

As shown in FIG. 9, TF-mAb-SC1 has a significant inhibitory effect on BxPC-3 subcutaneous xenograft tumor growth compared to IgG, and has an inhibition rate of up to 80%. And at the end of the experiment, the inhibition rate of tumor growth was 55%.

As shown in FIG. 10, TF-mAb-SC1 has a significant inhibitory effect on U87MG subcutaneous xenograft tumor growth and has an inhibition rate of up to 60% compared to IgG. And at the end of the experiment, the inhibition rate of tumor growth was 36%.

As shown in FIG. 11, TF-mAb-SC1 has a significant inhibitory effect on HCC1806 subcutaneous xenograft tumor growth compared to IgG, and has an inhibition rate of up to 90% or more. And at the end of the experiment, the inhibition rate of tumor growth was 83%.

Example 8: Significant inhibition of tumor matrix collagen accumulation by TF-mAb-SC1 BxPC-3 tumors from Example 7 were collected and fixed in 4% neutral formaldehyde, followed by paraffin embedding. It was then fragmented, routinely waxed in water and stained with Masson. For each stained sample, 5-10 fields were imaged at 100x magnification (100 μm in the description) and statistically analyzed.

The results shown in FIG. 12 show that TF-mAb-SC1 significantly inhibits the accumulation of tumor matrix collagen (blue region) that can contribute to tumor growth inhibition, where the right panel uses Image-pro plus. The results of the statistical analysis that had been performed are shown.

Example 9: Significant reduction of the lumen region of the tumor vessel by TF-mAb-SC1 BxPC-3 tumors from Example 7 were collected and fixed in 4% neutral formaldehyde, followed by paraffin embedding. , And fragmented, routinely waxed to water, and stained with CD31 immunohistologically. For each immunohistologically stained sample, 5-10 fields were imaged at 200x magnification (50 μm in the description) and statistically analyzed.

The results shown in FIG. 13 show that TF-mAb-SC1 significantly reduced the luminal region of the tumor vessel, where the right figure shows the statistical results of the luminal region of the blood vessel.

Example 10: Determination of TF-mAb-SC1 for its activity of inhibiting tumor cell migration The efficiency of TF-mAb for tumor cell migration levels was assessed in vitro using a transwell chamber system: 1 × 10 ⁵ MDA-MB-231 or 8x10 ⁴ BxPc-3 cells in 200 μl serum-free medium at specific concentrations of TF-mAb-SC1 (100 nM, 33.3 nM or 11.1 nM) or mouse IgG. Mix separately with (shown as IgG in the figure); the resulting mixture is added to the upper chamber, and 600 μl complete medium containing 10% FBS is added to the lower chamber; cells are 5% CO ₂ Incubated at 37 ° C. in an incubator; after 8 hours, cells on the upper surface of the chamber membrane were wiped with a damp cotton swab. The cells on the underside of the membrane were fixed with 95% ethanol for 30 minutes and then stained with 0.2% crystal violet for 30 minutes and washed with distilled water to remove excess crystal violet. After drying at room temperature, five representative fields were randomly selected under a microscope, and the number of cells migrating to the underside of the chamber membrane was counted and analyzed. In this experiment, both TF knockout cells (sh-TF) and corresponding vector control cells (sh-NT) were used to confirm the activity of TF-mAb-SC1 to inhibit tumor cell migration.

As shown in FIG. 14, migration of MDA-MB-231 (FIG. 14A) and BxPC-3 (FIG. 14B) cells was significantly inhibited by knocking out the TF gene.

As shown in FIG. 15, TF-mAb-SC1 was able to significantly inhibit the migration levels of MDA-MB-231 (FIG. 15A) and BxPC-3 (FIG. 15B) in a concentration-dependent manner.

The results showed that the migration of tumor cells could be effectively inhibited by inhibiting TF.

Example 11: Significant inhibition of in vivo hematogenous metastasis of tumor cells by TF-mAb-SC1 The effect of TF-mAb-SC1 on tumor cell migration levels was evaluated in vivo using an experimental model of hematogenous metastasis. : 2 × 10 ⁶ luciferase-labeled MDA-MB-213 cells (MDA-MB-231-luc) were mixed in 200 μl PBS with 100 μg TF-mAb-SC1 or mouse IgG and 20 on ice. Incubate for minutes and then slowly inject into the tail vein of 6-week-old Balb / c female nude mice; 4 hours later, the nude mice are anesthetized and a solution of D-luciferin potassium salt in PBS. Nude mice were intraperitoneally injected at a volume of 150 mg / kg; after 6 minutes, the mice were exposed to a small animal in vivo imaging system (IVIS SPECTRUM) for 1 minute, and fluorescence intensity was measured; and statistical analysis was performed on each. This was done using 5 nude mice per group.

As shown in FIG. 16, knockout of the TF gene was able to significantly inhibit the hematogenous mobility of MDA-MB-231-luc. Similarly, as shown in FIG. 17, TF-mAb-SC1 can also significantly inhibit the hematogenous migration capacity of MDA-MB-231-luc cells, where the right figure shows cells that are migrated to the lung. The statistical results of the fluorescence intensity of are shown.

3 × 10 ⁶ MDA-MB-231 cells were mixed in 200 μl PBS with 100 μg TF-mAb-SC1 or IgG, incubated on ice for 20 minutes, and then 6 week old female SCID. It was slowly injected into the tail vein of Beige mice. After 6 weeks, mice were sacrificed, lungs were imaged and fixed in Bouin solution, and then photographed and weighed. The number of metastases on each lung was recorded using 7 mice per group.

The results shown in FIG. 18 suggested that TF-mAb-SC1 significantly inhibited the formation and growth of tumor metastases on the mouse lung.

Example 12: Rapid and high rate internalization of TF-mAb-SC1 into lysosomes MDA-MB-231 cells with a density of 50% in a culture dish specific for use in a laser confocal microscope. Plated. After about 16 hours, 10 μg / ml TF-mAb-SC1 was added and cells were incubated at 38 ° C or 4 ° C for 1 hour and then fixed at room temperature for 30 minutes with 4% paraformaldehyde. After washing 3 times with PBS, cells were incubated with Lamp-2 (rabbit anti-human) antibody for 1 hour at 37 ° C. to label the location of lysosomes and unbound antibody was removed by washing with PBS. .. After 30 minutes of incubation at 37 ° C. with Alexa Floor 594-labeled donkey anti-mouse secondary antibody and Alexa Fluor 488-labeled donkey anti-rabbit secondary antibody, terminal binding antibody was removed by washing. The location of the nucleus was labeled with DAPI staining, and then the internalization of the antibody was observed under a laser confocal microscope.

As shown in FIG. 19, TF-mAb-SC1 can be internalized in lysosomes.

Example 13; Determination of Biological Activity of TF-mAb-Ch This experimental method also refers to step 4 of Example 1.

The results shown in FIG. 20 suggest that TF-mAb-Ch has a strong affinity for TF cell domain proteins and has an EC50 of about 0.011 _nM .

Example 14 :; Determination of TF-mAb-Ch binding affinity for TF-positive tumor cells This experimental method refers to Example 3.

The results show that TF-mAb-C has good binding affinities for both MDA-MB-231 (FIG. 21A) and H1806 (FIG. 21B) cells, and 2.3 nM and 2.1 nM EDs, respectively. It was shown to have ₅₀ .

Example 15: Effect of TF-mAb-Ch on TF-PAR2 intracellular signaling pathway This experimental method refers to Example 4.

The results shown in FIG. 22 showed that TF-mAb-Ch inhibited FVIIa-induced phosphorylation levels of MAPK / ERK in a concentration-dependent manner.

Example 16: Rapid and efficient internalization of TF-mAb-Ch into lysosomes This experimental method refers to Example 12.

The results shown in FIG. 23 indicate that TF-mAb-Ch could be internalized in lysosomes.

In the above experiment, since the TF-mAb antibody of the present invention can be easily internalized, it is for the progression to antibody-drug conjugate (ADC) and for the treatment of high TF expression-related tumors. It was appropriate.

Example 17: Humanization and determination of activity of TF-mAb-SC1 by reference to the sequences of the heavy chain variable region (SEQ ID NO: 7) and light chain variable region (SEQ ID NO: 8) of antibody TF-mAb-SC1. The humanized template that best matched the non-CDR regions was selected in the Germline database. The CDR regions of the murine antibody TF-mAb-SC1 were then transplanted into selected humanized templates to replace the CDR regions of the humanized templates, followed by recombination with IgG1 / κ invariant regions. On the other hand, based on the three-dimensional structure of the murine antibody, reversion mutations are performed on the residues that directly interacted with the CDR regions and the residues that had an important effect on the conformation of VL and VH. , Thereby obtaining five humanized heavy chain variable regions (SEQ ID NOs: 9, 10, 11, 12 and 13) and four humanized light chain variable regions (SEQ ID NOs: 14, 15, 16 and 17).

Based on the engineered VHs and VLs, their humanized heavy and light chains are expressed in separate associations and finally a total of 20 humanized antibodies, namely TF-mAb-H29 to TF-mAb-H48. Got The corresponding combinations of heavy and light chains for each antibody are shown in the table below:

First, 20 humanized antibodies were determined by ELISA binding assay for their affinity for extracellular domain proteins of TF (see Step 4 of Example 1 for experimental methods). The results are shown in Table 1. The binding affinity curves for TF-mAb-H39 and TF-mAb-H44 to the extracellular domain protein of TF are shown in FIGS. 24A and 24B, respectively.

The 20 humanized antibodies were determined by flow cytometry for their binding affinity for MDA-MB-231 cells at 10 μg / ml and 1 μg / ml. The experimental method was referred to Example 3, and the results are shown in Table 2.

The effects of nine humanized antibodies on the TF-PAR2 intracellular signaling pathway were also determined. For the experimental method, refer to Example 4.

The experimental results shown in FIG. 25 showed that each humanized antibody inhibited FVIIa-induced phosphorylation levels of MAPK / ERK to varying degrees in a concentration-dependent manner.

In addition, after incubating 6 humanized antibodies with tumor cells HCC1806, their inhibitory effect on tumors by inoculating the breast fat pad of nude mice with each of the 6 humanized antibodies (100 μg for each antibody). Decided about. For the experimental method, refer to Example 17.

The experimental results shown in FIG. 26 show that all human antibodies show significant activity in inhibiting tumor growth, among which TF-mAb-H35, TF-mAb-H39, TF-mAb-H40, TF. It was suggested that -mAb-H44 and TF-mAb-H45 showed outstanding activity in inhibiting tumor growth.

In addition, the humanized antibody TF-mAb-H39 and the chimeric antibody TF-mAb-Ch were also incubated with the tumor cell HCC1806 and then in different volumes (100 μg, 30 μg and 10 μg) with the humanized antibody TF-mAb-H39 and the chimera. Their effect of inhibiting the growth of HCC1806 heterologous transplants by another inoculation of the breast fat pad of nude mice with the antibody TF-mAb-Ch was also determined. For the experimental method, refer to Example 7. The experimental results are shown in FIG. 27. The results showed that the humanized antibody TF-mAb-H39 had a tumor growth inhibitory activity comparable to that of the chimeric antibody TF-mAb-Ch.

Example 18: Very Abnormal Activation of TF in Basal-like and Triple Negative Breast Cancer First, whole cell proteins were prepared for various tumor cells from different tissues. After accurate quantification, the expression level of TF protein was determined by Western blot.

The results show that highly aberrant activation and expression of the TF protein is present in some highly invasive and highly metastatic basal-like or basal A / B and triple-negative breast cancer (as shown in FIG. 28) cell lines. However, high expression of the TF protein occurred only in some low-grade intraluminal breast cancer cell lines.

Next, the expression level of TF mRNA in the breast cancer cell lineage in the database E-TABM-157 (European Molecular Biology Laboratory-European Bioinformatics Institute, EMBL-EBI) was analyzed. The results show that the expression level of TF mRNA in highly invasive and highly metastatic basal A / B and triple-negative breast cancer (particularly triple-negative breast cancer) cell lines is generally higher than that in lumen subtype breast cancer cell lines. This was shown to be statistically significant (Fig. 29). Therefore, the TF-targeting antibody of the present invention had a more pronounced effect in the diagnosis, prevention and treatment of triple-negative breast cancer.

Example 19: Very Abnormal Activation of TF in Pancreatic Cancer First, whole cell proteins were prepared for various tumor cell lines from different tissues. After accurate quantification, the expression level of TF protein was determined by Western blot. The results showed that highly aberrant activation and expression of the TF protein was present in highly invasive and highly metastatic pancreatic cancer (as shown in FIG. 30) cell line.

Next, the expression level of TF mRNA in pancreatic tumors and normal tissues in the database GSE15471 (Gene Expression Omnibus, GEO) was analyzed. The results showed that TF mRNA was generally expressed at high levels in pancreatic tumor tissue (Fig. 31). Therefore, the TF target antibody of the present invention had a more remarkable effect in the diagnosis, prevention and treatment of pancreatic cancer.

All documents referred to in the present invention are incorporated into the present application by reference as if each individual document were specifically and individually indicated to be incorporated by reference. Further, after reading the content taught in the present invention, various modifications and modifications to the present invention may be made by those skilled in the art, and their equivalents are also included in the scope of the claims. It should be understood that

Claims (12)

An anti-human tissue factor monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody is:
HCDR1, having the sequence set forth in SEQ ID NO: 1.
HCDR2 having the sequence set forth in SEQ ID NO: 2 and HCDR3 having the sequence set forth in SEQ ID NO: 3;
Heavy chain variable region including, and:
LCDR1 having the sequence shown in SEQ ID NO: 4
LCDR2 having the sequence set forth in SEQ ID NO: 5 and LCDR3 having the sequence set forth in SEQ ID NO: 6.
The antibody has the light chain variable region, which comprises:
TF-mAb-SC1, having the heavy chain variable region set forth in SEQ ID NO: 7 and the light chain variable region set forth in SEQ ID NO: 8.
TF-mAb-H29, having the heavy chain variable region set forth in SEQ ID NO: 9 and the light chain variable region set forth in SEQ ID NO: 14.
TF-mAb-H30 having the heavy chain variable region set forth in SEQ ID NO: 10 and the light chain variable region set forth in SEQ ID NO: 14.
TF-mAb-H31, having the heavy chain variable region set forth in SEQ ID NO: 11 and the light chain variable region set forth in SEQ ID NO: 14.
TF-mAb-H32 having the heavy chain variable region set forth in SEQ ID NO: 12 and the light chain variable region set forth in SEQ ID NO: 14.
TF-mAb-H33 having the heavy chain variable region set forth in SEQ ID NO: 13 and the light chain variable region set forth in SEQ ID NO: 14.
TF-mAb-H34, having the heavy chain variable region set forth in SEQ ID NO: 9 and the light chain variable region set forth in SEQ ID NO: 15.
TF-mAb-H35, having the heavy chain variable region set forth in SEQ ID NO: 10 and the light chain variable region set forth in SEQ ID NO: 15.
TF-mAb-H36 having the heavy chain variable region set forth in SEQ ID NO: 11 and the light chain variable region set forth in SEQ ID NO: 15.
TF-mAb-H37, having the heavy chain variable region set forth in SEQ ID NO: 12 and the light chain variable region set forth in SEQ ID NO: 15.
TF-mAb-H38, which has a heavy chain variable region set forth in SEQ ID NO: 13 and a light chain variable region set forth in SEQ ID NO: 15.
TF-mAb-H39, having the heavy chain variable region set forth in SEQ ID NO: 9 and the light chain variable region set forth in SEQ ID NO: 16.
TF-mAb-H40, having the heavy chain variable region set forth in SEQ ID NO: 10 and the light chain variable region set forth in SEQ ID NO: 16.
TF-mAb-H41, having the heavy chain variable region set forth in SEQ ID NO: 11 and the light chain variable region set forth in SEQ ID NO: 16.
TF-mAb-H42, having the heavy chain variable region set forth in SEQ ID NO: 12 and the light chain variable region set forth in SEQ ID NO: 16.
TF-mAb-H43, having the heavy chain variable region set forth in SEQ ID NO: 13 and the light chain variable region set forth in SEQ ID NO: 16.
TF-mAb-H44, having the heavy chain variable region set forth in SEQ ID NO: 9 and the light chain variable region set forth in SEQ ID NO: 17.
TF-mAb-H45, having the heavy chain variable region set forth in SEQ ID NO: 10 and the light chain variable region set forth in SEQ ID NO: 17.
TF-mAb-H46, having the heavy chain variable region set forth in SEQ ID NO: 11 and the light chain variable region set forth in SEQ ID NO: 17.
TF-mAb-H47 having the heavy chain variable region set forth in SEQ ID NO: 12 and the light chain variable region set forth in SEQ ID NO: 17, and
TF-mAb-H48 having the heavy chain variable region set forth in SEQ ID NO: 13 and the light chain variable region set forth in SEQ ID NO: 17.
An antibody or antigen-binding fragment thereof selected from the group consisting of . The antibody according to claim 1, wherein the EC50 of affinity for a human TF protein is 0.01 to 0.03 _nM . Use of the antibody according to any one of claims 1-2 for the manufacture of a pharmaceutical for the prevention and / or treatment of TF-related diseases. The TF-related diseases are as follows:
Tumor development, growth and / or metastasis;
Thrombosis-related diseases;
The use according to claim 3 , selected from the group consisting of inflammation; and metabolism-related diseases. The use according to claim 4 , wherein the tumor is a tumor with high TF-expression. An isolated nucleic acid comprising the nucleic acid encoding the heavy chain variable region of the anti-human tissue factor monoclonal antibody according to any one of claims 1 to 2 and the nucleic acid encoding the light chain variable region of the monoclonal antibody. Polynucleotide. A vector comprising the isolated polynucleotide according to claim 6. A genetically engineered host cell comprising the vector according to claim 7 . An immune cell expressing the antibody according to any one of claims 1 and 2 . A pharmaceutical composition comprising the antibody according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier. A method for determining the TF protein in a sample in vitro, including:
(1) The step of contacting the sample with the antibody according to any one of claims 1 and 2 in vitro; and (2) the step of determining whether or not an antigen-antibody complex is formed;
A method, wherein the formation of the complex indicates the presence of a TF protein in a sample. It is a method of preparing an antibody.
(A) The step of culturing the host cell according to claim 8 under appropriate conditions for expression; and (b) the step of separating the antibody from the culture;
How to include.

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