WO2017028823A1 - 靶向于组织因子的抗体、其制备方法和用途 - Google Patents

靶向于组织因子的抗体、其制备方法和用途 Download PDF

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WO2017028823A1
WO2017028823A1 PCT/CN2016/096291 CN2016096291W WO2017028823A1 WO 2017028823 A1 WO2017028823 A1 WO 2017028823A1 CN 2016096291 W CN2016096291 W CN 2016096291W WO 2017028823 A1 WO2017028823 A1 WO 2017028823A1
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antibody
variable region
light chain
heavy chain
chain variable
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French (fr)
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余科
张学赛
林卿
李晴柔
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复旦大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to the field of medicine, and in particular to antibodies, human preparation methods and uses thereof that target human tissue factor.
  • Tissue factor is a 47 kDa transmembrane glycoprotein. Under normal physiological conditions, TF expression is mainly shielded from the subendothelial cell layer. Once the body's blood vessels are traumatized, TF is exposed to the bloodstream, and the exogenous coagulation reaction is initiated by binding and activating factor VII.
  • TF is abnormally activated and expressed in many tumor tissues and plays an important role in the occurrence and development of tumors.
  • patients are mostly accompanied by spontaneous thrombosis, such as Deep-vein thrombosis (DVT), Disseminated intravascular coagulation (DIC) and Pulmonary embolism (PE).
  • DVD Deep-vein thrombosis
  • DIC Disseminated intravascular coagulation
  • PE Pulmonary embolism
  • TF/FVIIa complexes In addition to initiating the exogenous coagulation pathway, TF/FVIIa complexes directly bind to and induce activation of the transmembrane G protein-coupled receptor Protease-activated receptor 2 (PAR2).
  • PAR2 is an important signaling pathway regulating inflammatory response. Although there are few studies on PAR2 in the field of tumors, it is conceivable that TF can affect a series of tumor function signals in cells through PAR2.
  • TF-PAR2 phosphorylates MAPK/ERK and induces gene expression of key growth factors, immunoregulators, and chemokines (such as VEGF, CSF1/2, IL8, CXCL1, etc.) to promote neovascularization.
  • TF can also enhance the migration and adhesion of tumor cells through interaction with the Rac1 and ⁇ 1 family-associated integrins, thereby enhancing the ability of tumor cells to metastasize as a whole (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).
  • TF-induced hypercoagulable state directly contributes to the survival and hematogenous metastasis of tumor cells (Blood, 2008, 111: 190-9; Cancer Res., 2015, 75 (1 Suppl): Abstract nr B19), ie
  • the clotting reaction initiated by TF/FVIIa leads to the formation of thrombin and fibrin deposition, which not only causes tumor cells to escape immune attack, but also increases the interaction between tumor cells and endothelial cells, and helps the spread and penetration of tumor cells, which is beneficial to the spread and penetration of tumor cells.
  • the occurrence of hematogenous metastasis which is also an important reason for the current difficult treatment of cancer.
  • TF also plays a role in thrombotic diseases.
  • TF or MPTF Mesoparticle tissue factor
  • coagulation is also an important cause of Venous thromboembolism (VTE), and its content in blood is more than VTE.
  • Severity is directly proportional, and there are many studies showing that TF can be used as an important marker for clinical diagnosis and assessment of VTE patients and a potential target for VTE therapy (Thrombosis research, 2010, 125: 511-512; Lupus., 2010, 19: 370-378; Annual review of physiology, 2011, 73: 515-525).
  • TF also plays a role in inflammation and metabolic diseases. Studies have shown that inflammatory diseases are accompanied by abnormal angiogenesis and blood clotting. Studies by Maria I Bokarewa and others have shown that various inflammatory stimuli promote the expression of TF on the surface of endothelial cells and monocytes, and their results show that overexpression of TF is also a major factor in inducing and promoting inflammation (Arthritis Res 2002). 4:190-195).
  • Studies such as Leylla Badeanlou have shown that inhibition of TF-PAR2 signaling pathway by targeting specific antibodies to TF or knocking out TF can significantly inhibit Diet-induced obesity and adipose tissue inflammation can significantly improve the therapeutic effect of insulin on diabetes (Nature medicine, 2011, 17(11): 1490-1497).
  • the object of the present invention is to provide a TF antibody which has specific properties for targeting human TF, inhibits tumor growth and metastasis activity, and has anticoagulant and FXa-producing activity.
  • a heavy chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes at least one amino acid and is capable of retaining TF binding affinity.
  • a heavy chain of an antibody having the heavy chain variable region of the first aspect.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 7.
  • a light chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • Any one of the above amino acid sequences is subjected to addition, deletion, modification and/or substitution of at least one amino acid derivative sequence having TF binding affinity.
  • a light chain of an antibody having a light chain variable region of the third aspect.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 8.
  • an antibody having:
  • the antibody has: the heavy chain of the second aspect; and/or the light chain of the fourth aspect.
  • the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
  • the number of amino acids added, deleted, modified and/or substituted does not exceed 40% of the total amino acid number of the initial amino acid sequence.
  • the number of amino acids added, deleted, modified and/or substituted is from 1 to 7.
  • the at least one amino acid sequence added, deleted, modified and/or substituted is an amino acid sequence having a homology of at least 80%.
  • the addition, deletion, modification, and/or substitution of at least one amino acid has any one or more of an activity of inhibiting a TF-related signaling pathway, an anticoagulant activity, and an anti-FXa production activity.
  • an antibody according to the invention for (a) preparing a diagnostic agent; and/or (b) preparing a medicament for preventing and/or treating a TF-related disease .
  • the TF-related disease is selected from the group consisting of tumor development, growth and/or metastasis; thrombosis-related diseases; inflammation; metabolic-related diseases;
  • the tumor is a tumor with high expression of TF.
  • the high expression of TF refers to the ratio of the level L1 of the TF transcript and/or protein in the tumor tissue to the level L0 of the transcript and/or protein in the normal tissue, L1/L0 ⁇ 2, Good land ⁇ 3.
  • the tumor is selected from the group consisting of triple negative breast cancer, pancreatic cancer, lung cancer, and malignant glioma.
  • the drug is an antibody drug conjugate.
  • a recombinant protein having:
  • a polynucleotide which encodes a polypeptide selected from the group consisting of:
  • a vector comprising the polynucleotide of the eighth aspect is provided.
  • a genetically engineered host cell comprising the vector of the ninth aspect or the polynucleotide having the eighth aspect integrated in the genome is provided.
  • an antibody drug conjugate comprising:
  • an antibody moiety selected from the group consisting of a heavy chain variable region of the first aspect, a heavy chain of the second aspect, a light chain variable region of the third aspect, a light chain of the fourth aspect, Four aspects of antibodies, or a combination thereof;
  • a coupling moiety coupled to the antibody moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
  • an immune cell which expresses or is exposed to the antibody of the fifth aspect of the invention outside the cell membrane.
  • the immune cells include NK cells, T cells.
  • the immune cells are human.
  • the antibody is a single chain antibody.
  • a pharmaceutical composition comprising:
  • an active ingredient selected from the group consisting of a heavy chain variable region of the first aspect, a heavy chain of the second aspect, a light chain variable region of the third aspect, a light chain of the fourth aspect, or The antibody of the fourth aspect, the recombinant protein of the seventh aspect, the antibody drug conjugate of the eleventh aspect, the immune cell of the twelfth aspect, or a combination thereof;
  • an active ingredient selected from the group consisting of the heavy chain variable region of the first aspect, the heavy chain of the second aspect, and the light chain of the third aspect a variable region, a light chain of the fourth aspect, or an antibody of the fourth aspect, a recombinant protein of the seventh aspect, an antibody drug conjugate of the eleventh aspect, an immune cell of the twelfth aspect, or a combination thereof, wherein
  • the active ingredient is used to prepare a medicament, reagent, test plate or kit.
  • the reagent, test plate or kit is used to:
  • the agent is used for treating or preventing diseases such as tumors, thrombotic diseases, obesity, and diabetes which express TF protein.
  • a method of detecting a TF protein in a sample comprising the steps of:
  • test panel comprising: a substrate (support plate) and a test strip, the test strip comprising the antibody of the fifth aspect or the eleventh aspect Immunoconjugate.
  • a kit is provided, characterized in that the kit comprises:
  • a first container comprising the antibody of the present invention.
  • the kit contains the test plate of the sixteenth aspect.
  • a method for preparing a recombinant polypeptide comprising:
  • a method for treating a tumor, a thrombotic disease, an inflammatory disease, and/or a metabolic disease comprising: using (e.g., administering to a subject in need thereof) the antibody of the fifth aspect, An antibody-drug conjugate of the antibody, or a CAR-T cell expressing the antibody, or a combination thereof.
  • the metabolic diseases include: obesity, or diabetes.
  • an anti-TF antibody wherein the antibody has an EC 50 affinity for human TF protein of 0.005-0.10 nM, preferably 0.005-0.05 nM, more preferably 0.01-0.03 nM or 0.01-0.02 nM.
  • the antibody does not bind to a wild-type murine TF protein.
  • the antibody has one or more characteristics selected from the group consisting of:
  • the antibody is TF-mAb-SC1, TF-mAb-Ch or TF-mAb-H29 to TF-mAb-H48.
  • a method of preparing a humanized or chimeric antibody comprising the steps of:
  • a human-mouse chimeric antibody is expressed by transfecting an animal cell.
  • the humanized antibody is expressed by transfecting the animal cell.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • a method for inhibiting migration of tumor cells comprising the steps of: administering an antibody of the present invention, an antibody-drug conjugate of the antibody, or The CAR-T cells expressing the antibody.
  • Figure 1 shows the binding activity of a series of original anti-human TF monoclonal antibodies to human TF-positive (MDA-MB-231 and BxPC-3), murine TF-positive (B16-F10) cells (Cell The binding activity showed that TF-mAb-SC1 showed the best binding activity at a concentration of 10 ⁇ g/mL.
  • Figure 2 is a ELISA assay for Binding affinity of TF-mAb-SC1 to TF extracellular domain protein.
  • Figure 3 shows the inhibition of BxPC-3 intracellular signaling pathway TF-PAR2 by a series of original monoclonal antibodies by Western blot.
  • Figure 4 shows the results of single enzyme digestion and double enzyme digestion of human-mouse chimeric antibody (TF-mAb-Ch) expression plasmid.
  • Figure 4A shows the results of variable restriction enzyme digestion and double enzyme digestion of heavy chain plasmid.
  • Figure 4B shows the results of single-enzyme digestion and double-enzyme digestion of the variable region expressing the light chain plasmid.
  • Figure 5 is a result of Binding affinity of TF-mAb-SC1 to cell surface TF, wherein Figure 5A shows binding affinity to BxPC-3, Figure 5B shows binding affinity to MDA-MB-231, and Figure 5C shows binding U87MG binds affinity, and Figure 5D shows binding affinity for H1975.
  • Figure 6 shows the results of the effect of TF-mAb-SC1 on the intracellular signal transduction pathway TF-PAR2 of FVIIa-activated BxPC-3 cells.
  • Figure 7 shows the results of anti-clotting activity test of TF-mAb-SC1 with BxPC-3 (Fig. 7A) and MDA-MB-231 (Fig. 7B) cell surface TF as TF sources, respectively.
  • Figure 8 shows the results of TF-mAb-SC1 anti-FXa production activity, with BxPC-3 (8A) and MDA-MB-231 (8B) cell surface TF as TF sources, respectively.
  • Figure 9 shows the results of TF-mAb-SC1 inhibition of tumor growth of subcutaneous BxPC-3 in nude mice (arrows indicate the time of administration).
  • Figure 10 shows the results of TF-mAb-SC1 inhibition of tumor growth of subcutaneous U87MG in nude mice (arrows indicate the time of administration).
  • Figure 11 shows the results of TF-mAb-SC1 inhibition of tumor growth of subcutaneous HCC1806 in nude mice (arrows indicate the time of administration).
  • Figure 12 shows the results of TF-mAb-SC1 inhibition of BxPC-3 tumor matrix collagen accumulation and statistical analysis of Image-pro plus.
  • Figure 13 shows that TF-mAb-SC1 reduces the vascular lumen area of BxPC-3 tumors and its statistical results.
  • Figure 14 is a graph showing the migration levels of MDA-MB-231 (Figure 14A) and BxPC-3 ( Figure 14B) cells in the case of TF gene knockout.
  • Figure 15 is a graph showing the anti-tumor cell migration activity of TF-mAb-SC1, wherein Figure 15A is the inhibition of TF-mAb-SC1 Determination of migration level of MDA-MB-231 cells; Figure 15B is an assay for inhibition of migration level of BxPC-3 cells by TF-mAb-SC1;
  • Figure 16 shows the results of detection of hematogenous migration level and fluorescence intensity of MDA-MB-231-luc cells in mice after TF gene knockout.
  • Figure 17 shows the results of TF-mAb-SC1 inhibition of hematopoietic migration of MDA-MB-231-luc cells in mice and fluorescence intensity statistics.
  • Figure 18A shows the results of MDA-MB-231 tumor metastases on the lungs and their statistical analysis
  • Figure 18B shows the results of weight comparison of the lungs.
  • Figure 19 shows the results of laser confocal microscopy of TF-mAb-SC1 by intracellular endocytosis to lysosomes.
  • Figure 20 is a ELISA assay for the binding affinity of the chimeric antibody TF-mAb-Ch to the TF extracellular domain protein.
  • Figure 21 is a measurement of the binding affinity of TF-mAb-Ch to TF-positive tumor cell MDA-MB-231.
  • Figure 22 shows the results of the effect of TF-mAb-Ch on the intracellular signaling pathway TF-PAR2 of BxPC-3.
  • Figure 23 shows the detection of TF-mAb-Ch by cell internalization to lysosomes.
  • Figure 24 shows the results of detection of the effect of humanized antibody on BxPC-3 intracellular signaling pathway TF-PAR2.
  • Figure 25 is a graph showing the results of detection of humanized antibody inhibiting tumor growth of subcutaneous HCC1806 in nude mice (arrows indicate the time of administration).
  • Figure 26 is a comparison of the expression of TF protein in a highly invasive, highly metastatic Basal-type breast cancer (especially triple negative breast cancer) cell line and in a Luminal-type breast cancer cell line.
  • Figure 27 is a graph showing the analysis of TF mRNA in a highly invasive, highly metastatic Basal-type breast cancer (especially triple negative breast cancer) cell line and in a Luminal-type breast cancer cell line according to the Cancer Cell Line Encyclopedia (CCLE) database. Comparison of expression results.
  • Figure 28 shows the results of detection of TF protein expression in different pancreatic cancer cell lines.
  • Figure 29 is a graph showing the expression levels of TF mRNA in different pancreatic cancer cell lines according to the CCLE database.
  • the inventors have unexpectedly obtained an anti-TF monoclonal antibody TF-mAb-SC1 through extensive screening, and the experimental results show that the monoclonal antibody against the TF protein is an IgG2b type antibody.
  • the highly specific antibody capable of binding TF antigen with high affinity ELISA which measured about 0.019 nM EC 50
  • the antibodies have significant anti-tumor activity, but does not have visible mammals
  • the toxic side effects ELISA which measured about 0.019 nM EC 50
  • the chimeric antibody, the humanized antibody, and the corresponding ADC obtained based on the TF-mAb-SC1 also have excellent characteristics.
  • the present invention has been completed on this basis.
  • antibody or "immunoglobulin” is a heterotetramer of about 150,000 daltons having the same structural features.
  • a glycan protein consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds.
  • Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions.
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR).
  • the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • the "light chain" of a vertebrate antibody can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region.
  • Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • variable regions which are divided into four framework regions (FR), four
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and is not directly involved in the binding reaction.
  • CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard DNA recombination techniques, all of which are useful antibodies.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as a variable region having a monoclonal antibody from a murine, and a chimeric antibody from a constant region of a human immunoglobulin (see, e.g., U.S. Patent 4,816,567 and U.S. Patent No. 4,816,397, incorporated herein by reference in its entirety herein.
  • a humanized antibody refers to an antibody molecule derived from a non-human species having one or more complementarity determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see US patent) 5,585,089, herein incorporated by reference in its entirety.
  • CDRs complementarity determining regions
  • the antibody may be monospecific, bispecific, trispecific, or more multiple specificity.
  • the antibody of the present invention further includes a conservative variant thereof, which means that there are up to 10, preferably up to 8, more preferably up to 5, optimally compared to the amino acid sequence of the antibody of the present invention. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • the present invention provides a highly specific and high affinity antibody against TF comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) amino acid sequence, the light chain comprising a light chain variable Region (VL) amino acid sequence.
  • VH heavy chain variable region
  • VL light chain variable Region
  • the respective CDRs of the heavy chain variable region (VH) amino acid sequence and the light chain variable region (VL) amino acid sequence are selected from the group consisting of:
  • the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90. %, optimally at least 95% of the amino acid sequence.
  • the antibody has an activity of inhibiting a TF-related signaling pathway; has anticoagulant activity; has anti-FXa production activity, or a combination thereof.
  • the antibody of the present invention may be a double-stranded or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and more preferably a whole human. Sourced antibodies.
  • the antibody derivative of the present invention may be a single chain antibody, and/or an antibody fragment such as Fab, Fab', (Fab') 2 or other known antibody derivatives in the field, and IgA, IgD, IgE. Any one or more of IgG and IgM antibodies or antibodies of other subtypes.
  • the animal is preferably a mammal, such as a mouse.
  • the antibody of the invention may be a chimeric antibody, a humanized antibody, a CDR grafted and/or a modified antibody that targets human TF.
  • any one or more of the above SEQ ID No.: 1 - SEQ ID No.: 3, or they are added, deleted, modified and/or substituted for at least one amino acid A sequence having TF binding affinity, located in the CDR region of the heavy chain variable region (VH).
  • any one or more of the above SEQ ID No.: 4-SEQ ID No.: 6, or they are added, deleted, modified and/or substituted for at least one amino acid A sequence having TF binding affinity, located in the CDR region of the light chain variable region (VL).
  • VH CDR1, CDR2, CDR3 are each independently selected from any one or more of SEQ ID No.: 1 - SEQ ID No.: 3, or they are added, A sequence having TF binding affinity that lacks, modifies, and/or substituted at least one amino acid;
  • VL CDR1, CDR2, CDR3 are each independently selected from any one or more of SEQ ID No.: 4-SEQ ID No.: Or a sequence having TF binding affinity for addition, deletion, modification and/or substitution of at least one amino acid.
  • the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1-33% of the total amino acid number of the initial amino acid sequence. More preferably, it is 5-30%, more preferably 10-25%, and still more preferably 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted may be It is 1-7, more preferably 1-5, more preferably 1-3, still more preferably 1-2.
  • the antibody that targets TF is TF-mAb-SC1 (formerly known as TF-mAb).
  • the heavy chain variable region (VH) amino acid sequence of the antibody TF-mAb-SC1 is the amino acid sequence set forth in SEQ ID NO.: 7.
  • the light chain variable region (V-Kappa) amino acid sequence of the antibody TF-mAb-SC1 is the amino acid sequence set forth in SEQ ID NO.: 8.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as PCR amplification or genomic library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form a single chain antibody.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • 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.
  • the resulting host cells are cultured under conditions suitable for expression of the antibody of the invention.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antibodies of the present invention are purified by conventional separation and purification means well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • the binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al, Anal. Biochem., 107: 220 (1980).
  • the antibodies of the invention can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • ADC Antibody-drug conjugate
  • the invention also provides an antibody-drug conjugate (ADC) based on an antibody of the invention.
  • ADC antibody-drug conjugate
  • the antibody-conjugated drug comprises the antibody, and an effector molecule, which is coupled to the effector molecule, and is preferably chemically coupled.
  • the effector molecule is preferably a therapeutically active drug.
  • the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.
  • the antibody of the present invention and the effector molecule may be coupled by a coupling agent.
  • the coupling agent may be any one or a combination of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between an effector molecule and an antibody, such as glutaraldehyde or the like.
  • the coupling agent using a carboxyl group may be any one or more of an cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acyl hydrazine coupling agent (coupling site is an acylhydrazine).
  • Certain residues on the antibody are used to link to a variety of functional groups, including imaging agents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes). , stabilizers (such as ethylene glycol polymers) and therapeutic agents.
  • imaging agents such as chromophores and fluorophores
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers such as ethylene glycol polymers
  • therapeutic agents such as ethylene glycol polymers
  • the antibody can be conjugated to a functional agent to form a conjugate of the antibody-functional agent.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • the functional agent can be attached to the antibody either directly or indirectly via a linker.
  • Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC comprises a linker between the drug and the antibody.
  • the linker can be a degradable or non-degradable linker.
  • Degradable linkers are typically susceptible to degradation under the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (eg, lysosomal proteases or endosomal proteases), or sugar linkers, for example, which can be glucuronide Enzymatically degraded glucuronide-containing linker.
  • 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 (e.g., linkers that hydrolyze at pH less than 5.5, such as barium splices) and linkers that degrade under reducing conditions (e.g., disulfide bond linkers).
  • Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.
  • the linker Prior to attachment to an antibody, the linker has an reactive reactive group capable of reacting with certain amino acid residues, and attachment is achieved by reactive reactive groups.
  • Sulfhydryl-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (eg, iodine, bromine or chlorinated); haloesters (eg, iodine, bromine or chlorinated) Halogenated methyl ketone (eg iodine, bromine or chlorinated), benzyl halide (eg iodine, bromine or chlorinated); vinyl sulfone, pyridyl disulfide; mercury derivative such as 3,6- Di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • the linker can include, for example, a maleimide attached to the antibody via
  • the drug can be any cytotoxic, cytostatic or immunosuppressive drug.
  • the linker binds the antibody to the drug, and the drug has a functional group that can bond to the linker.
  • the drug may have an amino group, a carboxyl group, a thiol group, a hydroxyl group, or a ketone group which may be bonded to a linker.
  • the drug is directly attached to the linker, the drug has a reactive group that is reactive prior to attachment to the antibody.
  • Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors , vinca alkaloids, etc.
  • cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs including, for example, auristatin, camptothecin (camptothecins), docamycin/duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), taxanes ( Taxanes), benzodiazepines or benzodiazepine containing drugs (eg pyrrolo[1,4]benzodiazepines (PBDs), porphyrin benzodiazepines Classes (indolinobenzodiazepines) and oxazolidinobenzodiazepines and vinca alkaloids.
  • typical cytotoxic drugs including, for example, auristatin, camptothecin (camptothecins), docamycin/duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), tax
  • the drug-linker can be used to form an ADC in a simple step.
  • the bifunctional linker compound can be used to form an ADC in a two or more step process. For example, a cysteine residue is reacted with a reactive moiety of the linker in a first step, and in a subsequent step, a functional group on the linker reacts with the drug to form an ADC.
  • a functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety.
  • a portion based on an azide compound can be used to specifically react with a reactive alkynyl group on a drug moiety.
  • the drug is covalently bound to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for amines) And alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols).
  • the invention also provides a method of making an ADC, which can further comprise: binding the antibody to a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the invention comprise: binding an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further comprise: binding the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via a linker.
  • the antibody drug conjugate ADC is represented by the following formula:
  • Ab is an antibody
  • D is a drug
  • the antibodies of the invention or their ADCs can be used in detection applications, for example for detecting samples, to provide diagnostic information.
  • the sample (sample) used includes cells, tissue samples, and biopsy specimens.
  • biopsy shall include all types of biopsies known to those skilled in the art.
  • the biopsy used in the present invention may include, for example, a resected sample of a tumor, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the invention also provides a kit comprising an antibody (or a fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.
  • the antibody of the invention may be immobilized on a test plate.
  • the invention also provides the use of an antibody of the invention, for example for the preparation of a diagnostic preparation, or for the preparation of a medicament for the prevention and/or treatment of a TF-related disease.
  • TF-related diseases include tumorigenesis, growth and/or metastasis, thrombosis-related diseases, inflammation, metabolic-related diseases, and the like.
  • the tumor includes (but is not limited to): breast cancer (such as triple negative breast cancer), pancreatic cancer, lung cancer, glioblastoma, gastric cancer, liver cancer, esophageal cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer , endometrial cancer, ovarian cancer, cervical cancer, leukemia, bone marrow cancer, angiosarcoma, etc.; especially triple-negative breast cancer, pancreatic cancer, malignant glioma and lung cancer, more preferably triple-negative breast cancer and/or pancreatic cancer .
  • breast cancer such as triple negative breast cancer
  • pancreatic cancer lung cancer
  • glioblastoma gastric cancer
  • liver cancer liver cancer
  • esophageal cancer kidney cancer
  • colorectal cancer bladder cancer
  • prostate cancer endometrial cancer
  • ovarian cancer cervical cancer
  • leukemia bone marrow cancer
  • angiosarcoma etc.
  • triple-negative breast cancer pancreatic cancer
  • thrombosis-related diseases include, but are not limited to, atherosclerosis, acute coronary syndrome, acute myocardial infarction, stroke, hypertension, deep vein thrombosis, pulmonary embolism, renal embolism and arterial surgery, adjacent to the coronary artery Thrombosis caused by road transplantation.
  • the inflammation includes (but is not limited to): rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Lytle syndrome, psoriasis arthropathy, infectious arthritis, tuberculous arthritis, viral joints Inflammation, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis.
  • the metabolic related diseases include, but are not limited to, diabetes, foodborne obesity, and fat inflammation.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition comprising the above An antibody or active fragment thereof or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the antibody of the present invention may also be a cell therapy for expression of a nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention can be directly used for binding to a TF protein molecule, and thus can be used for the prevention and treatment of diseases such as tumors.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-mentioned monoclonal antibody (or a conjugate thereof) of the present invention and pharmacy An acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the antibody of the present invention has excellent biological activity and specificity and has a high affinity (the EC 50 can be as high as about 0.01 to 0.03 nM by ELISA). In addition, it has a good binding affinity to cell surface TF and can be used as an antibody targeting TF.
  • the humanized antibody of the present invention has not only activity comparable to that of a murine antibody but also lower immunogenicity.
  • the antibody and ADC of the present invention not only have significant therapeutic effects in a plurality of tumor models, but are also applicable to other diseases associated with high expression of TF, such as thrombotic diseases, metabolic diseases and the like.
  • Step 1 preparation of hybridoma cells:
  • TF extracellular domain protein was immunized into Balb/c mice, and the amount of TF extracellular domain protein was 100 ⁇ g/head.
  • the spleen cells and SP2/0 cells were fused by PEG-mediated fusion, PEG was removed, resuspended in HAT complete medium containing feeder cells, inoculated into 96-well plates, and cultured by ELISA. Hole screening. Finally, the cells of the positive wells were cloned and cultured by limiting dilution method. The cells with high titer, good morphology and monoclonal growth were screened by ELSIA or immunofluorescence to continue subcloning screening until the positive clones were screened for three consecutive times. At 100%, the cell strain can be expanded and constructed.
  • Step 2 Preparation of human TF murine monoclonal antibody ascites:
  • the hybridoma cells screened in step 1 were expanded and cultured, and the mice were intraperitoneally injected with pristane (0.5 mL/mouse) to provide a favorable environment for the growth of hybridoma cells. After 7-10 days, each intraperitoneal injection was 10 ⁇ . From the 7th day, the hybridoma cells of 10 6 were observed daily for the ascites production and mental state of the mice, and ascites was taken and centrifuged to remove the oil at -80 ° C for purification.
  • pristane 0.5 mL/mouse
  • step 2 The ascites frozen in step 2 was thawed on ice, filtered through a 0.45 ⁇ m filter, dialyzed against PBS overnight at 4 ° C, and finally purified by FPLC technique and concentrated to the desired concentration by ultrafiltration, and dispensed, Store at -80 °C for later use.
  • Step 4 Determination of the biological activity and targeting specificity of a monoclonal antibody directed against human TF mice:
  • the results are shown in Figure 1.
  • the antibodies tested can specifically bind to human TF (MDA-MB-231 and BxPC-3 cells) without targeting murine TF (B16-F10 cells), of which TF- mAb-SC1 has higher affinity for human TF than the other 5 antibodies.
  • the antigen (TF extracellular domain protein) was diluted to 0.5 ⁇ g/mL with a coating solution, coated with an ELISA plate, 100 ⁇ L/well, 4 ° C, overnight. Wash off the excess antigen, block with 2% BSA for 2h at room temperature, then add 3 times serial dilution of each monoclonal antibody, 100 ⁇ L/well, incubate for 2h at room temperature; wash away unbound antibody, add appropriate concentration of horseradish peroxidase Labeled anti-mouse secondary antibody, 100 ⁇ L/well, incubated for 1 h at room temperature. The unbound secondary antibody was washed away, TMB coloring solution was added, and the color was developed to the appropriate color depth.
  • TF-mAb-SC1 has a strong affinity for the TF extracellular domain, EC 50 of about 0.019nM.
  • pancreatic cancer cells BxPC-3 were plated in 12-well plates. After 12 hours, the cells were washed 3 times with sterile PBS, then added to serum-free medium at 37 ° C, 5% CO 2 incubator starved for 4 h. Subsequently, each monoclonal antibody was diluted in a 3-fold gradient and incubated with BxPC-3 in an incubator for 1 h, followed by activation of BxPC-3 intracellular PAR2 signaling pathway with 25 nM FVIIa, and after pre-cooled PBS for 15 min at 37 °C.
  • the cells were washed once and the cell proteins were collected on ice and the effect of TF-mAb-SC1 on the phosphorylation level of downstream MAPK/ERK was identified by Western blot. Only FVIIa stimulation was performed, and cells not incubated with monoclonal antibody were used as positive controls. The effect of these six antibodies on the intracellular signaling pathway TF-PAR2 of BxPC-3 was determined by Western blot.
  • TF-mAb-SC1 Since TF-mAb-SC1 exhibits very high specificity, very high affinity, and significant inhibition of the phosphorylation level of MAPK/ERK, it was selected for sequencing and subsequent studies.
  • the CDR amino acid sequence of the heavy chain variable region is:
  • SEQ ID No.: 1 SYWMN;
  • SEQ ID No.: 2 MIYPADSETRLNQKFKD;
  • SEQ ID No.: 3 EDYGSSDY.
  • VH amino acid sequence is set forth in SEQ ID NO.: 7.
  • the CDR amino acid sequence of the light chain variable region is:
  • SEQ ID No.: 4 SASSSVSYMN;
  • SEQ ID No.: 5 GISNLAS
  • SEQ ID No.: 6 QQKSSFPWT.
  • VL amino acid sequence is shown in SEQ ID NO.: 8:
  • a human-mouse chimeric antibody was constructed based on the obtained highly active and specific murine TF-mAb-SC1.
  • the CDR amino acid sequence of the heavy chain variable region was determined by correlation database analysis:
  • SEQ ID No.: 18 MIYPXDSETRLNXKFKD (X is selected from any one of A, D, E, Q, and Y)
  • SEQ ID No.: 19 GYSFXSYWMN (X is selected from any one of A, I, Y, Q, and W)
  • SEQ ID No.: 20 AREDYGXSDY (X is selected from any one of S, P, G, D, M, and N).
  • the relevant CDR analysis determines the CDR amino acid sequence of the light chain variable region as:
  • SEQ ID No.: 21 QQXSSFXWT (X is selected from any one of S, P, K, G, and H);
  • SEQ ID No.: 22 SASSXVSYMN (X is selected from any one of A, P, D, S);
  • SEQ ID No.: 23 GXSNLAS (X is selected from any one of P, D, I, and S).
  • Primers were designed to introduce EcoR I and Nhe I in the heavy chain variable region, and to introduce the Age I and BsiW I restriction endonuclease sites in the light chain variable region, and then the above-mentioned antibody heavy and light chains were obtained.
  • the variable region sequences were cloned into the vector containing the human IgG1 heavy chain constant region and the Kappa chain constant region, respectively, after identification (Fig. 4A shows the results of heavy chain digestion, and Fig.
  • sample 4B shows the results of light chain digestion, wherein sample 1 is Corresponding heavy/light chain blank vector, sample 2 is a vector cloned into the heavy chain/light chain variable region, 3 and 4 are single-cut samples, and 5 are double-digested samples, using transfection
  • the chimeric antibody constructed by the technique and mammalian expression system (CHO-S or HEK-293 cells) was expressed and purified, and the obtained human-mouse chimeric antibody was designated as TF-mAb-Ch.
  • TF-mAb-SC1 diluted from 333.33 nM to 0.15 nM as a primary antibody, mixed with 3 ⁇ 10 5 MDA-MB-231 or BxPC-3 suspended in 100 ⁇ L of RPMI-1640 serum-free medium, respectively, or 100 ⁇ L of TF-mAb-SC1 diluted from 66.67 nM to 0.03 nM in a 3-fold gradient as a primary antibody mixed with 3 ⁇ 10 5 U87MG suspended in 100 ⁇ L of MEM serum-free medium, or 100 ⁇ L of 33.33 nM and 3.33 nM TF-mAb-SC1 was mixed as a primary antibody with 3 ⁇ 10 5 H1975 suspended in 100 ⁇ L of RPMI-1640 serum-free medium, then incubated at 4 ° C for 1 h, and the cells were washed twice with PBS to remove unbound primary antibody.
  • the target cells were incubated with 200 ⁇ L, 2 ⁇ g/mL, PE-labeled secondary antibody at 4 ° C for 30 min, and the cells were washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 400 ⁇ L of PBS and flow cytometry. Binding affinity of TF-mAb-SC1 to the corresponding cell surface TF was determined.
  • TF-mAb-SC1 has good binding affinity to BxPC-3, MDA-MB-231 and U87MG with EC 50 of 2.6 nM (Fig. 5A), 2.5 nM (Fig. 5B) and 1.6, respectively.
  • nM Fig. 5C
  • Fig. 5D shows that TF-mAb-SC1 also has a good binding affinity for H1975.
  • BxPC-3 3 ⁇ 10 5 pancreatic cancer cells
  • BxPC-3 were plated in 12-well plates. After 12 hours, the cells were washed 3 times with sterile PBS, then added to serum-free medium at 37 ° C in a 5% CO 2 incubator for 4 h, followed by TF-mAb-SC1 was diluted from 100 nM to 1.2 nM in a 3-fold gradient and incubated with BxPC-3 in an incubator for 1 h, followed by activation of BxPC-3 intracellular PAR2 signaling pathway with 25 nM FVIIa, and after 15 min at 37 °C, The cells were washed once with pre-cooled PBS, and the cell proteins were collected on ice and the effect of TF-mAb-SC1 on the phosphorylation level of downstream MAPK/ERK was identified by Western blot. Only FVIIa stimulation was performed and not incubated with TF-mAb-SC1. The cells were used as positive controls.
  • TF-mAb-SC1 significantly inhibited the phosphorylation level of downstream MAPK/ERK in a dose-dependent manner.
  • TF-mAb-SC1 100 nM of TF-mAb-SC1 was diluted to 1.5625 nM (final volume 50 ⁇ L) and separately with 3 ⁇ 10 4 MDA suspended in 50 ⁇ L of Hanks Balanced Salt Solution (HBSS) containing 5 mM CaCl 2 -MB-231 and BxPC-3, incubate for 15 min at room temperature, then add 50 ⁇ L of citrate human plasma, mix rapidly, and measure the absorbance at 405 nm every 15 sec over the next 2 h to calculate the TF on the cell surface.
  • HBSS Hanks Balanced Salt Solution
  • Fig. 7 the ordinate represents the time of the coagulation rate
  • the abscissa represents the concentration of TF-mAb-SC1
  • the cell surface TF of BxPC-3 Fig. 7A
  • MDA-MB-231 Fig. 7B
  • the experimental results show that the concentration of TF-mAb-SC1 ⁇ 12.5 nM has significant anticoagulant activity.
  • TF-mAb-SC1 100 nM TF-mAb-SC1 was diluted to 1.5625 nM (final volume 50 ⁇ L) and separately with 1.5 ⁇ 10 4 BxPC-3 and MDA-MB-231 cells suspended in 50 ⁇ L HBSS (containing 3 nM FVIIa) at room temperature. Incubate for 20 min with shaking, then add 50 ⁇ L FX (final concentration 50 nM) to start the reaction. After 5 min, add 25 ⁇ L of 1 M EDTA to stop the reaction; then add 25 ⁇ L of 3 mM S2765, mix quickly and measure the kinetics every 15 s for the next 60 min. The reaction curve was studied to calculate the activity against FXa production.
  • TF-mAb-SC1 showed better anti-FXa production activity with IC 50 of 9.0 nM (Fig. 8A) and 6.4 nM (Fig. 8B), respectively.
  • Nude mice were randomly divided into two groups, 10 in each group. Tumor cells (1 ⁇ 10 7 BxPC-3 or 5 ⁇ 10 6 U87MG or 2.5 ⁇ 10 6 HCC1806) and 20 mg/kg dose of TF- were firstly used. mAb-SC1 was mixed and incubated for 30 min at room temperature, co-inoculated into the back or breast pad of immunodeficient mice (Balb/c nude mice) to observe the inhibition of BxPC-3 subcutaneous tumor growth, and the other group used normal small Murine IgG (abbreviated as murine IgG) served as a control. Body weight and tumor size of nude mice were measured periodically, tumor growth curves were drawn, and activity was assessed.
  • Tumor cells 1 ⁇ 10 7 BxPC-3 or 5 ⁇ 10 6 U87MG or 2.5 ⁇ 10 6 HCC1806) and 20 mg/kg dose of TF- were firstly used.
  • mAb-SC1 was mixed and incubated for 30 min at room temperature, co-inoculated into the back or breast pad of immunode
  • TF-mAb-SC1 inhibited the growth of BxPC-3 subcutaneous tumors more significantly than the mouse lgG, and the inhibition rate was up to 80%.
  • TF-mAb-SC1 inhibited the growth of U87MG subcutaneous tumors more significantly, with an inhibition rate of up to 60%.
  • TF-mAb-SC1 significantly inhibited HCC1806 subcutaneous tumor growth compared to murine lgG, with inhibition rates up to >90%.
  • Example 8 TF-mAb-SC1 significantly inhibits tumor matrix collagen accumulation
  • BxPC-3 xenografts from Example 7 were harvested, 4% neutral formaldehyde fixed, paraffin embedded sections, routinely dewaxed to water and subjected to Masson staining. For each sample stained at 100 ⁇ magnification (100 ⁇ m in the legend), 5 to 10 fields of view were collected and statistically analyzed.
  • TF-mAb-SC1 significantly inhibited the accumulation of tumor matrix collagen (blue region), resulting in tumor growth inhibition, and the right panel shows the results of statistical analysis using Image-pro plus.
  • Example 9 TF-mAb-SC1 significantly reduces tumor vascular lumen area
  • the BxPC-3 xenografts from Example 7 were collected, fixed in 4% neutral formaldehyde, embedded in paraffin sections, routinely dewaxed to water and subjected to CD31 immunohistochemical staining. For each sample stained by immunohistochemistry, at a magnification of 200 ⁇ (50 ⁇ m in the legend), 5 to 10 fields of view were collected and statistically analyzed.
  • TF-mAb-SC1 can significantly reduce the tumor area of the tumor, and the right picture shows the statistical results of the vascular lumen area.
  • TF-mAb-SC1 100 nM, 33.3 nM and 11.1 nM
  • Mouse IgG mouse IgG
  • 600 ⁇ L of complete medium containing 10% FBS was added to the lower layer of the chamber at 37 ° C, 5%.
  • the cells were cultured in a CO 2 incubator. After 8 hours, the cells on the upper surface of the chamber membrane were wiped with a wet cotton swab.
  • TF-mAb-SC1 The cells on the surface of the membrane were fixed with 95% ethanol for 30 min, then stained in 0.2% crystal violet for 30 min, and distilled water was washed away to remove excess crystal violet. After drying at room temperature, five representative fields of view were randomly selected under a microscope, and the number of cells migrating to the lower surface of the small chamber was statistically analyzed.
  • the anti-tumor cell migration activity of TF-mAb-SC1 was further confirmed by the TF gene knockout cells (sh-TF) and the corresponding vector control cells (sh-NT).
  • TF-mAb-SC1 significantly inhibited the migration levels of MDA-MB-231 (Fig. 15A) and BxPC-3 (Fig. 15B) cells in a concentration-dependent manner.
  • Example 11 TF-mAb-SC1 significantly inhibits in vivo hematogenous metastasis of tumor cells
  • TF-mAb-SC1 In vivo evaluation of the effect of TF-mAb-SC1 on tumor cell migration levels using an experimental hematogenous metastasis model: 2 x 106 luciferase-labeled MDA-MB-231 cells (MDA-MB-231-luc) and 0.1 mg
  • the TF-mAb-SC1 or IgG was mixed in 200 ⁇ L PBS, incubated on ice for 20 min, and slowly injected into the female nude mice of 6-8 weeks old by tail vein; after 4 h, the nude mice were anesthetized with fluorescein potassium salt PBS.
  • the solution was intraperitoneally injected into nude mice at a dose of 150 mg/kg. After 6 minutes, it was exposed to a small animal live imager (IVIS SPECTRUM) for 1 min, and the fluorescence intensity was measured.
  • IVIS SPECTRUM small animal live imager
  • MDA-MB-231 cells 3 ⁇ 10 6 MDA-MB-231 cells were mixed with 0.1 mg of TF-mAb-SC1 or IgG in 200 ⁇ L PBS, incubated on ice for 20 min, and slowly injected into the 6-week-old female SCID Beige mice through the tail vein. In vivo, after 6 weeks, the mice were sacrificed, and the lungs were removed by Bouin's solution, photographed and weighed, and the number of metastases on each lung was recorded.
  • TF-mAb-SC1 significantly inhibited the formation of tumor metastases on the lungs of mice.
  • Example 12 TF-mAb-SC1 can be rapidly and efficiently internalized by cells to lysosomes
  • 50% density MDA-MB-231 cells were plated in a laser confocal culture dish. After about 16 h, 10 ⁇ g/mL TF-mAb-SC1 was added and incubated at 37 ° C or 4 ° C for 1 h, and washed three times with pre-warmed PBS. The antibody not bound to the cells was removed, and then fixed with 4% paraformaldehyde for 30 min at room temperature.
  • Lamp-2 (rabbit anti-human) antibody was incubated at 37 ° C for 1 h to label the position of the cell lysosome, wash the unbound antibody with PBS, and incubate Alexa Fluor 594-labeled scorpion anti-mouse and Alexa Fluor at 37 °C. 488-labeled donkey anti-rabbit secondary antibody for 30 min. The unbound antibody was washed away, stained with DAPI to label the nuclear position, and then the endocytosis of the antibody was observed by laser confocal microscopy.
  • TF-mAb-SC1 can be endocytosed to lysosomes by cells.
  • Example 1 The experimental method is referred to Example 1, Step 4.
  • TF-mAb-Ch had a strong affinity for the TF extracellular domain protein, and the EC 50 was about 0.011 nM.
  • TF-mAb-Ch had a good binding affinity to MDA-MB-231 cells with an EC 50 of 2.3 nM (Fig. 21).
  • Example 4 The experimental method is referred to in Example 4.
  • Example 16 TF-mAb-Ch can be rapidly and efficiently internalized by cells to lysosomes
  • Example 12 The experimental method is referred to in Example 12.
  • TF-mAb-Ch can be endocytosed to lysosomes by cells.
  • TF-mAb antibody of the present invention is easily endocytosed, it is suitable for development as an antibody-drug conjugate (ADC) and is applied to the treatment of a tumor associated with high expression of TF.
  • ADC antibody-drug conjugate
  • the CDR region of the murine antibody TF-mAb-SC1 is then transplanted onto the selected humanized template, the CDR region of the human template is replaced, and then recombined with the IgG1 constant region, and based on the three-dimensional structure of the murine antibody, Residues with embedded residues, direct interactions with CDR regions, and residues that have important effects on the conformation of VL and VH are subjected to back mutation to obtain variable regions of 5 humanized heavy chains (SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13) and the variable region of the 4 humanized light chain (SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17).
  • the binding affinities of the 20 humanized antibodies to MDA-MB-231 cells at 10 ⁇ g/mL and 1 ⁇ g/mL were measured by flow cytometry.
  • the experimental results are shown in Table 3.
  • the results are shown in Table 2.
  • Example 18 TF is highly abnormally activated in triple negative mammary glands
  • the total cell protein was prepared from a variety of tumor cell lines with different tissue sources. After accurate quantification, the expression level of TF protein was detected by Western blot.
  • TF protein was highly aberrantly activated in some highly invasive, high-metastatic triple-negative breast cancer (Fig. 26), but only a few highly expressed in non-triple negative breast cancer cell lines with relatively low malignancy. .
  • the expression level of TF mRNA of breast cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) database was analyzed.
  • the results showed that the expression level of TF mRNA in the high-invasive and high-metastatic Basal-type breast cancer (especially triple-negative breast cancer) cell population was generally higher than that of the Luminal-type breast cancer cell line (Fig. 27). ). Therefore, the antibody targeting TF of the present invention has a more remarkable effect in the diagnosis, prevention and treatment of triple negative breast cancer.
  • Example 19 TF is highly abnormally activated in pancreatic cancer
  • the total cell protein was prepared from a variety of tumor cell lines with different tissue sources. After accurate quantification, the expression level of TF protein was detected by Western blot. The results showed that TF protein was highly aberrantly activated in high-invasive, highly metastatic pancreatic cancer (Fig. 28).
  • the expression level of TF mRNA in pancreatic cancer cell lines in the CCLE database was analyzed, and the results showed that TF mRNA was generally expressed at a high level in pancreatic cancer cell lines (Fig. 29). Therefore, the antibody targeting TF of the present invention has a more remarkable effect in the application for diagnosing, preventing and treating pancreatic cancer.

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Abstract

本发明提供一种新的组织因子单克隆抗体、其制备方法和用途。

Description

靶向于组织因子的抗体、其制备方法和用途 技术领域
本发明涉及医药领域，具体地涉及靶向于人组织因子的抗体、其制备方法和用途。
背景技术
组织因子(Tissue factor，TF)是一个47kDa的跨膜糖蛋白。正常生理状态下TF表达主要屏蔽于血管内皮下细胞层，一旦机体血管受到创伤，TF暴露于血流，通过结合并激活VII因子从而启动外源性凝血反应。
研究发现，TF在众多肿瘤组织中异常激活表达，在肿瘤的发生和发展过程中起着重要作用。特别是在癌症晚期，病人大多伴随自发性血栓，如深度静脉血栓(Deep-vein thrombosis，DVT)、弥漫性血管内凝血(Disseminated intravascular coagulation，DIC)和肺栓塞(Pulmonary embolism，PE)等(Thrombosis research,2013,131:S59-S62；Journal of Thrombosis and Haemostasis,2011,9(s1):306-315)；而TF在肿瘤细胞中的异常表达则是这些症状发生的主要诱因。对众多肿瘤临床样本分析表明，TF的表达水平直接影响肿瘤的转移、病人血栓的发生等恶化指标，如在乳腺癌中TF异常表达率为85.8％，在胰腺癌中为88.5％，在肺癌中为83.6％，食道癌中为91.3％等(Blood,2012,119:924-932)。
TF除了起始外源性凝血途径，TF/FVIIa复合物还能直接结合和诱导跨膜G蛋白偶联受体Protease-activated receptor 2(PAR2)的活化。PAR2是调控炎症反应的重要信号通路，虽然对PAR2在肿瘤领域的研究目前还比较少，但可以想象，TF通过PAR2能影响细胞内一系列肿瘤功能信号。概而言之，TF-PAR2通过MAPK/ERK磷酸化，诱导关键生长因子、免疫调节因子和趋化因子的基因表达(如VEGF、CSF1/2、IL8、CXCL1等)，促进新生血管的形成，为肿瘤的生长提供了充足的养分、能量和适宜的微环境。此外，TF还可以通过与Rac1、β1家族相关整合素的相互作用，以提高肿瘤细胞的迁移性和粘附性，从而在整体上增强肿瘤细胞的血行性转移能力(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)。
同时，TF-诱导的高凝状态又直接有助于肿瘤细胞的生存和血行性转移(Blood,2008,111:190-9；Cancer Res.,2015,75(1Suppl):Abstract nr B19)，即TF/FVIIa起始的凝血反应，导致凝血酶的生成、纤维蛋白的沉积，这不仅使肿瘤细胞逃逸免疫攻击，还增加了肿瘤细胞与内皮细胞的相互作用，帮助肿瘤细胞的扩散和渗透，利于血行性转移的发生，这也正是当前癌症难治疗的重要原因。
研究表明，TF在血栓类疾病中也起作用。除了在肿瘤发生、发展中的作用，TF或MPTF(Microparticle tissue factor)起始的凝血作用也是引发静脉血栓栓塞症(Venous thromboembolism,VTE)的重要原因，其在血液中的含量更是与VTE的严重程度成正比，目 前更是有很多研究表明TF可以作为VTE病人临床诊断、评估病情的重要标志物和VTE治疗的潜在靶点(Thrombosis research,2010,125:511-512；Lupus.,2010,19:370-378；Annual review of physiology,2011,73:515-525)。同样，TF在动脉血栓类疾病中的作用也不容忽视，众多临床数据表明TF在动脉粥样硬化的形成和发展过程中起重要作用，2009年，Steppich B A，Braun S L等对174位不稳定型心绞痛病人(unstable Angina pectoris，uAP)和112位急性心肌梗塞(Acute myocardial infarction，AMI)进行研究，结果表明血浆中TF的活性直接影响心血管病病人的死亡率，而且TF可以作为心血管类疾病的诊断和预后判断的标志物(Thrombosis research,2012,129:279-284；Thromb J.,2009,7(11):1-9)；2014年，Jiang P,Xue D等通过光化学法致血栓模型和FeCl3致血栓模型研究表明，与內源凝血途径相比，TF起始的外源凝血途径在动脉血栓类疾病的发生、发展中起更重要的作用，而且实验结果更证明TF起始的外源凝血途径可以作为动脉血栓类疾病治疗的靶标(Thrombosis research,2014,133(4):657-666)。
TF在炎症及代谢疾病中也起作用。研究表明，炎症性疾病的发生都伴随有异常的血管生成和凝血。Maria I Bokarewa等研究表明，多种炎症刺激物都会促进TF在内皮细胞和单核细胞表面的表达，而且他们的实验结果显示TF的过表达也是诱导和促进炎症发生的一个主要因子(Arthritis Res 2002,4:190-195)。
同样，研究表明TF在肥胖和糖尿病的治疗中起显著的调控作用，如Leylla Badeanlou等研究表明，通过靶向TF的特异性抗体或敲除TF以阻断TF-PAR2信号通路，可明显的抑制饮食诱导的肥胖性疾病及脂肪组织炎症的发生并可以显著改善胰岛素对糖尿病的治疗效果(Nature medicine,2011,17(11):1490-1497)。
因此，鉴于TF在各类相关疾病中作用和功能，开发靶向TF的特异性治疗抗体，对TF在癌症、血栓、炎症等各类疾病中引起的血管增生、异常凝血等所导致的病理特征的诊断、治疗和预防是极其有益的。
发明内容
本发明目的就是提供了一种TF抗体，它具有特异性靶向于人TF、具有抑制肿瘤生长和转移活性，并具有抗凝血和抑制FXa生成活性等特性。
在本发明第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO:1所示的CDR1，
SEQ ID NO:2所示的CDR2，和
SEQ ID NO:3所示的CDR3；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TF结合亲和力的衍生序列。
在本发明的第二方面，提供了一种抗体的重链，所述的重链具有第一方面的重链可变区。
在另一优选例中，所述重链可变区具有SEQ ID NO:7所示的氨基酸序列。
在本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO:4所示的CDR1'，
SEQ ID NO:5所示的CDR2'，和
SEQ ID NO:6所示的CDR3'；
上述氨基酸序列中任意一种氨基酸序列经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的衍生序列。
在本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有第三方面的轻链可变区。
在另一优选例中，所述轻链可变区具有SEQ ID NO:8所示的氨基酸序列。
在本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)第一方面的重链可变区；和/或
(2)第三方面的轻链可变区；
或者，所述抗体具有：第二方面的重链；和/或第四方面的轻链。
在另一优选例中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量，不超过初始氨基酸序列总氨基酸数量的40％。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-7个。
在另一优选例中，所述经过添加、缺失、修饰和/或取代的至少一个氨基酸序列为同源性为至少80％的氨基酸序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸具有抑制TF相关信号通路的活性、抗凝血活性、抗FXa生成活性中的任意一种或几种。
在本发明的第六方面，提供了一种本发明所述抗体的应用，所述抗体用于(a)制备诊断试剂；和/或(b)制备预防和/或治疗TF相关的疾病的药物。
在另一优选例中，所述TF相关的疾病选自下组：肿瘤的发生、生长和/或转移；血栓类相关疾病；炎症；代谢相关疾病；或其组合。
在另一优选例中，所述肿瘤为TF高表达的肿瘤。
在另一优选例中，所述的TF高表达指肿瘤组织中TF转录本和/或蛋白的水平L1与正常组织中转录本和/或蛋白的水平L0之比，L1/L0≥2，较佳地≥3。
在另一优选例中，所述的肿瘤选自下组：三阴性乳腺癌、胰腺癌、肺癌和恶性胶质瘤。
在另一优选例中，所述药物为抗体药物偶联物。
在本发明的第七方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)第一方面的重链可变区、第二方面的重链、第三方面的轻链可变区、第四方面的轻链、或第四方面的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在本发明的第八方面，提供了一种多核苷酸，它编码选自下组的多肽：
(1)第一方面的重链可变区、第二方面的重链、第三方面的轻链可变区、第四方面的轻链、或第四方面的抗体；或
(2)第七方面的重组蛋白。
在本发明的第九方面，提供了一种载体，它含有第八方面的多核苷酸。
在本发明的第十方面，提供了一种遗传工程化的宿主细胞，它含有第九方面的载体或基因组中整合有第八方面的多核苷酸。
在本发明的第十一方面，提供了一种抗体药物偶联物，该抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：第一方面的重链可变区、第二方面的重链、第三方面的轻链可变区、第四方面的轻链、第四方面的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
在本发明的第十二方面，提供了一种免疫细胞，所述免疫细胞表达或在细胞膜外暴露有本发明第五方面的抗体。
在另一优选例中，所述的免疫细胞包括NK细胞、T细胞。
在另一优选例中，所述的免疫细胞为人的。
在另一优选例中，所述的抗体为单链抗体。
在本发明的第十三方面，提供了一种药物组合物，它含有：
(i)活性成分，所述活性成分选自下组：第一方面的重链可变区、第二方面的重链、第三方面的轻链可变区、第四方面的轻链、或第四方面的抗体、第七方面的重组蛋白、第十一方面的抗体药物偶联物、第十二方面的免疫细胞、或其组合；以及
(ii)药学上可接受的载体。
在本发明的第十四方面，提供了一种活性成分的用途，所述活性成分选自下组：第一方面的重链可变区、第二方面的重链、第三方面的轻链可变区、第四方面的轻链、或第四方面的抗体、第七方面的重组蛋白、第十一方面的抗体药物偶联物、第十二方面的免疫细胞、或其组合，其中所述活性成分被用于制备药剂、试剂、检测板或试剂盒。
在另一优选例中，所述试剂、检测板或试剂盒用于：
(1)检测样品中TF蛋白；和/或
(2)检测肿瘤细胞中内源性的TF蛋白；和/或
(3)检测表达TF蛋白的肿瘤细胞；
而所述药剂用于治疗或预防表达TF蛋白的肿瘤、血栓类疾病、肥胖及糖尿病等疾病。
在本发明的第十五方面，提供了一种体外检测(包括诊断性或非诊断性)样品中TF蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与本发明抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在TF蛋白。
在本发明的第十六方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有第五方面的抗体或第十一方面的免疫偶联物。
在本发明的第十七方面，提供了一种试剂盒，其特征在于，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有本发明的抗体；和/或
(2)第二容器，所述第二容器中含有抗本发明抗体的二抗；
或者，
所述试剂盒含有第十六方面的检测板。
在本发明的第十八方面，提供了一种重组多肽的制备方法，该方法包括：
(a)在适合表达的条件下，培养第十方面的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是第五方面的抗体或第七方面的重组蛋白。
在本发明的第十九方面，提供了一种治疗肿瘤、血栓类疾病、炎症性疾病和/或代谢类疾病的方法，包括：使用(如给需要的对象施用)第五方面的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
在另一优选例中，所述的代谢类疾病包括：肥胖、或糖尿病。
在本发明的第二十方面，提供了一种抗TF抗体，其中该抗体对人TF蛋白的亲和力的EC50为0.005-0.10nM，较佳地为0.005-0.05nM，更佳地0.01-0.03nM或0.01-0.02nM。
在另一优选例中，所述抗体不结合于野生型的鼠TF蛋白。
在另一优选例中，所述抗体具有选自下组的一个或多个特性：
(a)抑制肿瘤细胞迁移或转移；
(b)抑制肿瘤生长。
在另一优选例中，所述的抗体为TF-mAb-SC1、TF-mAb-Ch或TF-mAb-H29至TF-mAb-H48。
在本发明的第二十一方面，提供了一种制备人源化或嵌合抗体的方法，包括步骤：
将本发明的鼠源抗体可变区的核苷酸序列克隆入含有人抗体恒定区的表达载体后，通过转染动物细胞表达人-鼠嵌合抗体。
将本发明的含人源FR区的抗体可变区的核苷酸序列克隆入含有人抗体恒定区的表达载体后，通过转染动物细胞表达人源化抗体。
在另一优选例中，所述的抗体是部分或全人源化的单克隆抗体。
在本发明的第二十二方面，提供了一种抑制肿瘤细胞迁移的方法，其特征在于，包括步骤：给需要的对象施用本发明的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为一系列原始抗人TF单克隆抗体(original hybridoma)对人源TF-阳性(MDA-MB-231和BxPC-3)、鼠源TF-阳性(B16-F10)细胞的结合活性(Cell binding activity)检测结果，在10μg/mL浓度条件下，TF-mAb-SC1显示最佳结合活性。
图2为ELISA测定TF-mAb-SC1对TF胞外区蛋白的结合亲和力(Binding affinity)。
图3为Western blot测定一系列原始单克隆抗体对BxPC-3胞内信号通路TF-PAR2的抑制作用。
图4为人-鼠嵌合抗体(TF-mAb-Ch)表达质粒的单酶切、双酶切鉴定结果，其中，图4A为表达重链质粒的可变区单酶切、双酶切鉴定结果，图4B为表达轻链质粒的可变区单酶切、双酶切鉴定结果。
图5为TF-mAb-SC1对细胞表面TF的结合亲和力(Binding affinity)检测结果，其中，图5A为对BxPC-3结合亲和力，图5B为对MDA-MB-231结合亲和力，图5C为对U87MG结合亲和力，图5D为对H1975的结合亲和活性。
图6为TF-mAb-SC1对FVIIa激活的BxPC-3细胞胞内信号通路TF-PAR2的影响检测结果。
图7为TF-mAb-SC1抗凝血活性检测结果，分别以BxPC-3(图7A)和MDA-MB-231(图7B)细胞表面TF作为TF来源。
图8为TF-mAb-SC1抗FXa生成活性检测结果，分别以BxPC-3(8A)和MDA-MB-231(8B)细胞表面TF作为TF来源。
图9为TF-mAb-SC1抑制裸鼠皮下BxPC-3肿瘤生长的检测结果(箭头所指为开始给药时间)。
图10为TF-mAb-SC1抑制裸鼠皮下U87MG肿瘤生长的检测结果(箭头所指为开始给药时间)。
图11为TF-mAb-SC1抑制裸鼠皮下HCC1806肿瘤生长的检测结果(箭头所指为开始给药时间)。
图12为TF-mAb-SC1抑制BxPC-3肿瘤基质胶原的堆积的检测结果及其Image-pro plus进行统计分析的结果。
图13为TF-mAb-SC1减小BxPC-3肿瘤血管管腔面积及其统计结果。
图14为TF基因敲除情况下，MDA-MB-231(图14A)和BxPC-3(图14B)细胞的迁移水平的测定。
图15为TF-mAb-SC1抗肿瘤细胞迁移活性测定，其中图15A为TF-mAb-SC1抑制 MDA-MB-231细胞的迁移水平的测定；图15B为TF-mAb-SC1抑制BxPC-3细胞的迁移水平的测定；
图16为TF基因敲除后，MDA-MB-231-luc细胞在小鼠体内血行性迁移水平检测及其荧光强度统计结果。
图17为TF-mAb-SC1抑制MDA-MB-231-luc细胞在小鼠体内血行性迁移能力检测及其荧光强度统计结果。
图18A为肺上的MDA-MB-231肿瘤转移灶及其统计分析结果；图18B为肺的重量比较结果。
图19为TF-mAb-SC1被细胞内吞(Internalization)至溶酶体的激光共聚焦显微镜观察结果。
图20为ELISA测定嵌合型抗体TF-mAb-Ch对TF胞外区蛋白的结合亲和力。
图21为TF-mAb-Ch对TF-阳性肿瘤细胞MDA-MB-231的结合亲和力的测定。
图22为TF-mAb-Ch对BxPC-3胞内信号通路TF-PAR2影响的检测结果。
图23为TF-mAb-Ch被细胞内吞(Internalization)至溶酶体的检测。
图24为人源化抗体对BxPC-3胞内信号通路TF-PAR2影响的检测结果。
图25为人源化抗体抑制裸鼠皮下HCC1806肿瘤生长的检测结果(箭头所指为开始给药时间)。
图26为TF蛋白在高侵袭、高转移的Basal-type乳腺癌(尤其是三阴性乳腺癌)细胞株群中和Luminal-type乳腺癌细胞株群中的表达结果对比。
图27为根据Cancer Cell Line Encyclopedia(CCLE)数据库分析TF mRNA在高侵袭、高转移的Basal-type乳腺癌(尤其是三阴性乳腺癌)细胞株群中和Luminal-type乳腺癌细胞株群中的表达结果对比。
图28为TF蛋白在不同胰腺癌细胞株中的表达检测结果。
图29为根据CCLE数据库分析TF mRNA在不同胰腺癌细胞株中的表达水平。
具体实施方式
本发明人通过广泛而深入的研究，经过大量筛选，意外地获得一种抗TF单克隆抗体TF-mAb-SC1，实验结果表明，该针对TF蛋白的单克隆抗体为IgG2b型抗体。所述的抗体能够高特异性地结合TF抗原，其具有很高的亲和力(ELISA测定其EC50约为0.019nM)并且，所述的抗体具有显著的抗肿瘤活性，而对于哺乳动物本身没有可见的毒副作用。此外，基于该TF-mAb-SC1而获得的嵌合抗体、人源化抗体以及相应的ADC也具有优异的特性。在此基础上完成了本发明。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四 聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以通过标准的DNA重组技术获得，它们都是有用的抗体。嵌合抗体是一个分子，其中不同的部分来自不同的动物种，例如具有来自鼠的单克隆抗体的可变区，和来自人免疫球蛋白的恒定区的嵌合抗体(见例如美国专利4,816,567和美国专利4,816,397,在此通过引用方式整体引入本文)。人源化的抗体是指来源于非人物种的抗体分子，具有一个或多个来源于非人物种的互补决定区(CDRs)和来源于人免疫球蛋白分子的框架区域(见美国专利 5,585,089，在此通过引用方式整体引入本文)。这些嵌合和人源化的单克隆抗体可以采用本领域熟知的DNA重组技术制备。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
抗TF的抗体
本发明提供一种针对TF的高特异性和高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
优选地，重链可变区(VH)氨基酸序列和轻链可变区(VL)氨基酸序列的各自CDR选自下组：
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)上述氨基酸序列中任意一种氨基酸序列经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
优选地，所述的抗体具有抑制TF相关信号通路的活性；具有抗凝血活性；具有抗FXa生成活性、或其组合。
本发明的抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab')2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
其中，所述动物优选为哺乳动物，如鼠。
本发明抗体可以是靶向人TF的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
在本发明的一种优选实施例中，上述SEQ ID No.:1-SEQ ID No.:3中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的序列，位于重链可变区(VH)的CDR区。
在本发明的一种优选实施例中，上述SEQ ID No.:4-SEQ ID No.:6中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的序列，位于轻链可变区(VL)的CDR区。
在本发明的一种更优选实施例中，VH CDR1、CDR2、CDR3分别独立地选自SEQ ID No.:1-SEQ ID No.:3中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的序列；VL CDR1、CDR2、CDR3分别独立地选自SEQ ID No.:4-SEQ ID No.:6中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的序列。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
本发明上述内容中，更优选地，所述添加、缺失、修饰和/或取代的氨基酸数量，可 以是1-7个，更优选为1-5个，更优选为1-3个，更优选为1-2个。
在另一优选例中，所述靶向TF的抗体为TF-mAb-SC1(原名称为TF-mAb)。
在另一优选例中，所述抗体TF-mAb-SC1的重链可变区(VH)氨基酸序列为如SEQ ID NO.:7所示的氨基酸序列。
在另一优选例中，所述抗体TF-mAb-SC1的轻链可变区(V-Kappa)氨基酸序列为如SEQ ID NO.:8所示的氨基酸序列。
抗体的制备
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、HEK-293细胞。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)。
在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2016096291-appb-000001
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1到8的值。
检测用途和试剂盒
本发明的抗体或其ADC可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如肿瘤的切除样本、通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
应用
本发明还提供了本发明抗体的用途，例如用于制备诊断制剂、或制备用于预防和/或治疗TF相关的疾病的药物。所述TF相关的疾病包括肿瘤发生、生长和/或转移、血栓类相关疾病、炎症、代谢相关疾病等。
本发明抗体、ADC或CAR-T等的用途，包括(但并不限于)：
(i)诊断、预防和/或治疗肿瘤发生、生长和/或转移，尤其是TF高表达的肿瘤。所述肿瘤包括(但并不限于)：乳腺癌(如三阴性乳腺癌)、胰腺癌、肺癌、恶性胶质瘤、胃癌、肝癌、食道癌、肾癌、结直肠癌、膀胱癌、前列腺癌、子宫内膜癌、卵巢癌、宫颈癌、白血病、骨髓癌、血管肉瘤等；尤其是三阴性乳腺癌、胰腺癌、恶性胶质瘤和肺癌，更优选为三阴性乳腺癌和/或胰腺癌。
(ii)诊断、预防和/或治疗血栓类相关疾病。所述血栓类相关疾病包括(但并不限于)：动脉粥样硬化、急性冠状动脉综合症、急性心肌梗塞、中风、高血压、深静脉血栓、肺栓塞、肾栓塞及动脉手术、冠状动脉旁路移植引起的血栓等。
(iii)诊断、预防和/或治疗炎症。所述炎症包括(但并不限于)：风湿性关节炎、骨关节炎、强直性脊柱炎、痛风、莱特尔综合征、牛皮癣性关节病、感染性关节炎、结核性关节炎、病毒性关节炎、真菌性关节炎、肾小球性肾炎、全身性红斑狼疮、克罗恩病、溃疡性结肠炎、急性肺损伤、慢性阻塞性肺疾病、特发性肺纤维化。
(iv)诊断、预防和/或治代谢相关疾病。所述代谢相关疾病包括(但并不限于)：糖尿病、食源性肥胖和脂肪炎症等。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述 的抗体或其活性片段或其融合蛋白或其ADC或相应的CAR-T细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明的药物组合物可直接用于结合TF蛋白分子，因而可用于预防和治疗肿瘤等疾病。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括：
(a)本发明抗体具有优异的生物活性和特异性，并具有很高的亲和力(ELISA测定其EC50可高达为约0.01-0.03nM)。此外，对细胞表面TF具有良好的结合亲合力，可用做靶向TF的抗体。
(b)本发明的人源化抗体不仅具有与鼠源抗体相当的活性，而且具有更低的免疫原性。
(c)本发明抗体和ADC均具有显著的抗肿瘤活性，而对于哺乳动物本身没有可见的毒副作用。
(d)本发明抗体和ADC不仅在多个肿瘤模型中有显著的治疗效应，而且还适用于其他与TF高表达有关的疾病，如血栓类疾病、代谢类疾病等。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比 和份数是重量百分比和重量份数。细胞株为常规的市售产品或购自ATCC，质粒均为市售产品。
实施例1  靶向人TF单克隆抗体的发现和制备
步骤①，杂交瘤细胞的制备：
首先将人TF蛋白的胞外区部分(UniProtKB/Swiss-Prot:P13726.1，从第34位到第251位氨基酸)免疫Balb/c小鼠，TF胞外区蛋白的用量为100μg/只，以制备免疫脾细胞；适时的制备鼠骨髓瘤细胞(SP2/0)和饲养细胞以备融合之需。
待上述三种细胞准备完毕，通过PEG介导融合免疫脾细胞和SP2/0细胞，去除PEG，用含有饲养细胞的HAT完全培养基重悬，接种到96孔板中培养，通过ELISA法进行阳性孔筛选。最后再对阳性孔的细胞通过有限稀释法进行克隆化培养，通过ELSIA或免疫荧光法筛选效价高、形态好、呈单克隆生长的细胞继续进行亚克隆筛选，直到连续三次筛选阳性克隆率全为100％，即可对该细胞株进行扩大培养和建库。
步骤②，靶向人TF鼠源单克隆抗体腹水的制备：
将步骤①中筛选出来的杂交瘤细胞扩大培养，小鼠适应性培养后腹腔注射降植烷(0.5mL/只)以为杂交瘤细胞生长提供有利环境，7-10d后，每只腹腔注射10×106的杂交瘤细胞，自第7天起，每天观察小鼠腹水产生状况和精神状态，采取腹水，离心去除油脂-80℃冻存，以备纯化。
步骤③，靶向人TF鼠源单克隆抗体的纯化：
将步骤②中冻存的腹水于冰上融化，经0.45μm滤膜过滤后，用PBS于4℃透析过夜，最后通过FPLC技术对该抗体进行纯化并超滤浓缩至所需浓度，分装、-80℃冻存备用。
步骤④，靶向人TF鼠源单克隆抗体的生物活性和靶向特异性确定：
经过初步筛选，我们选定约30个杂交瘤细胞进行二次有限稀释克隆筛选，然后在其中选定6个抗体，进行大量表达纯化，并在10μg/mL的浓度下利用流式细胞仪测定各个抗体对人乳腺癌细胞MDA-MB-231，人胰腺癌细胞BxPC-3和鼠黑色素瘤细胞B16-F10的亲和力。
结果如图1所示，测试的抗体可以特异性的靶向结合人源TF(MDA-MB-231和BxPC-3细胞)而不靶向结合鼠源TF(B16-F10细胞)，其中TF-mAb-SC1对人TF的亲和力比其他5个抗体都要高。
用包被液将抗原(TF胞外区蛋白)稀释成0.5μg/mL，包被ELISA板，100μL/孔，4℃，过夜。洗去多余抗原，用2％BSA于室温封闭2h，然后加入3倍梯度稀释的各单克隆抗体，100μL/孔，室温孵育2h；洗去未结合的抗体，加入合适浓度辣根过氧化物酶标记的抗鼠的二抗，100μL/孔，室温孵育1h。洗去未结合的二抗，加入TMB显色液，显色至适当颜色深浅，加入2M H2SO4，50μL/孔，终止显色反应，然后在450nm处测定其吸光度，并分析数据。如图2所示，TF-mAb-SC1对TF胞外区蛋白有很强的亲和性，EC50约为0.019nM。
同时，铺3×105个胰腺癌细胞BxPC-3于12孔板中，12小时之后，用无菌PBS洗涤细胞3 次，然后加入无血清培养基于37℃，5％CO2培养箱饥饿4h，随后将各单克隆抗体按3倍梯度稀释，并与BxPC-3于培养箱中孵育1h，随后用25nM FVIIa激活BxPC-3胞内PAR2信号通路，37℃作用15min后，用预冷的PBS洗涤细胞一次，并于冰上收集细胞蛋白并通过Western blot鉴定TF-mAb-SC1对下游MAPK/ERK的磷酸化水平的影响，只做FVIIa刺激，未与单克隆抗体孵育的细胞作为阳性对照。通过Western blot测定这6个抗体对BxPC-3胞内信号通路TF-PAR2的影响。
结果如图3所示，只有TF-mAb-SC1可以显著抑制下游MAPK/ERK的磷酸化水平并呈一定的剂量依赖性。
由于TF-mAb-SC1表现出非常高的特异性、非常高的亲和力以及对MAPK/ERK的磷酸化水平的显著抑制作用，故被选定用于测序以及后续研究。
采用常规测序，并通过Kabat数据库分析，得到以下序列信息：
重链可变区的CDR氨基酸序列为：
SEQ ID No.:1：SYWMN；
SEQ ID No.:2：MIYPADSETRLNQKFKD；
SEQ ID No.:3：EDYGSSDY。
完整的VH氨基酸序列如SEQ ID NO.:7所示。
Figure PCTCN2016096291-appb-000002
轻链可变区的CDR氨基酸序列为：
SEQ ID No.:4：SASSSVSYMN；
SEQ ID No.:5：GISNLAS；
SEQ ID No.:6：QQKSSFPWT。
完整的VL氨基酸序列如SEQ ID NO.:8所示：
Figure PCTCN2016096291-appb-000003
实施例2  人-鼠嵌合抗体的制备
在已获得的活性高、特异性强的鼠源TF-mAb-SC1的基础上，构建人-鼠嵌合抗体。
通过相关数据库分析，确定重链可变区的CDR氨基酸序列为：
SEQ ID No.:18：MIYPXDSETRLNXKFKD(X选自A、D、E、Q、Y中的任意一种)
SEQ ID No.:19：GYSFXSYWMN(X选自A、I、Y、Q、W中的任意一种)
SEQ ID No.:20：AREDYGXSDY(X选自S、P、G、D、M、N中的任意一种)。
通过相关数据库分析，确定轻链可变区的CDR氨基酸序列为：
SEQ ID No.:21：QQXSSFXWT(X选自S、P、K、G、H中的任意一种)；
SEQ ID No.:22：SASSXVSYMN(X选自A、P、D、S中的任意一种)；
SEQ ID No.:23：GXSNLAS(X选自P、D、I、S中的任意一种)。
设计引物在重链可变区引入EcoR I和Nhe I，在轻链可变区引入Age I和BsiW I限制性内切酶酶切位点，然后将上述所获得抗体重链和轻链的可变区序列分别克隆入含有人IgG1重链恒定区和Kappa链恒定区的载体，经鉴定无误后(图4A为重链酶切鉴定结果，图4B为轻链酶切鉴定结果，其中样品1为对应的重链/轻链空白载体，样品2为克隆入重链/轻链可变区的载体，3和4为单酶切后的样品，5为双酶切后的样品)，利用转染技术和哺乳动物表达系统(CHO-S或HEK-293细胞)将构建的嵌合型抗体表达、纯化，所获得的人-鼠嵌合型抗体，命名为TF-mAb-Ch。
实施例3  TF-mAb-SC1对TF-阳性肿瘤细胞的结合亲和力(Cell binding affinity)的测定
本实验以细胞表面TF高表达的三阴性乳腺癌细胞MDA-MB-231、胰腺癌细胞BxPC-3、恶性胶质瘤细胞U87MG和非小细胞肺癌细胞H1975作为靶细胞，将100μL按照3倍梯度从333.33nM稀释到0.15nM的TF-mAb-SC1作为一抗，分别与悬浮于100μL RPMI-1640无血清培养基中的3×105个MDA-MB-231或BxPC-3混匀，或将100μL按照3倍梯度从66.67nM稀释到0.03nM的TF-mAb-SC1作为一抗与悬浮于100μL MEM无血清培养基中的3×105个U87MG混匀，或将100μL的33.33nM和3.33nM的TF-mAb-SC1作为一抗与悬浮于100μL RPMI-1640无血清培养基中的3×105个H1975混匀，然后在4℃孵育1h，PBS洗涤细胞两次以去除未结合的一抗，再将靶细胞与200μL，2μg/mL，PE标记的二抗4℃孵育30min，PBS洗涤细胞两次以去除未结合的二抗，最后将细胞重悬在400μL PBS中，通过流式细胞仪测定TF-mAb-SC1对相应细胞表面TF的结合亲和力(Binding affinity)。
如图5所示，TF-mAb-SC1对BxPC-3、MDA-MB-231和U87MG均有较好的结合亲和力，EC50分别为2.6nM(图5A)、2.5nM(图5B)和1.6nM(图5C)，图5D显示TF-mAb-SC1对H1975也有较好的结合亲和力。
这说明，本实施例单克隆抗体能够以人源TF为作用靶点。
实施例4  TF-mAb-SC1对胞内信号通路TF-PAR2的影响
铺3×105个胰腺癌细胞BxPC-3于12孔板中，12小时之后，用无菌PBS洗涤细胞3次，然后加入无血清培养基于37℃，5％CO2培养箱饥饿4h，随后将TF-mAb-SC1按3倍梯度从100nM稀释到1.2nM，并与BxPC-3于培养箱中孵育1h，随后用25nM FVIIa激活BxPC-3胞内PAR2信号通路，37℃作用15min后，用预冷的PBS洗涤细胞一次，并于冰上收集细胞蛋白并通过Western blot鉴定TF-mAb-SC1对下游MAPK/ERK的磷酸化水平的影响，只做FVIIa刺激，未与TF-mAb-SC1孵育的细胞作为阳性对照。
检测结果如图6所示，TF-mAb-SC1显著抑制下游MAPK/ERK的磷酸化水平并呈一定的剂量依赖性。
实施例5  TF-mAb-SC1抗凝血活性测定
将100nM的TF-mAb-SC1倍比稀释到1.5625nM(终体积50μL)并分别与悬浮于50μL含有5mM CaCl2的Hanks平衡盐溶液(Hanks Balanced Salt Solution,HBSS)中的3×104个MDA-MB-231和BxPC-3，室温孵育15min，然后加入50μL柠檬酸盐人血浆，迅速混匀，并在接下来2h内每隔15sec于405nm处测定吸光值，以计算对细胞表面TF起始凝血的抗凝作用。
图7中纵坐标代表凝血速率的时间，横坐标代表TF-mAb-SC1的浓度，以BxPC-3(图7A)和MDA-MB-231(图7B)细胞表面TF作为TF来源。实验结果均显示TF-mAb-SC1浓度≥12.5nM时便具有显著抗凝血活性。
实施例6  TF-mAb-SC1抗FXa生成活性的测定
将100nM的TF-mAb-SC1倍比稀释到1.5625nM(终体积50μL)并分别与悬浮于50μL HBSS(含有3nM FVIIa)中的1.5×104个BxPC-3和MDA-MB-231细胞于室温振荡孵育20min，然后加入50μL FX(终浓度50nM)来起始反应，5min后加入25μL 1M的EDTA终止反应；随后加入25μL 3mM的S2765，迅速混匀并在接下来60min内每隔15s测定其动力学反应曲线，从而计算抗FXa的生成活性。
如图8所示，TF-mAb-SC1显示出较好的抗FXa生成活性，IC50分别为9.0nM(图8A)和6.4nM(图8B)。
实施例7  TF-mAb-SC1对体内肿瘤生长抑制活性的评定
随机将裸鼠分为两组，每组10只，首先将肿瘤细胞(1×107的BxPC-3或5×106的U87MG或2.5×106的HCC1806)与20mg/kg剂量的TF-mAb-SC1混合，室温孵育30min后，共同接种到免疫缺陷型小鼠(Balb/c裸鼠)背部或乳垫，观察对BxPC-3皮下瘤生长的抑制作用，另一组小鼠使用正常小鼠IgG(简称鼠IgG)作为对照。定期测量裸鼠体重及肿瘤大小，绘制肿瘤生长曲线，评定活性。
如图9所示，相比于鼠lgG，TF-mAb-SC1对BxPC-3皮下瘤生长抑制效果更为显著，抑制率最高可达80％。
如图10所示，相比于鼠lgG，TF-mAb-SC1对U87MG皮下瘤生长抑制效果更为显著，抑制率最高可达60％。
如图11所示，相比于鼠lgG，TF-mAb-SC1可以显著抑制HCC1806皮下瘤生长，抑制率最高可达>90％。
实施例8  TF-mAb-SC1显著抑制肿瘤基质胶原堆积
收集来自实施例7的BxPC-3移植瘤，4％中性甲醛固定后，石蜡包埋切片，常规脱蜡至水并进行Masson染色。对经染色的每个样本在100×的放大倍数下(图例为100μm)，采集5到10个视野并进行统计分析。
结果如图12所示，TF-mAb-SC1可以显著抑制肿瘤基质胶原的堆积(蓝色区域)，从而导致肿瘤的生长抑制，右图为利用Image-pro plus进行统计分析的结果。
实施例9  TF-mAb-SC1显著减小肿瘤血管管腔面积
收集来自实施例7中的BxPC-3移植瘤，4％中性甲醛固定后，石蜡包埋切片，常规脱蜡至水并进行CD31免疫组化染色。对经免疫组化染色的每个样本在200×的放大倍数下(图例为50μm)，采集5到10个视野并进行统计分析。
结果如图13所示，TF-mAb-SC1可以显著减小肿瘤血管官腔面积，右图为血管管腔面积的统计结果。
实施例10  TF-mAb-SC1抗肿瘤细胞迁移活性测定
利用transwell小室系统在体外评价TF-mAb对肿瘤细胞迁移水平的影响：将1×105个MDA-MB-231或8×104个BxPC-3细胞分别与一定浓度的TF-mAb-SC1(100nM，33.3nM和11.1nM)或Mouse IgG(简称IgG)混合于200μL的无血清培养基中，加到上层小室中，小室下层加入600μL含10％FBS的完全培养基，于37℃，5％CO2培养箱中培养，8h后，用湿棉签擦去小室膜上表面细胞，膜下表面细胞用95％乙醇固定30min后，于0.2％的结晶紫中染色30min，蒸馏水洗去多余的结晶紫，室温晾干后，在显微镜下，随机选取5个代表性的视野，分析统计迁移到小室膜下表面的细胞数。本实验同时以TF基因敲除的细胞(sh-TF)和相应载体对照细胞(sh-NT)进一步确认TF-mAb-SC1的抗肿瘤细胞迁移活性。
如图14所示，敲除TF基因，显著抑制MDA-MB-231(图14A)和BxPC-3(图14B)细胞的迁移。
如图15所示，TF-mAb-SC1可以显著的抑制MDA-MB-231(图15A)和BxPC-3(图15B)细胞的迁移水平，并呈浓度依赖性。
上述结果提示，通过抑制TF，可以有效抑制肿瘤细胞的迁移。
实施例11  TF-mAb-SC1显著抑制肿瘤细胞的体内血行转移
利用实验性血行转移模型在体内评价TF-mAb-SC1对肿瘤细胞迁移水平的影响：将2×106荧光素酶标记的MDA-MB-231细胞(MDA-MB-231-luc)与0.1mg的TF-mAb-SC1或IgG混合于200μL PBS中，冰上孵育20min后，通过尾静脉缓慢注射到6到8周龄的雌性裸鼠体内；4h后，麻醉裸鼠，将荧光素钾盐PBS溶液，按照150mg/kg的剂量，腹腔注射到裸鼠体内，6min后于小动物活体成像仪中(IVIS SPECTRUM)曝光1min，测定荧光强度， 统计分析，每组5只裸鼠。
如图16所示，敲除TF基因可以显著抑制MDA-MB-231-luc的血行性迁移能力。同样，如图17所示，TF-mAb-SC1也可以显著的抑制MDA-MB-231-luc细胞的血行性迁移能力，右图为迁移到肺细胞荧光强度的统计结果。
将3×106的MDA-MB-231细胞与0.1mg的TF-mAb-SC1或IgG混合于200μL PBS中，冰上孵育20min后，通过尾静脉缓慢注射到6周龄的雌性SCID Beige小鼠体内，6周后，处死小鼠，取出肺经Bouin's液固定后，拍照并称重，并记录每个肺上转移灶的个数。
结果如图18所示，TF-mAb-SC1显著抑制小鼠肺上肿瘤转移灶的形成。
实施例12  TF-mAb-SC1可以迅速高效的被细胞内吞(Internalization)至溶酶体
铺50％密度MDA-MB-231细胞于激光共聚焦专用培养皿中，约16h后，加入10μg/mL的TF-mAb-SC1分别于37℃或4℃孵育1h，用预热的PBS洗涤三次去除未与细胞结合的抗体，之后用4％的多聚甲醛于室温固定30min。PBS洗涤三次后，37℃孵育Lamp-2(兔抗人)抗体1h，以标记细胞溶酶体的位置，PBS洗去未结合的抗体，37℃孵育Alexa Fluor 594标记的驴抗鼠和Alexa Fluor 488标记的驴抗兔二抗30min。洗去未结合抗体，用DAPI染色以标记细胞核位置，之后用激光共聚焦显微镜观察抗体的细胞内吞情况。
如图19所示，TF-mAb-SC1可以被细胞内吞至溶酶体。
实施例13  TF-mAb-Ch的生物活性测定：
实验方法参照实施例1，步骤④。
结果如图20所示，TF-mAb-Ch对TF胞外区蛋白有很强的亲和性，EC50约为0.011nM。
实施例14  TF-mAb-Ch对TF-阳性肿瘤细胞的结合亲和力(Cell binding affinity)的测定
参照实施例3中的实验方法。
结果表明，TF-mAb-Ch对MDA-MB-231细胞有较好的结合亲和力，EC50为2.3nM(图21)。
实施例15  TF-mAb-Ch对胞内信号通路TF-PAR2的影响
实验方法参照实施例4。
结果如图22所示，TF-mAb-Ch浓度依赖性的抑制FVIIa诱导的MAPK/ERK的磷酸化水平。
实施例16  TF-mAb-Ch可以迅速高效的被细胞内吞(Internalization)至溶酶体
实验方法参照实施例12。
结果如图23所示，TF-mAb-Ch可以被细胞内吞至溶酶体。
上述实验提示，由于本发明TF-mAb抗体易于被内吞，因此适合被开发成抗体偶联药物(Antibody–drug conjugate，ADC)并应用于TF高表达的相关肿瘤的治疗。
实施例17  TF-mAb-SC1的人源化及活性测定
参照TF-mAb-SC1的抗体重链可变区序列(SEQ ID NO:7)和轻链可变区序列(SEQ ID NO:8)，在Germline数据库中选取与其非CDR区匹配最好的人源化模板。然后将鼠源抗体TF-mAb-SC1的CDR区移植到所选择的人源化模板上，替换人源模板的CDR区，再与IgG1恒定区重组，同时以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，得到5个人源化重链的可变区(SEQ ID NO:9，SEQ ID NO:10，SEQ ID NO:11，SEQ ID NO:12，SEQ ID NO:13)及4个人源化轻链的可变区(SEQ ID NO:14，SEQ ID NO:15，SEQ ID NO:16，SEQ ID NO:17)。
表B
Figure PCTCN2016096291-appb-000004
Figure PCTCN2016096291-appb-000005
基于所述改造的VH和VL，分别组合表达这些人源化的重链及轻链，最终共得到的20个人源化抗体，即TF-mAb-H29至TF-mAb-H48。各抗体相应的重链和轻链组合如下表所示：
表C
序列编号 SEQ ID NO:9 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13
SEQ ID NO:14 TF-mAb-H29 TF-mAb-H30 TF-mAb-H31 TF-mAb-H32 TF-mAb-H33
SEQ ID NO:15 TF-mAb-H34 TF-mAb-H35 TF-mAb-H36 TF-mAb-H37 TF-mAb-H38
SEQ ID NO:16 TF-mAb-H39 TF-mAb-H40 TF-mAb-H41 TF-mAb-H42 TF-mAb-H43
SEQ ID NO:17 TF-mAb-H44 TF-mAb-H45 TF-mAb-H46 TF-mAb-H47 TF-mAb-H48
首先通过ELISA结合实验测定这20个人源化抗体对TF胞外区蛋白的亲和活性(实验方法参照实施例1，步骤④)，结果如表1所示。
表1  人源化抗体对TF胞外区蛋白的结合亲和力
抗体 EC50(nM) 抗体 EC50(nM)
TF-mAb-Ch 0.0100 TF-mAb-H39 0.0125
TF-mAb-H29 0.0178 TF-mAb-H40 0.0131
TF-mAb-H30 0.0147 TF-mAb-H41 0.0134
TF-mAb-H31 0.0145 TF-mAb-H42 0.0128
TF-mAb-H32 0.0168 TF-mAb-H43 0.0116
TF-mAb-H33 0.0189 TF-mAb-H44 0.0120
TF-mAb-H34 0.0154 TF-mAb-H45 0.0138
TF-mAb-H35 0.0105 TF-mAb-H46 0.0119
TF-mAb-H36 0.0234 TF-mAb-H47 0.0130
TF-mAb-H37 0.0173 TF-mAb-H48 0.0153
TF-mAb-H38 0.0178 IgG(阴性对照) ＞6.67
通过流式细胞仪测定这20个人源化抗体分别在10μg/mL和1μg/mL浓度下，对MDA-MB-231细胞的结合亲和力，实验方法参照实施例3，结果如表2所示。
表2  人源化抗体对MDA-MB-231的结合亲和力；
Figure PCTCN2016096291-appb-000006
还测定了9个人源化抗体对胞内信号通路TF-PAR2的影响，实验方法参照实施例4。
实验结果如图24所示，各人源化抗体不同程度的抑制FVIIa诱导的MAPK/ERK的磷酸化水平，并呈浓度依赖性。
此外，还测定6个人源化抗体在10mg/kg剂量下对HCC1806皮下移植瘤的生长抑制作用，实验方法参照实施例7。
实验结果如图25所示，所有人源化抗体均表现出显著抑制肿瘤生长活性，其中TF-mAb-35、TF-mAb-39、TF-mAb-40、TF-mAb-44和TF-mAb-45表现出优异的抑制肿瘤生长活性。
实施例18，TF在三阴性乳腺中高度异常激活
首先针对多种不同组织来源的肿瘤细胞株，制备细胞总蛋白，精确定量后，通过Western blot检测TF蛋白的表达水平。
结果显示，TF蛋白在一些高侵袭、高转移的三阴性乳腺癌(如图26)细胞株呈高度异常激活表达，而在相对恶性程度较低的非三阴性乳腺癌细胞株中仅少数高表达。
然后，对Cancer Cell Line Encyclopedia(CCLE)数据库中乳腺癌细胞株的TF mRNA表达水平进行分析。结果表明，高侵袭、高转移的Basal-type乳腺癌(尤其是三阴性乳腺癌)细胞株群中TF mRNA的表达水平普遍高于Luminal-type乳腺癌细胞株群且具有统计学意义(图27)。因此，本发明以TF为靶点的抗体，在诊断、预防和治疗三阴性乳腺癌的应用中具有更为显著的效果。
实施例19  TF在胰腺癌中高度异常激活
首先针对多种不同组织来源的肿瘤细胞株，制备细胞总蛋白，精确定量后，通过Western blot检测TF蛋白的表达水平。结果显示，TF蛋白在高侵袭、高转移的胰腺癌(如图28)细胞株普遍呈高度异常激活表达。
然后，对CCLE数据库中胰腺癌细胞株的TF mRNA表达水平进行分析，结果表明，TF mRNA在胰腺癌细胞株中普遍呈较高水平的表达(图29)。因此，本发明以TF为靶点的抗体，在诊断、预防和治疗胰腺癌的应用中具有更为显著的效果。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (25)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:1所示的CDR1，
    SEQ ID NO:2所示的CDR2，和
    SEQ ID NO:3所示的CDR3；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留TF结合亲和力的衍生序列。
  2. 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。
  3. 一种抗体的轻链可变区，其特征在于，所述的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:4所示的CDR1'，
    SEQ ID NO:5所示的CDR2'，和
    SEQ ID NO:6所示的CDR3'；
    上述氨基酸序列中任意一种氨基酸序列经过添加、缺失、修饰和/或取代至少一个氨基酸的具有TF结合亲和力的衍生序列。
  4. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区。
  5. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链。
  6. 根据权利要求5所述的抗体，其特征在于，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
  7. 根据权利要求5中所述的抗体，其特征在于，所述抗体的重链序列选自下组：SEQ ID NO.:7、9、10、11、12、或13；和/或
    所述的抗体的轻链序列选自下组：SEQ ID NO.:8、14、15、16、或17。
  8. 根据权利要求5所述的抗体，其特征在于，所述添加、缺失、修饰和/或取代的氨基酸数量为1-7个。
  9. 根据权利要求5所述的抗体，其特征在于，所述经过添加、缺失、修饰和/或取代的至少一个氨基酸序列为同源性为至少80％的氨基酸序列。
  10. 一种权利要求5所述抗体的应用，其特征在于，所述抗体用于(a)制备诊断试剂；和/或(b)制备预防和/或治疗TF相关的疾病的药物。
  11. 根据权利要求10所述的应用，其特征在于，所述TF相关的疾病选自下组：
    肿瘤的发生、生长和/或转移；
    血栓类相关疾病；
    炎症；和
    代谢相关疾病。
  12. 根据权利要求11所述的应用，其特征在于，所述肿瘤为TF高表达的肿瘤。
  13. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  14. 一种多核苷酸，其特征在于，它编码选自下组的多肽：
    (1)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体；或
    (2)如权利要求13所述的重组蛋白。
  15. 一种载体，其特征在于，它含有本发明权利要求14所述的多核苷酸。
  16. 一种遗传工程化的宿主细胞，其特征在于，它含有权利要求15所述的载体或基因组中整合有权利要求14所述的多核苷酸。
  17. 一种抗体药物偶联物，其特征在于，该抗体药物偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、如权利要求5所述的抗体、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
  18. 一种免疫细胞，其特征在于，所述免疫细胞表达或在细胞膜外暴露有权利要求5所述的抗体。
  19. 一种药物组合物，其特征在于，它含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、如权利要求13所述的重组蛋白、如权利要求17所述的抗体药物偶联物、权利要求18所述的免疫细胞、或其组合；以及
    (ii)药学上可接受的载体。
  20. 一种活性成分的用途，其特征在于，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、如权利要求13所述的重组蛋白、或如权利要求17所述的抗体药物偶联物、权利要求18所述的免疫细胞、或其组合，其中所述活性成分被用于制备药剂、试剂、检测板或试剂盒。
  21. 一种体外检测样品中TF蛋白的方法，其特征在于，所述方法包括步骤：
    (1)在体外，将所述样品与权利要求5所述的抗体接触；
    (2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在TF蛋白。
  22. 一种检测板，其特征在于，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有权利要求5所述的抗体或权利要求17所述的免疫偶联物。
  23. 一种重组多肽的制备方法，该方法包括：
    (a)在适合表达的条件下，培养权利要求16所述的宿主细胞；
    (b)从培养物中分离出重组多肽，所述的重组多肽是权利要求5所述的抗体或权利要求13所述的重组蛋白。
  24. 一种治疗肿瘤、血栓类疾病、炎症性疾病和/或代谢类疾病的方法，其特征在于，包括：使用如权利要求5所述抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞。
  25. 一种抗TF抗体，其特征在于，该抗体对人TF蛋白的亲和力的EC50为0.005-0.10nM。
PCT/CN2016/096291 2015-08-20 2016-08-22 靶向于组织因子的抗体、其制备方法和用途 WO2017028823A1 (zh)

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