TW200407427A - Human tissue factor antibodies - Google Patents

Abstract

The present invention relates to isolated fully human antibodies that immunoreacts with human tissue factor (TF) to inhibit the binding of coagulation factor VIIa (FVIIa).

Description

200407427 发明 Description of the invention [Technical field to which the invention belongs] The present invention relates to an isolated antibody that performs an immune response with human tissue factor (TF) to inhibit the binding of the clotting factor Vila (FVIIa), and a human antibody inhibition using anti-TF Thrombosis associated with surgery, microsurgery, angioplasty, or trauma, or inhibition that occurs in association with factors such as deep venous thrombosis, disseminated intravascular coagulation (DIC), coronary artery disease, sepsis, inflammation, atherosclerosis Or immunotherapy for thrombosis and other TF functions in abnormal hemostatic conditions related to diseases such as cancer. The invention also discloses an antibody preparation method and a cell line for preparing the human monoclonal antibodies (Mabs). [Prior art] Blood coagulation is a process involving complex interactions between various blood components (or factors), which eventually forms a fibrin clot. In general, the blood components involved in what is known as the "cascade" of blood coagulation are enzymes or zymogens, which are proteins that did not have enzyme activity in the past through the action of activators (which are activated coagulation factors themselves). It is then converted into proteolytic enzymes. Coagulation factors known to perform such transformations are commonly referred to as "activating factors" with a lower case &quot; a 〃 at the end of the term (eg, factor Vila). Activated factor X (&quot; Xa ") is Necessary for the conversion of prothrombin into thrombin (which then converts fibrinogen to fibrin as the final stage in the formation of a fibrin clot). There are two systems (or pathways) that promote factor X activation. "Endogenous pathway" means those reactions that promote thrombin formation by utilizing factors that are only found in plasma in 200407427. A series of activations involving proteases ultimately produce factor IXa, which combines with factor Villa to bind factors X breaks down to form Xa. In the "exogenous pathway" of blood coagulation, the same proteolysis is accomplished by FVIIa and its cofactor (TF). TF is a membrane-bound protein and usually does not circulate in activated form In plasma. However, when the blood vessel is damaged, TF can complex with FVIIa and catalyze factor X activation or factor IX activation in the presence of Ca2 + and phospholipids, and the two kinds of coagulation Given the importance of coagulation, Factor VII and TF have been found to play important roles in triggering blood coagulation. It is often necessary to selectively block the coagulation cascade in patients. Such as heparin, fragrant Anticoagulants such as legumin, coumarin derivatives, indane dione derivatives, or other agents can be used, for example, during kidney dialysis, or to treat deep venous thrombosis, disseminated intravascular coagulation (DIC ) And patients with other medical conditions. For example, heparin therapy with citrate ions or in vitro therapy can be used for dialysis to prevent coagulation during treatment. Heparin is also used to prevent deep venous thrombosis in patients undergoing surgery However, treatments with heparin or other anticoagulants may have undesired side effects. The available anticoagulants usually work systemically, rather than specifically at the site of injury. For example, heparin may cause massive bleeding. And because the half-life of heparin is about 80 minutes, it will be quickly eliminated from the blood and needs to be performed frequently. Because heparin acts as a cofactor for antithrombin III (ATIII) and ATIII is rapidly depleted in DIC treatment, it is often difficult to maintain an appropriate heparin dose, and continuous detection of 200,407,427 ATIII and heparin is necessary Content and concentration. Heparin is also ineffective if ATIII is considerably depleted. In addition, long-term use of heparin may also increase platelet aggregation and reduce platelet number, and has been expected to develop thrombocytopenia induced by heparin. Indandione Derivatives may also have toxic side effects. In addition to the anticoagulants briefly described above, several natural proteins have also been found to have anticoagulant activity. Furthermore, ATIII has been considered a therapeutic anticoagulant. International Patent application WO 92/15686 relates to an inactivation factor Vila for inhibiting blood coagulation. Antibodies are specific immunoglobulins (Ig) produced by the vertebrate immune system in response to stimuli produced by foreign proteins, glycoproteins, cells, or other antigenic foreign substances. The sequence of events that prompt organisms to defeat the invasion of foreign cells, or cause the system to exclude foreign matter, is at least partially unknown. An important part of this process is the production of antibodies that specifically bind to specific foreign substances. The specificity of binding of such polypeptides to specific antigens is very precise, and the numerous specificities that can be produced by individual vertebrates are of particular interest for their complexity and variability. There are millions of antigens that can elicit antibody responses, and individual antibodies react almost exclusively with specific antigens that elicit their production. Two main sources of vertebrate antibodies are used today, produced in situ by mammalian B lymphocytes, and produced by B-cell fusion tumors in cell cultures. The resistance system arises in situ due to the differentiation of immature B lymphocytes into plasma cells, which occur in response to stimuli produced by specific antigens. In undifferentiated B cells, portions of the DNA encoding various regions on the immunoglobulin chain are located separately in the genomic DNA. The sequences will be assembled in sequence before presentation. The aligned genes can be expressed in mature B lymphocytes to produce the desired antibodies. However, when a particular mammal is only exposed to a single antigen, it will not produce only a single population of antibodies. The in situ immune response to any particular antigen is limited to hybrid reactions to various epitopes present on the antigen. Each group of homology resistance system is produced by a single population of B cells, so the antibody produced in situ is "multiple strains of this limited but inherent heterogeneity. In many special cases, by using fusion tumor technology, Cell fusion tumors are solved by producing "single-clone" antibodies in cell culture. In this method, relatively short-lived (or dying) spleen cells or lymphocytes from mammals that have been injected with the antigen are combined with Immortal tumor cell lines are fused, and as a result, hybrid cells or "fusion tumors" with genetically encoded antibodies capable of producing B cells that are both immortal are produced. The hybrids formed by this are divided by screening, dilution, and regeneration A single genetic strain, and each strain thus represents a single genetic pure line. They can then produce a homogeneous line against the desired antigen. These antibodies (due to their pure genetic parentage) are called "single strains" Monoclonal antibodies with single-specificity have a great impact on immunology, and their availability has been used in sciences such as biology, morphology, biochemistry and others It has been confirmed that such monoclonal antibodies have been widely used not only as diagnostic reagents but also for therapeutic purposes (see, for example, Liz and Schlossmann, Blood, 59: 1-11, (1982)). Although the single antibody produced by the fusion tumor is effective in treating 200407427 as mentioned above, and it is obviously better than multiple antibodies because of its specificity, it still has an important disadvantage. In many applications, when these antibodies are intended to be used The use of monoclonal antibodies produced in non-human mammals in humans is severely limited. Repeated injections of "foreign" antibodies into humans (such as administering mouse antibodies) can cause harmful allergic reactions. Human-derived monoclonal antibodies, when injected into humans, cause anti-non-human antibody responses. The therapeutic use of anti-TF mouse Mabs has been disclosed in US Patent Nos. 6,001,978 and 5,223,427. International Patent Applications WO 99/51743 is a human / mouse chimeric monoclonal antibody against human TF. European Patent Application No. 83391 1 is a CDR-grafted antibody against human TF. Presta L. et al. , Thrombogenesis and Coagulation, Volume 85 (3) pp. 379-389 (2001) is about humanized antibodies against TF. There is still a need for improved compositions with anticoagulant activity in this technology, which can be relatively low Dosage is administered without the undesirable side effects associated with traditional anticoagulant compositions. The present invention accomplishes this by providing anticoagulants that do not have the side effects associated with traditional antibodies containing non-human sequences As needed, they can specifically act on the injury site and further provide other related advantages. Moreover, the present invention also provides an effect to inhibit the cellular function of TF (which includes diseases such as sepsis, inflammation, atherosclerosis, restenosis or cancer, etc. [Abstract] The present invention relates to a non-immunogen high affinity human 200407427 antibody against human TF, which inhibits the binding of clotting factor Vll / VIIa, and is effective against human TF for screening treatment Of human antibodies. In a first aspect, the invention relates to an isolated antibody that can immunoreact with an antigenic epitope presented on human TF. The term "human tissue factor" or "human TF" is used herein to refer to a full-length polypeptide receptor that contains the amino acid sequence 1-263 of a natural human tissue factor. The term "antibody" is used herein to mean that it has Immunoglobulin molecules and fragments thereof capable of specifically binding to an antigen (eg, human TF). The full-length antibody contains four polypeptide chains, that is, two heavy (H) chains and two light (L) chains are linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (herein referred to as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three functional domains, CtH, CH2, and CH3. Each light chain consists of a light chain variable region (herein referred to as LCVR or LH) and a light chain constant region. The heavy chain constant region contains a functional domain, CL. The VH and VL regions can be further subdivided into regions with high variability (called complementarity determining regions (CDRs)), which are separated by more inherent regions (called framework regions (FRs)). Each segment of VH and VL is composed of three segments of CDRs and four segments of FRs. The sequence from amine terminal to carboxyl terminal is J: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Therefore, the definition of an antibody also includes one or more antibody fragments that still retain the ability to specifically bind to an antigen (such as human TF). It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments included in the term "antibody" include ⑴Fab fragments, which are monovalent fragments composed of VL, VH, CL, and CH1 g domains; (ii) F (ab) 2 200407427 and F (ab ') Fragment 2 is a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region; (out) an Fd fragment consisting of VH and CH1 functional domains; (iv) an Fv fragment consisting of a single-armed antibody Vl and VH; (v) a dAb fragment (Wade et al. (1989) Nature 341: 544-546), which consists of a VH functional domain; and (vi) a complementary complementation determining region (CDR) that is isolated. Moreover, although the two functional domains (VL and VH) of the Fv fragment are encoded by separate genes, they can use recombinant methods to join them by a synthetic linker peptide, which can make them the VL and VH The VH regions are paired to form a single protein chain of a monovalent molecule (known as a single-chain Fv (scFv); see, for example, Bode et al. (1988) Science 242: 423-426; and Huston et al. (1988)? 1 * 〇〇: he 11 · Acad. Sci USA 85: 5879-5883). Such single chain antibodies are also intended to be included in the term "antibody". Other forms of single chain antibodies are also covered, such as diabodies. Double-acting antibodies are bivalent and bispecific antibodies, in which the VH and VL functional domains are expressed on a single polypeptide chain, but a linking peptide that is too short to allow two functional domains on the same chain to pair with each other This forces these domains to be paired with complementary domains of another chain to produce two antigen-binding sites (see, eg, 'Hulig', P. et al. (1993) Proc. Natl. Acad · Sci USA 90: 6444- 6448; Boyac, R. et al. (1994) Structure 2: 1121-1123). It is understood that human TF may have one or more epitopes that include (1) a peptide epitope consisting of a single peptide chain in human TF, and (2) a human TF polypeptide sequence consisting of more than one of its individual amino acid sequences A configurational epitope composed of unconnected spatially adjacent peptide chains; and (3) a molecular structure covalently attached to human TF after translation, such as a carbohydrate group (all or a part) After the composition of the -translated antigen must be determined. The terms "human antibody", "human antibody (majority)", "human TF antibody" and "human TF antibody (majority)" are used herein to include those having variable and constant regions derived from human germline immunoglobulin sequences antibody. Human antibodies of the invention can be, for example, in CDRs and particularly in CDR3 ', including amino acid residues not encoded by human germline immunoglobulin sequences (e.g., by random or site-directed mutagenesis in vitro or by in vivo Somatic mutations). However, the term "human antibody" is used herein without intending to include such antibodies in which CDR sequences derived from a germline of another mammalian breed (eg, mouse) have been implanted on a human framework sequence, such as a so-called human Sourced antibodies or human / mouse chimeric antibodies. "Isolated human antibody" as used herein means that it is substantially free of other human antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to human TF does not substantially contain Antigen-binding antibodies other than human TF). However, isolated antibodies that specifically bind to human T F may be cross-reactive to other antigens, such as T-F molecules derived from other species (discussed in further detail below). Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals. The term "epitope" as used herein means any epitope located on an antigen to which the antibody binds. An epitope determinant is usually composed of a chemically active surface group of a molecule such as an amino acid or a sugar side chain, and usually has a specific three-dimensional structural feature and a special charge characteristic. The term "immunological response" or "performing an immune response" as used herein means 13 200407427 the binding of any antibody to its epitope with a dissociation constant Kd of less than 10_4 M. Where appropriate, the terms "immune response" or "performing an immune response" may be used interchangeably with the term "specifically combining". The term "inhibition" as used herein means any reduction compared to reference VII. For example, an antibody that inhibits the binding of human clotting factor Vila to human TF, which means that any binding ability of human clotting factor Vila to human TF is compared to the ability of human clotting factor Vila to bind to human TF in the absence of the antibody Reduced antibodies. The term "affinity" as used herein means the strength of the binding of an antibody to an epitope. The affinity of an antibody is measured by the dissociation constant Kd, which is defined as [Ab] X [Ag] / [Ab-Ag] where [Ab-Ag] is the molar concentration of the antibody-antigen complex, and [Ab] is the The molar concentration of the bound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined as 1 / Kd. A better method for determining the specificity and affinity of Mabs by competitive inhibition can be found in Harlow et al., Antibodies: Laboratory Manual, Published by Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988), Edited by Collingen et al. , Today's Immunology Process, Greene Publishing and Willy International Scientific Publishing, NY (1992, 1993), and Miao Le, Enzymatic Methods 92: 589-601 (1983), their disclosures are incorporated herein by reference. In a second aspect, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a human antibody capable of immunoreactive with an antigenic epitope presented on human TF. The term "therapeutically effective amount" is an effective dose that can be titrated by a licensed practitioner to achieve the desired response. Dosage considerations will be 14 200407427 including efficacy, bioavailability, desired pharmacokinetic / pharmacokinetic conditions, therapeutic conditions (such as trauma, inflammation, septic shock), patient-related factors (such as weight, Health, age, etc.), whether co-administration with other medicines, time of administration, or other factors known to the physician. The dose of anti-TF human antibody administered to a patient will vary depending on the type and severity of the condition to be treated, but is generally between 0.1-5.0 mg / kg body weight. The term "individual" as used herein means any animal, especially a mammal, such as a human, and may be used interchangeably with the term "patient" if appropriate. In a third aspect, the invention relates to a composition comprising a human antibody capable of immunoreactive with an antigenic epitope presented on human TF. In another aspect, the present invention relates to a method for treating a FVIIa / TF-related disorder in humans, the method comprising administering to a therapeutically effective amount of a human antibody capable of immunoreactive with an antigenic epitope present on human TF. The human. "Treatment" means administering an effective amount of a therapeutically active compound of the present invention for the purpose of preventing any signs or conditions from developing, or for the purpose of curing or slowing down those signs or conditions that have developed. The term "treatment" is therefore intended to include prophylactic treatment. The term "FVIIa / TF-related diseases" is used herein to mean diseases or disorders in which TF and FVIIa are involved. It covers diseases or disorders related to thrombosis or coagulopathy, including inflammatory reactions and chronic thromboembolic diseases or disorders associated with fibrin formation, including vascular diseases such as deep venous thrombosis, arterial thrombosis, surgery Postthrombosis, 15 200407427 Coronary Artery Bypass Graft (CABG), Percutaneous Coronary Angioplasty (PTCA), Stroke, Tumor Growth, Tumor Metastasis, Angiogenesis, Thrombosis, Atherosclerosis and Reconstruction After Angioplasty Narrow, acute and chronic indications such as inflammation, septic shock, sepsis, hypertension, adult respiratory distress (ARDS), disseminated intravascular coagulation (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or Prophylactic treatment of arteriosclerotic blood vessels and other diseases or conditions at risk of thrombosis. Illnesses related to FVIIa / TF are not limited to in vivo coagulation disorders such as those specified above, but also include in vivo treatment methods related to FVIIa / TF, which may be caused, for example, by extracorporeal blood circulation, including blood from patients during dialysis procedures. Coagulation caused by blood extraction or blood bypass of a patient during a patient's aspiration, hemofiltration. The term "factor Vila" or "FVIIa" means "two-chain" activated coagulation factor VII which is decomposed by a specific cleavage action at the Argl52-Ilel53 peptide bond. FVIIa can be purified from blood or produced by recombinant methods. Obviously the implementation of the method described herein has nothing to do with the source of the purified factor Vila, and therefore the present invention is intended to cover the use of any factor Vila formulation suitable for use in the present invention. It is preferably human FVIIa. The term &quot; FVII &quot; means &quot; single-chain &quot; coagulation factor VII. In another aspect, the invention relates to a method for preparing a human antibody, the method comprising: a) preparing a human antibody against human TF, b) Test the antibodies in TF-induced clot analysis and screen out that the IC5Q can be less than 1 nM (for example, less than 500 pM, preferably less than 16 200407427 to 200 pM, preferably less than 100 pM in this analysis, (Preferably less than 50 pM, preferably less than 10 pM, more preferably less than 5 pM) human antibodies that inhibit clot formation, or test the antibodies in FXa yield analysis and screen for IC50 of less than 100 nM (in Analysis with FVIIa concentration of 0.1 nM), for example, less than 10 nM, preferably less than 5 nM, preferably less than 1 nM, more preferably less than 0.1 nM, inhibit human antibodies produced by FXa, or in FVIIa / Test antibodies and screen for IC5Q 値 in TF amine decomposition analysis (analyzed at 10 nM FVIIa concentration), such as below 40 nM, preferably below 20 nM, more preferably below 10 nM, a human antibody that inhibits FVIIa amidolytic activity induced by TF, or competitive analysis in FVIIa Test antibodies and screen for human antibodies that can compete with FVIIa, or test antibodies and screen for human antibodies that can bind to human TF in a TF ELISA assay containing TF. It should be understood that this should be used for TF-induced clot analysis The specific IC50 in the description refers to when normal human plasma is used. In another aspect, the present invention relates to a method for preparing a human antibody, the method comprising: a) preparing a human antibody against human TF, b) in Test antibodies from TF-induced clot analysis and screen for IC5Q 値 IC5G 値 + 1 nM lower than FFR-rFVIIa in this analysis, such as IC5G 値 + 500 pM lower than FFR-rFVIIa, compared to Better than IC5 of FFR-rFVIIa. 値 + 200 pM, preferably lower than IC50 of FFR-rFVIIa + 100 pM, preferably lower than IC5G of FFR-rFVIIa + 50 pM, preferably lower 17 200407427 at FFR -rFVIIa IC5Q 値 + 10 pM, better than FFR-rFVIIa IC5Q 値 + 5 pM, better than FFR-rFVIIa IC5Q 値, human antibody that inhibits clot formation, or test antibody in FXa production analysis And screen out IC5Q + 100 whose IC50 is lower than FFR-rFVIIa nM (0.1 nM FVIIa is used in this analysis), for example, IC5G 低于 + 10 nM lower than FFR-rFVIIa, preferably IC5Q 低于 + 5 nM lower than FFR-rFVIIa, and preferably lower than IC5 of FFR-rFVIIa.値 + 1 nM, better than IC5G of FFR-rFVIIa 値 + 0.1 nM, better than IC5 () of FFR-rFVIIa, human antibodies that inhibit FXa production, or test in FVIIa / TF amine decomposition analysis Antibodies can be screened for IC5G 値 IC5Q 値 lower than FFR-rFVIIa + 100 nM (10 nM FVIIa used in this analysis), such as IC5G 値 +40 nM lower than FFR-rFVIIa, preferably lower than FFR -rFVIIa's IC5Q 値 + 20 nM, preferably lower than FFR-rFVIIa 1 (: 5 () 値 +10 nM, more preferably lower than FFR-rFVIIa's IC5 () 値, inhibits FVIIa induced by TF Human antibodies with amidolytic activity, or test antibodies in FVIIa competition assays and screen human antibodies that can compete with FVIIa, or test antibodies in TF ELISA assays and screen human antibodies that bind to human TF . It should be understood that the specific IC50 as described in TF-induced clot analysis refers to when normal human plasma is used. The term "TF-induced clot analysis" is used herein to mean any analysis in which clot time is measured in a sample containing blood plasma and TF. An example of a TF-induced clot analysis is shown in Example 1, Analysis 7. The term "FXa yield analysis" is used herein to mean any analysis in which the activation effect of FX is measured in a sample containing TF, FVIIa, FX, calcium, and phospholipids. An example of an FXa yield analysis is shown in Example 1, Analysis 5. The term "FVIIa / TF amidolytic analysis" is used herein to mean any analysis in which the amidolytic activity of FVIIa is measured in the presence of TF, i.e., the cleavage of a small peptide mass. An example of a FVIIa / TF amidine decomposition analysis is shown in Example 1, Analysis 4. The term "TF ELISA analysis" as used herein means any ELISA analysis that includes TF and anti-F antibody. An example of a TF ELISA analysis is the direct and indirect TF ELISA analysis listed in Example 1, Analysis 1 and 2. The term "direct TF ELISA analysis" as used herein means any TF ELISA analysis that includes immobilized TF. Examples of direct TF ELISA analysis are listed in Example 1, Analysis 1. The term "indirect TF ELISA analysis" as used herein means any TF ELISA analysis in which TF is present in solution. Examples of indirect TF ELISA analysis are listed in Example 1, Analysis 2. In another aspect, the present invention relates to an isolated antibody capable of immunoreacting with an epitope present on human TF and inhibiting the binding of human coagulation factor Vila to human TF, which can be obtained by a method comprising the following steps: a) Preparation of human antibodies against human TF 'b) Testing of antibodies in TF-induced clot analysis and screening can be performed with IC50 of less than 1 nM, for example less than 500 pM in this 19 200407427 analysis, Preferably it is less than 200 pM, preferably less than 100 pM, preferably less than 50 pM, preferably less than 10 pM, more preferably less than 5 pM, a human antibody that inhibits clot formation, or is used in FXa yield analysis Test the antibody and screen for IC5Q 値 below 100 nM (in analysis with FVIIa concentration of 0.1 nM), such as below 10 nM, preferably below 5 nM, preferably below 1 nM, more preferably below 0.1 nM, a human antibody that inhibits FXa production, or test the antibody in FVIIa / TF amidine decomposition analysis and screen for IC5Q <100 nM (in an analysis performed at a FVIIa concentration of 10 nM), such as low Below 40 nM, preferably below 20 nM, more preferably below 10 nM, inhibiting TF induction Human antibodies with FVIIa amidolytic activity, or test antibodies in FVIIa competition assays and screen for human antibodies that can compete with FVIIa, or test antibodies in TF ELISA assays and screen for antibodies that bind to human TF Human antibodies. It should be understood that the specific IC50 as described in TF-induced clot analysis refers to when normal human plasma is used. In another aspect, the present invention relates to an isolated antibody capable of immunoreacting with an epitope present on human TF and inhibiting the binding of human coagulation factor Vila to human TF, which can be obtained by a method comprising the following steps: a) Preparation of human antibodies against human TF, b) Testing of antibodies in TF-induced clot analysis and screening can be performed in this analysis with IC5G 値 lower than FFR-rFVIIa IC5Q 値 + 1 nM, for example IC5G 値 +500 pM lower than FFR-rFVIIa, preferably IC5 lower than FFR-20 200407427 rFVIIa.値 + 200 pM, preferably lower than IC50 of FFR-rFVIIa 値 + 100 pM, preferably lower than IC5Q of FFR-rFVIIa + 50 pM, preferably lower than IC5Q of FFR-rFVIIa 値 + 10 pM, more preferably lower IC5Q 値 + 5 pM in FFR-rFVIIa, better than IC5Q 値 in FFR-rFVIIaa 'human antibodies that inhibit clot formation, or test antibodies in FXa yield analysis and screen out IC5G 値 lower than FFR-rFVIIa IC5.値 + 100 nM (0.1 nM FVIIa used in this analysis), such as IC5G 低于 + 10 nM lower than FFR-rFVIIa, preferably IC5Q 低于 + 5 nM lower than FFR-rFVIIa, preferably lower than FFR-rFVIIa IC5Q 値 + 1 nM, more preferably lower than FFR-i * FVIIa IC5Q 値 + 0.1 nM, more preferably lower than FFR-rFVIIa IC5〇 値, inhibit human antibodies produced by FXa, or decompose in FVIIa / TF amidine The antibody is tested during the analysis and screened for IC50 値 lower than FFR-rFVIIa IC5G 値 + 100 nM (10 nM FVIIa used in this analysis), for example, IC5G 値 + 40 nM lower than FFR-rFVIIa, preferably IC5Q 値 +20 nM lower than FFR-rFVIIa, preferably IC5Q 値 +10 nM lower than FFR-rFVIIa, more preferably IC5〇 値 lower than FFR-rFVIIa, inhibiting the decomposition of FVIIa amine induced by TF Active human antibodies, or test antibodies in FVIIa competition analysis and screen for human antibodies that can compete with FVIIa, or test antibodies in TF ELISA assays containing TF and screen for human antibodies that can bind to human TF.

It should be understood that the specific IC5Q 21 200407427 as described in TF-induced clot analysis refers to when normal human plasma is used. In a specific aspect of the invention, the method for producing a human antibody against human TF therein comprises immunizing a mammal with human TF and isolating the antibody produced by the immunized mammal. In a preferred embodiment, the mammal is a mouse. It is understood that the immunized lactating animals or mice are capable of producing human antibodies. In another aspect, the present invention relates to a method for preparing a human antibody, the method comprising: a) immunizing a mouse with human TF, and b) isolating an antibody-producing cell from the immunized mouse and preparing a secretable human Immortalized cells of antibodies, c) Isolate the medium containing the produced antibodies from immortalized cells, d) test the antibodies in an indirect TF ELISA assay containing TF in solution, and screen for antibodies that can bind to human TF in solution Human antibodies, e) test antibodies in FVIIa competition analysis and screen for human antibodies that can compete with FVIIa, f) test antibodies in FVIIa / TF amidation analysis and screen for IC5.値 Less than 100 nM (in an analysis performed at a concentration of FVIIa of 10 nM), for example, less than 40 nM, preferably less than 20 nM, more preferably less than 10 nM, inhibiting FVIIa amide induced by TF Degradation activity of human antibodies, g) Test antibodies in FXa yield analysis and screen for IC5 () 値 less than 100 nM (in an analysis with FVIIa concentration of 0.1 nM), such as less than 10 nM, compared with Preferably less than 5 nM, preferably less than 1 nM, more preferably less than 0.1 nM, to inhibit human antibodies produced by FXa, 22 200407427 h) test the antibodies in the TF-induced clot analysis and screen for them- In the analysis, IC5Q 値 is less than 1 nM, such as less than 500 pM, preferably less than 200 pM, preferably less than 100 pM, preferably less than 50 pM, preferably less than 10 pM, and more preferably less than 5 pM, human antibody that inhibits clot formation, i) screen and grow the immortalized cells that can produce the antibodies screened in step dh in a suitable medium, j) single from the medium of the screened immortalized cells Isolate the screened antibodies. It should be understood that the specific IC5 (), described in TF-induced clot analysis, refers to when normal human plasma is used. In another aspect, the present invention relates to a method for preparing a human antibody, the method comprising: a) immunizing a mouse with human TF, and b) isolating an antibody-producing cell from the immunized mouse and preparing a secretable human Immortalized cells of antibodies, c) Isolate the medium containing the produced antibodies from immortalized cells, d) test the antibodies in an indirect TF ELISA assay containing TF in solution, and screen for antibodies that can bind to human TF in solution Human antibodies, e) test antibodies in FVIIa competition analysis and screen for human antibodies that can compete with FVIIa, f) test antibodies in FVIIa / TF scan analysis and screen for antibodies with IC5Q lower than FFR-rFVIIa IC5Q 値 + 100 nM (10 nM FVIIa is used in this analysis), for example, IC5G 値 + 40 nM lower than FFR-rFVIIa, preferably lower than IC5Q 値 + 20 nM, preferably lower than 23 for FFR-rFVIIa 200407427 IC5 of FFR-rFVIIa.値 + 10 nM, more preferably lower than the IC50 of FFR-rFVIIa 人类, a human antibody that inhibits the FVIIa amine degrading activity induced by TF, g) test the antibody in the FXa production analysis and screen out that its IC50 is lower than IC5Q 値 + 100 nM of FFR_rFVIIa (0.1 nM FVIIa is used in this analysis), for example, lower than IC5Q of FFR-rFVIIa ◦ + 10 nM, preferably lower than IC5Q of FFR-rFVIIa 値 + 5 nM, preferably lower IC5 in FFR-rFVIIa.値 + 1 nM, better than IC5 of FFR-rFVIIa.値 + 0.1 nM, better than IC5Q of FFR-rFVIIa 値, human antibodies that inhibit FXa production h) test antibodies in TF-induced clot analysis and screen for IC5G lower in this analysis IC5Q 値 + 1 nM 'at FFR-rFVIIa, such as IC5G 値 + 500 pM lower than FFR-rFVIIa, preferably IC5G 低于 + 200 pM lower than FFR-rFVIIa, preferably lower than IC50 F + of FFR-rFVIIa 100 pM, for example, IC5G 値 +50 pM lower than FFR-rFVIIa, preferably IC5Q 値 +10 pM lower than FFR-rFVIIa, more preferably IC5 () 値 + 5 pM lower than FFR-rFVIIa, more preferably lower than FFR-rFVIIa's IC5G 抑制, a human antibody that inhibits clot formation, i) Screen and grow the immortalized cells that can produce the antibodies screened in steps d_h in a suitable medium, J) Infinite proliferation from the screened The culture medium of the cells is isolated from the antibodies selected. It should be understood that the specific IC50 as described in TF-induced clot analysis refers to when normal human plasma is used. The term "antibody-producing cell" is used herein to mean any cell capable of producing an antibody. These include fusion tumors, transfected cell lines, and relatively short-lived (or dying) spleen or lymphocytes obtained from mammalian animals that have been injected with the antigen. In another aspect, the present invention relates to an isolated antibody capable of immunoreacting with an epitope present on human TF and inhibiting the binding of human coagulation factor Vila to human TF, which can be obtained by a method comprising the following steps: a) immunizing mice with human TF, b) isolating antibody-producing cells from the immunized mice and preparing immortalized cells capable of secreting human antibodies, c) separating the medium containing the produced antibodies from immortalized cells, d) Testing antibodies in an indirect TF ELISA assay containing TF in solution and screening for human antibodies that can bind to human TF in solution, e) Testing antibodies in FVIIa competition analysis and screening for competitive binding to FVIIa F) human antibodies, f) test antibodies in FVIIa / TF amidolysis analysis and screen for IC5Q 値 below 100 nM (in an analysis performed at a FVIIa concentration of 10 nM), such as below 40 nM, compared to Human antibodies that inhibit FVIIa amidolytic activity induced by TF, preferably less than 20 nM, more preferably less than 10 nM, g) test antibodies in FXa yield analysis and screen for IC5 () 値 less than 100 nM ( In an analysis with FVIIa concentration of 0.1 nM), for example, less than 10 nM, preferably less than 5 nM, preferably less than 1 nM, more preferably less than 0.1 nM, human antibodies that inhibit FXa production, h) Test the antibody in the TF-induced clot analysis and screen out. In this analysis, IC50 is below 1 nM, for example below 500 pM, preferably 25 200407427 at 200 PM, preferably below 200 PM. 100 PM, preferably less than 50 pM, preferably less than 10 PM, more preferably less than 5 PM, human antibodies that inhibit clot formation, i) screen and grow in a suitable medium to produce the product that can be produced in step dh Immortalized cells of the screened antibody, J) Isolate the screened antibody from the medium of the screened immortalized cell. It should be understood that the specific IC5 () as described in TF-induced clot analysis refers to when normal human plasma is used. In another aspect, the present invention relates to an isolated antibody capable of immunoreacting with an epitope present on human TF and inhibiting the binding of human coagulation factor Vila to human TF, which can be obtained by a method comprising the following steps: a) immunizing mice with human TF, b) isolating antibody-producing cells from the immunized mice and preparing immortalized cells capable of secreting human antibodies, c) separating the medium containing the produced antibodies from immortalized cells, d) Testing antibodies in an indirect TF ELISA assay containing TF in solution and screening for human antibodies that can bind to human TF in solution, e) Testing antibodies in FVIIa competition analysis and screening for competitive binding to FVIIa Human antibody, f) test the antibody in FVIIa / TF amidine decomposition analysis and screen out the IC5G which is lower than FFR-rFVIIa.値 + 100 nM (10 nM FVIIa used in this analysis), such as IC5Q 低于 + 40 nM lower than FFR-rFVIIa, preferably IC5G 値 + 20 nM lower than FFR-rFVIIa, preferably lower than FFR-rFVIIa IC5Q 値 + 10 nM, better than IC50 26 of FFR-rFVIIa 値, a human antibody that inhibits TF-induced FVIIa amine degradation activity of human antibodies' g) Test antibodies in FXa yield analysis and screen for 1C5 〇 値 IC5Q below FFR-rFVIIa + 100 nM (0.1 nM FVIIa used in this analysis), for example, IC5Q below FFR-rFVIIa 値 + 10 nM ′, preferably lower than IC5G of FFR-rFVIIa値 + 5 nM, preferably lower than ICFR0r of FFR-rFVIIa + 1 nM, more preferably lower than IC5Q of FFR-rFVIIa 値 + 〇 · 1 nM 'more preferably lower than IC5Qr of FFR-rFVIIa, inhibiting FXa production Human antibodies h) Test the antibodies in a TF-induced clot assay and screen them for an IC5Q of less than FFR-rFVIIa 1 in this assay &lt;: 5 () 値 + 1 nM 'for example, IC5G 値 +500 pM lower than FFR-rFVIIa, preferably IC5G 値 +200 pM lower than FFR-rFVIIa, preferably lower than IC50 値 + of FFR-rFVIIa 100 pM, for example, lower than IC5Q 50 + 50 pM of FFR-rFVIIa, preferably lower than IC5 of FFR-rFVIIa.値 + 10 pM, better than IC5Q of FFR-rFVIIa 値 + 5 pM, better than IC5G of FFR_i * FVIIa 値, human antibodies that inhibit clot formation, i) screen and incubate in a suitable medium The immortalized cells that can produce the antibodies screened in step dh can be produced. J) The screened antibodies are isolated from the medium that has been screened for immortalized cells. It should be understood that the specific IC50 described in the TF-induced clot analysis refers to when normal human plasma is used. In a specific aspect of the invention, the immortal proliferating cell fusion tumor cell. 27 200407427 In another aspect, the present invention relates to a cell that produces a human antibody that immunologically reacts with an epitope present on human TF and inhibits the binding of human coagulation factor Vila to human TF. In a specific aspect of the invention, the cells are isolated lymphocytes. In another specific aspect of the present invention, the cell line is isolated from a mouse. In another specific aspect of the present invention, the cell is a fusion tumor cell. In a specific aspect of the invention, the fusion tumor cell line obtains an antibody-producing fusion tumor cell by fusing antibody-producing lymphocytes with immortalized cells. In another specific aspect of the invention, the isolated human antibody inhibits binding of human clotting factor Vila to human TF. In another specific aspect of the invention, the isolated human antibody is a monoclonal antibody. The term "single antibody" as used herein means a homologous immunoglobulin group, i.e., individual molecules of the antibody group are identical except for naturally occurring mutations. Antibodies are normally produced by lymphocytes derived from B cells in the bone marrow. Immunoglobulins with a single amino acid sequence are produced from lymphocytes derived from the same pure line. Lymphocytes cannot be cultured directly for long periods of time to make specific antibodies of the actual quality. However, Kohler et al. (1975, Nature, 256: 495) demonstrated that the somatic cell fusion process (especially the fusion between lymphocytes and myeloma cells) can produce a special product that can grow in culture and make a so-called "single antibody" Antibody for fusion tumor cells. Myeloma cells are lymphocyte tumor cells that often produce antibodies (depending on the cell strain), but are known as "non-productive" strains. In another specific aspect of the invention, the isolated human antibody is a recombinant antibody. The term "recombinant antibody" is used herein to include all human antibodies prepared, expressed, produced, or isolated by recombinant methods, such as antibodies expressed using recombinant expression vectors transfected into host cells (further (Described in section Π below), antibodies isolated from recombinant, combinatorial antibody libraries (further described in section ΙΙΙ below), animals (eg, mice) isolated from genes transfected into human immunoglobulin genes ) Antibodies (see, for example, Taylor, L. D. et al. (1992) Nucleic Acid Research 20: 6287-6295) or any other human antibody prepared, expressed, produced, or isolated by any method that includes splicing the human immunoglobulin gene sequence to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some specific aspects, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using animals transgenic with human Ig sequences), and Therefore, the amino acid sequences of the VH and VL regions of these recombinant antibodies are derived from and related to the human germline VH and VL sequences, but all components of the human antibody germline may not naturally exist in vivo. In the sequence. In another embodiment of the present invention, the isolated human antibody is a Fab fragment. In another specific aspect of the present invention, the isolated human antibody 29 200407427 is a F (ab) 2 fragment. In another specific aspect of the invention, the isolated human antibody is a F (abf) 2 fragment. In another specific aspect of the invention, the isolated human antibody is a single-chain Fv fragment. In another specific aspect of the present invention, the isolated human antibody has a Kd 値 with human TF in the range of 10 · 15-1 (T8 M. It should be understood that the human antibody and human TF Kd 値 is determined in an assay in which the human antibody is immobilized (see Analysis 6). In another specific aspect of the invention, the isolated human antibody has a Kd that is comparable to human TF値 is in the range of 10 · 15-10 · 1 () M. In another specific aspect of the present invention, the isolated human antibody has a Kd ratio of less than 10 · 8 Μ with human TF. In another specific aspect of the present invention, the isolated human antibody has a Kd 値 ratio with human TF of less than 1 (T9 M. In another specific aspect of the present invention, the isolated The isolated human antibody has a Kd ratio that is lower than that of human TF. In another specific aspect of the present invention, the isolated human antibody has a relationship with human TF that is lower than 10_π Μ. In another specific aspect of the present invention, the isolated human antibody has a Kd ratio of less than 1 (T12 Μ) with human TF. In the present invention In another specific aspect, the isolated human antibody has a ratio of less than 1 (T13 M to human TF). In another specific aspect of the invention, the isolated human antibody has The Kd ratio with human TF is lower than HT14 M. In another specific aspect of the present invention, the isolated human antibody has a heart ratio with human TF below 1 (15M. 30 200407427 in the present invention In another specific aspect, the method for preparing a human antibody includes testing the antibody in a TF-induced clot analysis, and screening can be performed in this analysis with IC5 () 値 below 1 nM inhibition A clot-forming human antibody. In another embodiment, the IC5Q is below 500 pM. In another embodiment, the IC50 is below 200 pM. In another embodiment, In the sample, the IC50 is below 100 pM. In another embodiment, the IC5Q is below 50 pM. In another embodiment, the IC50 is below 10 PM. In another specific aspect, the IC5Q is less than 5 pM 0. In another specific aspect of the present invention, the IC5Q is used for preparing human antibodies. The method includes testing antibodies in a TF-induced clot analysis and screening for IC5Q 値 + 1 nM with IC5Q 値 lower than FFR-rFVIIa in this analysis, such as IC5G 値 + 500 lower than FFR-rFVIIa pM, preferably below IC5Q 値 + 200 pM of FFR-rFVIIa, preferably below IC5Q 値 + 100 pM of FFR-rFVIIa, such as IC5G 値 + 50 pM below FFR-rFVIIa, preferably below FFR- rFVIIa's IC5G + 10 pM is better than FFR-rFVIIa's IC5QF + 5 pM, more preferably lower than FFR-rFVIIa's IC5G 値, which inhibits the formation of clot-forming human antibodies. It should be understood that the specific IC50 as described in TF-induced clot analysis refers to when normal human plasma is used. In another embodiment of the present invention, the method for preparing human antibodies includes testing antibodies in FXa yield analysis and screening for IC50 値 less than 100 nM (in one case, the FVIIa concentration is 0 · 1 nM In the analysis performed) human antibodies that inhibit FXa production. In another specific aspect, the 31 200407427 IC5Q series is below 10 nM. In another specific aspect, the IC5Q series is lower than 5 nM. In another specific aspect, the IC5. Actinide is below 1 nM. In another specific aspect, the IC5Q ratio is lower than 0 · 1 nM. In another specific aspect of the present invention, the method for preparing human antibodies includes testing the antibodies in FXa yield analysis and screening for IC5G 値 lower than FFR-rFVIIa IC50G + 100 nM (in this analysis Use 0. 1 nM FVIIa), for example, IC5G 値 + 10 nM lower than FFR-rFVIIa, preferably lower than IC5G 値 + 5 nM lower than FFR-rFVIIa, more preferably IC5Q 値 + 1 nM lower than FFR-rFVIIa, more preferably lower than IC5Q of FFR-rFVIIa 値 + 0. 1 nM, more preferably IC5G 値 than FFR-rFVIIa, inhibits human antibodies produced by FXa. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a FVIIa / TF amidolysis assay and screening the IC5Q to be less than 100 nM (at a concentration of FVIIa of 10 nM (Analyzed below) Human antibodies that inhibit FVIIa amidinolytic activity induced by TF. In another specific aspect, the IC50 is below 40 nM. In another specific aspect, the IC5Q ratio is below 20 nM. In another specific aspect, the IC50 is less than 10 nM. In another specific aspect, the IC5Q is less than 5 nM. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a FVIIa / TF amidine decomposition assay and screening for IC5Q (lower than FFR-rFVIIa) IC5Q (+100 nM). (10 nM FVIIa is used in this analysis), for example, IC50 値 +40 nM lower than FFR-rFVIIa, preferably IC5G 値 +20 nM lower than FFR-rFVIIa, preferably 32 200407427 lower than FFR-rFVIIa The IC5Q 値 +10 nM is better than the IC50 値 of the FFR-rFVIIa, which inhibits the human antibody of the FVIIa amidolytic activity induced by TF. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a FVIIa competition assay and screening for a human antibody that can compete with FVIIa for binding. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a TF ELISA assay and screening for a human antibody that can bind to human TF. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a direct TF ELISA analysis including immobilized TF and screening for a human antibody that can bind to the immobilized human TF. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in an indirect TF ELISA analysis including TF in solution and screening for a human antibody that can bind to human TF in solution. In another specific aspect of the present invention, the method for preparing a human antibody includes testing the antibody in an FXa production analysis using TF-expressing cells, and screening the IC5 () 値 below 500 nM ( Human antibodies that inhibit FXa production in an analysis performed at a FVIIa concentration of 1 nM. In another specific aspect, the IC5 () is less than 100 nM. In another specific aspect, the IC5 () ratio is less than 50 nM. In another specific aspect, the IC5Q ratio is less than 10 nM. In another specific aspect, the IC50 is less than 5 nM. In another specific aspect of the present invention, the method for preparing human antibodies 33 200407427 includes testing antibodies in an FXa production analysis performed by TF-expressing cells, and screening for IC5Q that is IC5G 値 + 500 nM (1 nM FVIIa is used in this analysis), for example, IC5Q 低于 + 100 nM lower than FFR-rFVIIa, preferably lower than IC5〇 値 + 50 nM, lower than FFR-rFVIIa, preferably lower than FFR -rFVIIa's IC5Q 値 + 10 nM, better than FFR-rFVIIa's IC5QF + 5 nM, more preferably lower than FFR-rFVIIa's IC5Q 値, inhibit human antibodies produced by FXa. The term "TF-expressing cell" means any mammalian cell that can express human TF. In another specific aspect of the present invention, the method for preparing a human antibody includes testing the antibody in a complete cell TF binding assay, and screening for those that can compete with FVIIa for binding to human TF expressed on the surface of a complete cell Human antibodies. In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in a biosensor analysis, and screening for a human antibody having a Kd 値 of less than 100 nM that binds to human TF. In another specific aspect, the 1 line of binding to human TF is less than 10 nM. In another specific aspect, the Kd 値 system bound to human TF is less than 5 nM. In another specific aspect, the Kd 値 of human TF binding is below 1 nM. In another specific aspect, the Kd 値 associated with human TF is lower than 0.5 nM. In another specific aspect, the Kd 値 that binds to human TF is lower than M. In another specific aspect, the Kd 値 of the human TF binding is below 10.11M. In another specific aspect, the Kd 値 binding to human TF is lower than 10 · 12M. In another specific aspect, the Kd ratio of the 34 200407427 combined with human TF is lower than 1 (T13 M. In another specific aspect, the 1 ratio of the combined human TF is lower than 10 · 14 M. In another specific aspect, the Kd 値 binding to human TF is lower than M. In another specific aspect of the present invention, the method for preparing a human antibody includes MAPK signal analysis We tested antibodies and screened human antibodies that could inhibit FVIIa-induced activation of MAPK signals. In a specific aspect, the anti-system inhibited the activation of MAPK signals induced by FVIIa- by 98%. In a specific aspect, the resistance system inhibits the activation of MAPK signal induced by FVIIa- by 90%. In a specific aspect, the resistance system inhibits the activation of MAPK signal induced by FVIIa- by 70%. In a specific aspect, the resistance system inhibits the activation of MAPK signal induced by FVIIa- by 50%. In a specific aspect, the resistance system inhibits the activation of MAPK signal induced by FVIIa- by 30%. The term "MAPK signal" means that it mediates mitogenicity Activated protein kinase (MAPK) or its homologous protein cascades in the intracellular event of activation in response to various extracellular stimuli. Three different MAP kinases have been identified in mammalian cells: 1) Extracellular regulatory kinases (Erk 1/2 or P44 / 42), 2) c-Jun N-terminal kinase (JNK) and 3) p38 kinase. The Erkl / 2 pathway involves the phosphorylation of Erk 1 (p44) and / or Erk 2 (p42). Activated MAP kinases such as p44 / 42 MAPK can be translocated into the nucleus, where they can phosphorylate and activate transcription factors including (Elk 1) and signal transductors and activators (Stat) of transcription. Erkl / 2 can also phosphorylate the kinase P90RSK in the cytoplasm or in the nucleus, and then p90RSK can reactivate a number of 35 200407427 transcription factors. The term "protein kinase" is intended to include enzymes capable of phosphorylating serine and / or threonine and / or tyrosine present in peptides and / or proteins. The term "activation of MAPK signals induced by FVIIa-" means that FVII binds to TF in mammalian cells and thereby induces MAPK signals. In another specific aspect of the present invention, the method for preparing a human antibody includes testing the antibody in a gene expression analysis (Analysis 15), and screening to inhibit the induction by FVIIa- independently selected from the listed Fra_1, Human antibodies for the up-regulating effects of Id2 and Cyr61 genes. It will be appreciated that anti-TF antibodies that inhibit TF activity can bind to different epitopes present in TF and may simultaneously inhibit the binding of FVIIa or FX or FXa to human TF. An object of the present invention is to screen antibodies that inhibit the binding of FVIIa to human TF and thereby inhibit the generation of intracellular signals induced by FVIIa. In another aspect of the present invention, the method for preparing human antibodies includes testing antibodies in a human cancer analysis (Analysis 13) and selecting human antibodies that can inhibit the growth or metastasis of human cancer. In another specific aspect, the isolated human antibody can inhibit the up-regulation of genes independently selected from the listed Fra_1, Id2 and Cyr61 § induced by FVIIa-. _ In another specific aspect of the present invention, the isolated human antibody does not inhibit the binding of FX or FXa to human TF. In another specific aspect of the present invention, the isolated human antibody can inhibit cells TF activity. 200407427 In another embodiment of the present invention, the method for preparing a human antibody includes testing the antibody in an epitope mapping assay, and screening for a human antibody that can bind to a better antigenic epitope on TF. In a specific aspect of the invention, the preferred epitope comprises the residue Trp45. In a specific aspect of the invention, the preferred epitope comprises the residue Lys46. In a specific aspect of the invention, the preferred epitope comprises the residue Tyr49. In another embodiment of the present invention, the isolated human antibody can bind to an epitope located in the interface between TF and FVIIa. The residues determined by the X-ray structure (Beneng et al., 1996 Nature, 380: 41-46) that can perform the interaction between the FVIIa protease domain and TF are: Ser39, Gly43, Trp45, Ser47, Phe50, Arg74, Phe76, Tyr94, Pro92. This interface between TF and FVIIa is characterized by a complex interfacial region containing many intermolecular hydrogen bonds that can cause many intimate contacts between TF and FVII to obtain a high specificity during the FVIIa binding process. The invention also relates to high affinity human monoclonal antibodies to TF. The TF surface containing the contact interface with the FVIIa protease domain possesses a special topology that is easy to react and generate protein-protein interactions. Another type of protein-protein interactions forms a complex between an antibody and a protein ligand. Therefore, a monoclonal antibody against this antigenic epitope on human TF can provide high affinity Mabs. One aspect of the present invention is a high-affinity human monoclonal antibody capable of immunoreacting against the contact interface to the FVIIa protease domain. The human TF antibody of the present invention acts as an antagonist against TF-mediated coagulation induction 37 200407427, thereby inhibiting the binding of coagulation factor FVIIa to TF and thereby blocking thrombin production and subsequent fibrin deposition. Human TF antibodies are particularly useful for administration to humans for the treatment of various conditions involving intravascular coagulation. As such, human TF antibodies can be used to inhibit TF activity resulting in, for example, inhibition of blood coagulation, thrombosis or platelet deposition. Furthermore, the human TF antibody (acting to inhibit the intracellular function of TF, the signal function of TF) according to the present invention can be used in conditions such as sepsis, inflammation, atherosclerosis, restenosis, or cancer. Human TF antibodies are useful in a variety of diseases. It covers diseases or disorders related to thrombosis or coagulopathy, including inflammatory reactions and chronic thromboembolic diseases or disorders associated with fibrin formation, including vascular diseases such as deep venous thrombosis, arterial thrombosis, surgery Postthrombosis, coronary artery bypass graft (CABG), percutaneous coronary angioplasty (PTC A), stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, atherosclerosis and restenosis after angioplasty , Acute and chronic indications such as inflammation, septic shock, sepsis, hypertension, adult respiratory distress (ARDS), systemic inflammatory response syndrome (SIRS), diffuse intravascular coagulation (DIC), pulmonary embolism, platelets Deposition, myocardial infarction, or arteriosclerotic vessels at risk of thrombosis, venous occlusive disease caused by peripheral blood progenitor cell (PBPC) transplantation, hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura ( TTP) and other diseases or conditions for the preventive treatment of mammals. Human TF antibodies can be used to prevent thrombotic thrombosis in patients identified to be at high risk, such as in those undergoing surgery or suffering from congestive heart failure. Human TF anti-38 200407427 is particularly useful for the treatment of intimal hyperplasia or regeneration caused by acute vascular trauma.  narrow. Acute vascular trauma is injury that occurs quickly (that is, hours to months), rather than chronic vascular trauma (such as arterial atherosclerosis) that develops throughout life. Acute vascular trauma often results from surgical procedures such as vascular reconstruction using techniques such as angioplasty, endarterectomy, arterial atherosclerosis, and placement of vascular implants. Hyperplasia may occur as a delayed response to, for example, an implant placement or organ transplant response. Because human TF antibodies are more selective than heparin, they only bind to TF that has been exposed to the wound site, and because human TF antibodies do not destroy or inhibit other coagulation proteins, they are more effective and should be used to prevent deep vein thrombosis May be less likely to cause bleeding episodes than heparin. As shown in the following examples, the human TF antibody of the present invention can selectively bind to cell surface TF and limit its functional activity by inhibiting the binding of coagulation FVIIa and TF. Human TF antibodies that remain bound to TF can inhibit platelet accumulation at the site of vascular wounds by blocking thrombin production and subsequent fibrin deposition. Because human TF antibodies can block thrombin production and limit platelet deposition at the site of acute vascular trauma, human TF antibodies that maintain binding to TF and thereby inhibit FVIIa binding can be used to inhibit vascular restenosis. Compositions containing human TF antibodies are particularly useful in methods of treating patients when formulated into pharmaceutical compositions, where they can be administered to patients suffering from various disease states to treat coagulation-related conditions. Such human TF antibodies, which can be combined with TF to inhibit the binding of FVIIa and TF, have a longer plasma half-life and therefore have a longer anticoagulant activity time compared to other anticoagulants. In the medical instructions of the subject composition of the present invention, coagulants are generally used for treatment, such as deep venous thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIC), lung and kidney blood related to sepsis. Fibrin deposition, anti-phospholipid syndrome (APS), atherosclerosis and myocardial infarction. These compositions can be used to inhibit mechanical vascular trauma, such as by surgery, microsurgery, balloon angioplasty, endarterectomy, reductive atherosclerotic plaque, fixed mold placement, laser light treatment Or damage caused by rotational vibration, or secondary trauma to vascular implants, molds, bypass implants, or organ transplants. These compositions are therefore useful for inhibiting platelet deposition and related diseases. Thus, a method of inhibiting coagulation, vascular restenosis, or platelet deposition, for example, comprises administering to a patient a composition comprising a human TF antibody in an amount sufficient to effectively inhibit coagulation, vascular restenosis, or platelet deposition. These methods have also been found to be useful in the treatment of patients with acute coronary occlusion (such as acute myocardial infarction), which involves the administration of human TF antibodies in combination with histone plasminogen activator and can accelerate the thrombolytic effects induced by tPA . The human TF resistance system is administered before, at the same time or immediately after the administration of a thrombolytic agent (such as a tissue cytosinogen activator). According to the present invention, human monoclonal antibodies against human TF can be produced by immunizing transgenic mice (obtained from Medarex) carrying part of the human immune system instead of mouse system with human TF. Spleen cells obtained from these transgenic mice are used to make fusion tumors that secrete human monoclonal antibodies as described (see, for example, Wood et al., International Patent Application WO 91/00906, Kucherapie The et al., PCT Publication WO91 / 10741; Lombok et al., International Patent Application No. WO407 / 03918, 20040427, Kay et al., International Patent Application No.  92/03917, Lomberg, N. Et al. 1994 Nature 368: 856-859; Greene, L1. Et al. 1994 Natural Genetics 7: 13-21; Morrison, S. L. Et al. 1994, Proc.  Natl.  Acad.  Sci USA 81: 6851-6855; Brugeman et al. 1993 Immunology 7: 33-40; Tyrone et al. 1993 PNAS 90: 3720-3724; Brugeman et al. 1991 European Journal of Immunology 21: 1323- 1326) o human monoclonal antibodies against human TF can also be made by phage display technology. Human antibody libraries can be constructed from immunized humans and displayed on the surface of filamentous phage. In many cases, high affinity human single-chain Fv (ScFv) and Fab antibody fragments have been isolated using panning techniques in which the antigen of interest is immobilized on a solid surface, that is, a microtiter plate or bead (Babes C. F. , III and Burton, D. R. Biotechnology Trends 1996, 14: 230-234; Et al., Human Antibodies 1997, 8: 155-68). Phage display systems with large primary libraries may also directly isolate human antibodies without immunization (Dihard H. L et al., Biochemical Journal 1999, 18218-18230). [Embodiment] The present invention is further illustrated by the following examples, however, they are not intended to limit the protection scope of the present invention. The substance (individually or in combination with each other) disclosed in the foregoing description and the following embodiments is a form for understanding the preferred form of the present invention. Example 1 Preparation of Human Mab with Immunospecificity to Human TF 41 200407427 Reagent Human TF can be Leo, L. V. Μ. , Thrombosis Research, 51: 373-384 1988, isolated from the human brain. Lipidized recombinant human TF (Dade, Innovin, Beckster) can also be used as a human thromboplastin reagent. Rat, rabbit, baboon and porcine prothrombin kinase were prepared from brain tissue. Add twice the volume at 45 ° C to 0. 9% NaCl was added to the brain tissue, and the tissue was homogenized with a human glass homogenizer. After incubating at 45 ° C for 30 minutes with occasional shaking, the samples were centrifuged at 2000 Xg for 20 minutes. Discard the pellet and store a small aliquot of the supernatant at -80 ° C until use. Relipidated TF can be obtained by reconstructing a recombinant human full-length TF (American Diagnostic # 4500) in a phospholipid carrier (PC / PS 75/25). Biotinylated human TF was prepared as follows: Biotin-NHS (η-succinyl-based biotin, Sigma-Delta-1759) was dissolved in DMF (dimethylformamide) to a concentration of 1. 7 mg / ml. Add 60 μl of this biotin-NHS solution to 1 mg / ml dissolved in 0. 1 M NaHC03 buffer in human TF and allowed to react at room temperature for 4 hours. The solution containing biotinylated TF was dialyzed against PBS-buffer overnight. The FVIIa used is such as Tim, L. Recombinant human FVIIa prepared as described in Biochem 27: 7785-7793, 1988. sTF: Substantially like Furigad, P. -Ο. Recombinant human soluble and expressed in E. coli as described in Protein Science 5, 153M540 (1996), et al. 42 200407427 S2288: Reconstituted in H2O with a concentration of 17. 24 mg / ml (Chromogenix) FX: Purified human plasma FX (HFX, Enzyme Research Laboratories Co., Ltd.) FXa: Purified human plasma FX is activated with Russell Snake Venom (HFXa, Enzyme Research Laboratories Co., Ltd.) Co., Ltd.) Chromogenic enzyme X: dissolved in H20, the concentration is 1. 25 mg / ml (Bringa Ingram) 125I-FVIIa was prepared by standard radiometric calibration procedures. FFR-rFVIIa: The active site of FVIIa is blocked with D-Phe-L-Phe-L-Arg-chloromethyl ketone. Such as Sorison B. B. Et al., Journal of Biochemistry 272: 1 1863-1 1868, 1997. Immune effect Human TF is emulsified in Freund's complete adjuvant. HuMab mice or their hybrid offspring (Medarex) were given a 40 microgram dose via subcutaneous injection. After 14 and 28 days (and a substantial interval of 14 days), mice were similarly injected with another 20 micrograms of TF emulsified in incomplete Freund's adjuvant. Blood was sampled 10 days after the last injection and tested for the presence of human TF-specific antibodies in the blood pupa by TF ELISA (Analysis 1 and 2). Fusion Mice tested positive for blood clots in Analysis 1-3 were injected intravenously with an additional 20 micrograms of human TF and sacrificed three days later. The spleen was removed aseptically and dispersed into a single cell suspension. Fox-myeloma cells were grown in CD fusion tumor medium (Gibco 43 200407427 11279-023). In this fusion experiment, spleen cells and myeloma cells (P3x63 Ag8. 653, ATCC CRL-1580) and Sp2 / 0 (ATCC CRL-1581). Cells were seeded in microplates and incubated at 37 ° C. Then change the medium three times in two weeks. One hundred microliters of supernatant was removed from each tank of fused tumor cell culture and tested for the presence of TF-specific antibodies in a TF ELISA (Analysis 1 and 2). Example 2: Screening of various analytical methods used to screen for the presence of specific selected antibodies in blood slugs and culture supernatants are described below: ^ Direct TF ELISA analysis (Analysis 1): The g-immunoplate was coated with human sTF in 1 ug / ml in PBS. The plate was blocked with a blocking agent (TBS containing 5 mM CaCl2 and 2% BSA) and TBS + 0. Wash with 05% Tween 20 and add mash from the fusion tumor cells. After incubating at room temperature for 1 hour, the plate was washed and anti-human IgG labeled with horseradish peroxidase (HRPO) was added. After another one hour of incubation, the plate was washed and colorized with TMB-substrate (Kem-EN-Tec) according to the manufacturer's operating manual. The absorbance was measured on an ELISA-reader at 450 nm. Indirect TF ELISA analysis (Analysis 2): Nock-immunoplates were coated with 0. Goat anti-human IgG (Southern Biotechnology Association, Cat- # 2040-1) stored in PBS at 5 µg / ml and incubated overnight at 4 ° C 44 200407427. The plate was blocked with blocking buffer (TBS with 5 mM CaCl2 & 2% BSA) and blocked with TBS + 5 mM CaCl2 + 0. Wash in 05% Tween 20. The mash obtained from the fusion tumor cells was added, and the plates were incubated at room temperature for 1 hour. After another washing, biotinylated human sTF was added at a concentration of 1 μg / ml and incubated for 1 hour. After washing, the streptavidin-HRPO solution was added and incubated for one hour. The plate was colored as described in Analysis 1 with TMB-substance. FVIIa competition analysis (Analysis 3): The NOCK-immunoplate was incubated with human STF (5 μg / mL in PBS) overnight. The plate was washed and blocked in TBS buffer containing 5 mM 0:30 and 2% BSA. Anti-human TF Mabs were added and the plates were incubated for 2 hours. After the plate was washed, biotinylated human FVIIa was added (1 μg / ml in TBS buffer containing 5 mM CaCl2 and 2% BSA) and the plate was incubated for 1 hour. The plate was washed again and then HRPO-calibrated streptavidin was added and incubated for 45 minutes. After washing the flat plate again, the color was developed using TMB-substrate (Kem-EN-Tec) according to the manufacturer's operation manual. Inhibitory effect of FVIIa / sTF amidolytic activity (Analysis 4): Inhibition of amidolytic activity catalyzed by FVIIa-TF caused by anti-human TF Mabs uses soluble human TF (10 nM), recombinant human FVIIa (10 nM) and increasing concentrations of Mabs (0. 0122-50 nM). Various concentrations of anti-human TF Mabs or FFR-rFVIIa with 10 nM TF and 10 nM FVIIa in BSA buffer (50 mM Hepes, pH 7. 4, 100 mM NaCl, 5 mM 0 &amp; (: 12 and 1 mg / ml BSA) at room temperature 45 200407427 before incubation for 60 minutes, and then the substrate S2288 (1. 2 mM, Chromogenix). The color development process was continuously measured at 405 nm for 30 minutes. The amidolytic activity is expressed in mOD / min. IC5Q 値, which inhibits the inhibition of FVIia / TF amidolytic activity by Mabs. The IC50 F of FFR-rFVIIa in this analysis is 7 + / · 3 nM. Inhibition by FXa (Analysis 5): Before adding FX (50 nM), lipidated TF (10 pM), FVIIa (100 pM), and anti-TF Mabs or FFR-rFVIIa (0-50 nM) were added to Incubate in BAS buffer (see Analysis 4) for 60 minutes. After another 10 minutes of incubation, the reaction was stopped by adding 1/2 volume of stop buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 20 mM EDTA). Add substrate S2765 (0. 6 mM, Chromogenix) and continuous measurement of absorbance at 405 nm for 10 minutes to determine the amount of FXa produced. Mab can be calculated to inhibit IC5Q (R) that is activated by FVIIa / lipidated TF-. IC5 of FFR-rFVIIa in this analysis.値 is 51 +/- 26 pM. Biosensor analysis (Analysis 6): The antibody was tested on a Biacore instrument by passing a standard solution of anti-human TF Mab through a wafer with immobilized human IgG antibodies. Then pass different concentrations of sTF, which are stored in 150 mM NaCl ’10 mM CaCl2 and 0. 0003% Polysorbate 20 of 10 mM hepes pH 7. 4 in. Kd 値 was calculated from the sensor spectra using integrated Biacore estimation software. TF-dependent clot formation analysis (Analysis 7): This analysis was performed on an ACL300 research clot formation device (ILS Practice 46 200407427). Dilute to 50 mM imidazole, pH 7.4, 100 mM.  NaCL. 0. 1% BSA anti-human TF Mabs solution was mixed with 25 mM CaCl2 in a ratio of 2 to 5 and added to the sample cup of the clot forming device. Thromboprokinase from humans, rats, rabbits, baboons, or pigs was diluted with the imidazole buffer, and the antibody-free samples placed in the reagent tank 2 and The clot formation time of human, rat, rabbit, baboon or pig plasma is approximately 30 seconds. During the analysis, the antibody and CaCl2 mixture was transferred to 25 microliters of prothrombin kinase reagent and precultured for 900 seconds before adding 60 microliters of plasma. The maximum clot formation time was set to 400 seconds. This thromboplastin dilution was used as a standard curve to convert clot formation time to TF activity relative to a control group without added anti-TF Mabs or FFR-rFVIIa. IC5 of FFR-rFVIIa in this analysis.値 is 4. 4 +/- 0. 4 pM. Inhibition of FX activation catalyzed by FVIIa / cell surface TF by Mabs (Analysis 8): Cells expressing human TF using a single layer, such as human lung fibroblasts WI-38 (ATCC No.  CCL-75), human gallbladder cancer cell line J82 (ATCC No.  HTB-1), human keratinocyte cell line CCD 1102KerTr (ATCC no.  CRL-2310), human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231 serve as TF sources for FX activation catalyzed by FVIIa / TF. Place monolayers of cell-filled monolayers in 24-, 48-, or 96-well plates in buffer A (10 mM Hepes, pH 7. 45, 150 mM NaC, 4 mM KC1 and 11 mM glucose) and once in buffer B (buffer A supplemented with 1 mg / ml BSA and 5 mM Ca2 +). FVIIa (1 nM), FX (135 nM) and Mab at different concentrations were dissolved in 47 200407427 buffer B.  (Or FFR-rFVIIa) to the cells at the same time. Pre-incubate cells with anti-TF Mabs or FFR-rFVIIa for 15 minutes before adding rFVIIa and FX. Fxa formation was allowed to proceed at 37 ° C for 15 minutes. Remove 50-microliter aliquots from each well and add to 50 microliters of stop buffer (Buffer A supplemented with 10 mM EDTA and 1 mg / ml BSA). By transferring 50 μl of the above mixture to a microtiter pan and adding 25 μl of the chromogenic enzyme Chromozym X (final concentration 0. 6 mM) to each well to determine the total amount of FXa produced. The absorbance was measured continuously at 405 nm and the original color rendering rate was converted to FXa concentration using the FXa standard curve. In this analysis, the IC50 of FFR-rFVIIa is 1. 5 nM. Inhibition of 125rFVIIa binding to cell surface TF by Mabs (Analysis 9): Combining research uses cells expressing human TF, such as human lung fibroblasts WI-38 (ATCC No.  CCL-75), human gallbladder cancer cell line J82 (ATCC No.  HTB-1), human keratinocyte cell line CCD 1102KerTr (ATCC no.  CRL-2310), human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231. Monolayers in 24-well tissue culture plates were washed once with buffer A (see analysis 8) and with buffer B (see analysis 8). The monolayer was pre-incubated with 100 µl of ice buffer B for 2 minutes. Different concentrations of Mab (or FFR-rFVIIa) and FVIIa (0. 5 nM mI-FVIIa) were simultaneously added to the cells (final volume reached 200 μl). The flat plate was incubated at 4 ° C for 2 hours. After the incubation is completed, the unbound material is removed, and the cells are washed four times with ice-cooled buffer B 48 200407427 and 300 microliters of lysis buffer (200 mM NaOH, 1% SDS and -10 mM EDTA). Dissolve. Radioactivity was measured in a Garma meter (Cobra, Packard Instruments). GraFit4 (Ericas Software Limited, UK) was used to analyze the combined data and perform curve fit. IC5 of FFR-rFVIIa in this analysis.値 is 4 nM. Inhibition of p44 / 42 MAPK activation induced by FVIIa / TF by Mab (Analysis 10): Phosphorylated p44 / 42 MAPK was determined by quantitative detection of chemiluminescence (Fuji film LAS-1000) from Western blot analysis And / or Akt, and / or P90RSK. Prior to the experiment, cells expressing human TF, such as CCD 1102KerTr, NHEK P166, human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231 were cultured at 0-0. 1% FCS medium for 24 or 48 hours to allow cells to rest. The cells must be 70-80% confluent on the day of the experiment. This experiment was performed by pre-incubating the cells with excess Mab or FFR-rFVIIa in a blood-free medium at 37 t for 30 minutes, and then adding 10-100 nM FVIIa and incubating for 10 minutes. The cells were washed twice in ice-cooled PBS and then placed in lysis buffer (20 mM Tris, 0.1 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 10 mM β-glyceryl phosphate, 5 mM sodium pyrophosphate, 150 mM NaCl, pH 7. 5 contains 0. 1 mM 4- (2-aminoethyl) benzenesulfonium fluoride (AEBSF) and 1 mM benzamidine. Add before use: 1 mM sodium orthovanadate, 5 μg / ml Leprotinin, 10 μg / ml Aprotinin) to dissolve. The lysate was mixed with the SDS-sample buffer and loaded onto an SDS-polyacrylamide gel. A series of standard biological samples were loaded on each gel 49 200407427. Proteins to be separated on SDS-polyacrylamide gels.  It was transferred to the nitrocellulose membrane by electroporation, and the kinases P44 / 42 MAPK, Akt, and 90RSK were visualized with phosphate-specific antibodies, and the chemiluminescence was quantified by Fujifilm LAS-1000. Epitope Mapping Analysis (Analysis 11): Preparation of Soluble TF (sTF) Variants Using Reverse PCR (QuikChange, Starkins, La Joule ’

CA, USA) Constructed sTF variants (122C, W45C, K46C, Y94C, F140C, W158C, K201C) using wild-type plastids (Frigard et al., Protein Science 5,1531-1540, 1996) as templates . As previously described (Frigard et al., Protein Science 5,1531-1540, 1996) and with minor modifications, wild-type and these variants were expressed and purified in E. coli. Cell lysis was performed using X-crush (Biox, Sweden) technology in 10 mM Tris-HCl buffer, pH 7.5, then resuspended in the same buffer and 1 mg of DNAse was added. Centrifuge the solution at 11000 Xg at 4 ° C for 20 minutes, and place the endosomes in 75 ml of 6 M GnHCl, 0.5 M NaC and 20 mM

Tris-HCl, denatured at pH 8.0. After incubation at room temperature for 1 hour, the denatured protein was diluted dropwise in 1 liter of a solution containing 50 mM Tris-HCl, (U5 M NaCl, pH 8.0 and refolded with gentle stirring for about 2 hours. Purification was performed using Q-Sepharose Fast Flow (Famacia, Eusala, Sweden) and FVIIa affinity chromatography as described by Ferrigard et al. (1996). Protein homogeneity was examined by SDS-PAGE Degree. Measured under ^^^ and determined the concentration using the calculated extinction coefficient of 37440 Minr1 (Jill and Van Hieb, 1989). 200407427 MaxiSorp flat plate (Nunc-Immuno) coated with wild type dissolved in TBS sTF and these variants (10 μg / ml) were blocked with blocking buffer (TBS containing 0.1% Tween 20 and 0.5% BSA). The plate was washed with washing buffer (TBS and 0.1% Tween). 20) Rinse. Add anti-human TF Mabs in blocking buffer at a concentration of 1 ng / ml and incubate for one hour. Then wash the plate with washing buffer (6X). Then use the HRP-calibrated anti-human in blocking buffer at a dilution of 1: 2000 (Hillika Biosystems has The company) uses TMBplus-substrate (Kemtech Catalog No. 4390A) to detect antibody binding. The final ELISA signal (OD45q.62 ()) is used to measure the binding of each antibody to all STF variants. Tracing method (Analysis 12): Mix human prothrombin protokinase (for example, Innovin, Dade, Behring, final dilution 50,000 乂) with 0 &amp; (: 12 (final concentration 16.7 11 ^) and anti-1 Mabs and mix Incubate at room temperature for 15 minutes. Add citrate-stabilized human whole blood (280 microliters) to a roteg sample cup (Pentapharm) and preheat at 37 ° C for 5 minutes. Microliter prothrombin protokinase / CaCV anti-TF Mab mixture was added. Elastic clotting was subsequently performed in a RoTEG device (Pentapharm) for 1 hour. CoagPr0 software ™ (Medical Sciences, Assas, Denmark) A rate analysis graph was obtained from a coagulogram. Example 3 Analysis of human cancer. To investigate the therapeutic efficacy of human anti-Mabs on the growth and metastasis of human cancer in the mouse model (Analysis 13) 200407427 Treatment: administered via bolus injection iv Human anti-TF Mabs; 10 mg / kg = 0.1 mg / 10 g; the injection volume is 0.1 ml per gram of any of the following three treatment solutions: A. vehicle control group B. 1 mg / ml human FFR-rFVIIa C. 1 mg / ml anti- -TF Mabs mode description: I. Primary growth and liver metastasis of colon cancer

Healthy female athymic mice (UwAw) aged 7-8 weeks were used. In order to destroy the residual immune resistance generated by the implantation of human cells in nude mice, the mice were routinely irradiated with 5 Gy for 2 days before human tumor transplantation (Foger et al., 1997). As described (Li et al., Human Gene Therapy 10: 3045-3053, 1999) by implantation of LS1747T human colon cancer cells (ATCC CCL 188) cultured in RPMI 1640 containing 15% fetal bovine blood pupa (FCS) Free of mice. Briefly, cells were harvested with trypsin-EDTA, washed twice, and resuspended in bloodless RPMI supplemented with heparinized sodium solution (1 U / ml). A small subcostal incision was then made under anesthesia and 3 3 106 LS174T cells in 50 ml of phosphate buffered saline were injected into the spleen. After 3 to 5 minutes, the spleen blood vessels were ligated and the spleen was surgically removed. This procedure will cause stable liver metastases (more than 95%). Treatment was started immediately after implantation with anti-TF Mabs and continued until the full phase of investigation. Mice were sacrificed on days 15 and 30 of tumor cell inoculation, the liver was removed and weighed, and the number of visually detectable tumor nodes on the surface of the liver was counted. Liver samples were fixed in AFA 52 200407427 (5% acetic acid, 75% ethanol, 2% formalin, 18% water) overnight, transferred to 100% ethanol, and embedded in paraffin to prepare 5-mm sections for tissue Quantitative metastasis of nodules for quantitative analysis of immunohistology and apoptosis. Investigate 1-1: Objective: To inject iv injection of iv; 10 mg / kg anti-TF Mabs for LS174T colon tumors. The effect of macroscopic growth and liver metastasis in nude mice. Mice: 60 homozygous nu / nu 6-week-old NMRI males. Grouping: Mice were randomly divided into four groups of 15 mice each and treated with solutions A, B or C. Termination: Mice will be terminated when weight loss &gt; 20% or other obvious signs of severe poisoning appear. Parameters: Tumor size was recorded daily at two right-angle diameters during the growth phase. The weight was recorded immediately and 2-3 times a week. Autopsy measures Metastatic nodule formation in the liver. II. Primary Growth and Lung Metastasis of Breast Cancer Human breast cancer cells MDA-MB-231 (ATCC HTB26) were cultured in Duchenne's Modified Inger's Medium (DMEM) supplemented with 10% fetal bovine blood pupa (FCS). MDA-MB-231 cells (3 106) were injected subcutaneously into nude mice (7- or 8-week-old female mice). Primary tumor growth and metastasis were assessed as previously described (Li et al., Human Gene Therapy 12: 515-526, 2001). Study II-1:

Purpose: to inject i.v .; 10 mg / kg anti-TF 53 200407427

The effect of Mabs on the macroscopic growth and liver metastasis of MDA-MB-231 breast tumors in nude mice. Mice: 60 homozygous nu / nu 6-week-old NMRI males. Grouping: Mice were randomly divided into four groups of 15 mice each and treated with solutions A, B or C. Termination: Mice will be terminated when weight loss &gt; 20% or other obvious signs of severe poisoning appear. Parameters: Tumor size was recorded daily at two right-angle diameters during the growth phase. The weight was recorded immediately and 2-3 times a week. Autopsy measures Metastatic nodule formation in the lungs. III. Primary Growth of Glioblastoma Tumor Xenografts The tumor cell line MG U373 is a human glioblast polymorphic cell line with high angiogenic activity, high vascular density, and rapid growth in nude mice. Tumors are inoculated into the flank, followed by standard procedures (see proposals disclosed in experimental plans). Mice were observed twice daily for signs of poisoning and tumor size was measured daily with two vertical diameters. Tumors were transplanted into the ribs of nu / nu homozygous nude mice with an NMRI background. Mice were obtained from 7-week-old male mice from M &amp; B (Ry, Denmark). Animals are fed in a living organism environment and are allowed to receive an unlimited amount of sterile food pellets and water in water. Three different studies were performed using this glioblastoma tumor model: Study III-1: Objective: To inject iv to the study group administered iv; 10 mg / kg of anti-TF Mabs against U373 tumors in nude mice The effect of macroscopic growth. 54 200407427 Mice: 60 homozygous nu / nu 6-week-old NMRI males. Grouping: Mice were randomly divided into four groups of 15 mice each and treated with solutions A, B or C. Termination: Animals will be terminated when weight loss &gt; 20% or other obvious signs of severe poisoning appear. Parameters: Tumor size was recorded daily at two right-angle diameters during the growth phase. The weight was recorded immediately and 2-3 times a week. Study III-2: Objective: To study the effect of injection of i.v .; 10 mg / kg anti-TF Mabs on the macroscopic growth of U373 tumors in nude mice. After pre-treatment tumor growth has been established. Mice: 60 homozygous nu / nu 6-week-old NMRI males. Grouping: Mice were randomly divided into four groups of 15 mice each and treated with solutions A, B or C. Processing begins when 6 consecutive (daily) measurements indicate that Gambertz is growing. This is equivalent to the end of 100-200 mm3: the treatment is continued until the tumor grows to a maximum volume of about 1.0 cm3, that is, the tumor-free diameter is greater than 15 mm, or until 6 consecutive measurements have been established to regenerate Dagonbates. Tumor sections from each group will be obtained at the end for histological and immunochemical evaluation. Animals are about to end when weight loss &gt; 20% or other obvious signs of severe poisoning appear. Parameters: Tumor size was recorded daily at two right-angle diameters during the growth phase. The weight was recorded immediately and twice a week. Study III-3: Objective: To study the effect of anti-TF Mabs on the growth of intracranial U373 tumors in nude mice. Mice: 60 homozygous nu / nu 6 weeks 齢 NMRI male. Tumor: U373 was implanted through the eye into the right hemisphere according to standard procedures. Grouping: Mice were randomly divided into four groups of 15 mice each and treated with solutions A, B or C. End: euthanize mice that show signs of chronic nerve injury. Data: Quantitative survival (i.e. time versus nerve damage) via Kaplan-Meier statistics. Example 4 (Analysis 14) A 0.5 ml matrix gel plunger was placed subcutaneously under the abdominal skin of a mouse (mTF-KO / hTF-KI mouse) in which the TF gene has been knocked out and inserted into the human TF gene . B-FGF (5 ng) was incorporated into the matrix gel and the newly manifested angiogenesis (angiogenesis) in the gel was quantified by measuring the hemoglobin content after one week. Human anti-TF Mabs' inhibitory ability (inhibition percentage of hemoglobin) can be evaluated after single or repeated parenteral administration of these proteins. Example 5 (Analysis 15) To identify the gene expression of antibodies that prevent FVIIa from binding to TF and antibodies that prevent FX from binding to TF Analysis of FHKa-treated BHK-TF cells has been observed in cDNA microarray analysis Three genes are specifically upregulated. These include: Fra-1 (the gene encoding Fos-associated antigen 1), Id2 (the gene encoding a member of the helix-loop-helix protein), and Cyr61, which encodes an extracellular matrix signaling protein. The following analysis is designed for screening to prevent induction by FVIIa 56 200407427

Fra-1, Id2, or Cyr61 up-regulated anti-TF Mabs. Cell cultures Unless otherwise specified, reagents used were purchased from GIBCO-BRL Life Technologies. BHK-TF cells (produced as described by Purson LK et al., J. Biol. Chem. 273, 6228-6232, 1998) are grown in cells containing 10% FCS, 100 IU / ml penicillin, and 100 μg / ml streptomyces In Duchenne's modified Inger's medium to obtain a 95-100% confluence, it was washed and cultured in FCS-free medium for 16-18 hours. The cells were washed again and exposed to FCS-free medium containing 100 nM FVIIa. To select fragments for northern transfection analysis, the cells were treated as described below. BHK_TF cells were grown in Duchenne modified Ingrid medium containing 10% FCS, 100 IU / ml penicillin and 100 μg / ml streptomycin to obtain a 95-100% confluence, washed and re-fCS-free The medium was cultured for 16-18 hours. The cells were washed again and exposed to FCS-free medium containing 100 nM FVIIa for 1 hour. CRL2091 cells (ATCC) were grown in Ehrlich's modified Ingrid medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin to obtain 95-100% confluence. Next, the cells were starved for 16-18 hours and treated with FBS-free medium containing 100 nM FVIIa for 6 hours. Murine 3T3-L1 cells (ATCC) were maintained in Dugar's modified Ingrid medium supplemented with 10% fetal bovine blood pupa, 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells were grown to confluence and contained 1 μM dexamethasone (sigma), 10 μg / ml human insulin (Novo Nordisk A / S) and 1 μM 57 200407427 BRL49653 (Novo Nordisk A / S) The medium was induced for 1 hour. 〇Selected fragments for northern transfection analysis. Using the superscript II kit (Life Technology) according to the manufacturer's manual, reverse transcription PCR was performed from dexamethasone, insulin and The RNA of 3T3-L1 cells treated with BRL49653 for 1 hour was selected to produce Fra-1. Id2 and Cyr61 were cloned from RNA isolated from BHK-TF cells treated with FVIIa for 1 hour and CRL2091 cells treated with FVIIa for 6 hours by reverse transcription PCR. Upstream and downstream primers are: 5'-GCGGCCGCCATGTACCGAGACTACGGGGAACCG-3, and 5'-GCGGCCGCTCACAAAGCCAGGAGTGTAGG-3 'for Fra-1, 55- CAGCATGAAAGCCTTCAGAG-3-3 and 5'-CTCTGGTGATGCAGGCTGAC-3' for M2,5, -CGTCACCCT 3, and 5, -CTTGGTCTOCTGCATTTCT-3 for Cyr61. The parameters for PCR are: a cycle of denaturation at 94 ° C for 10 seconds, bonding at 65 ° C for 15 seconds, and extension at 72t for 1.5 minutes, denaturation at 94t for 10 seconds, bonding at 64 ° C for 15 seconds, and One cycle of extension for 1.5 minutes at 72 ° C, 10 seconds of denaturation at 94 ° C, 15 seconds of bonding at 63 ° C, and one cycle of 1.5 minutes extension at 72 ° C, 10 seconds of denaturation at 94t One cycle of bonding for 15 seconds at 62 ° C and extension of 1.5 minutes at 72 ° C, one cycle of denaturation at 94 ° C for 10 seconds, 15 seconds of bonding at 61 ° C, and 1.5 minute extension at 72 ° C, One cycle of denaturation at 94 ° C for 10 seconds, bonding at 60 ° C for 15 seconds and extension of 1.5 minutes at 72 ° C, denaturation at 94 ° C for 10 seconds, bonding at 55 ° C for 15 seconds and 72 Forty cycles of 1.5 minutes at ° C. All fragments were cloned into TOPO 2.1 (Invitokine) 58 200407427 and sequenced using a Megabase sequencer. Northern Transfection Analysis Use TriZol to follow the manufacturer's instructions to isolate total RNA from BHK-TF cells cultured with FVIIa, FX, ASIS, 1F44A1, or TF8-5G9. 20 micrograms of RNA were placed in a denaturing gel containing 1% agarose, 20 mM MOPS, 5 mM NaOAc, 6% formalin, and 1 mM EDTA for size fractionation, and transferred to a Hybond N + membrane (Ai (Mersen) and fixed by UV crosslinking. [A-32P] dATP (Emerson) The cDNA encoding Fra-Bu Id2 or Cyr61 was calibrated with Prime It kit (Starkin) and hybridized using Express Hyb (cloning technology) according to the manufacturer's instructions and radiated Autoradiography. Example 6 (Analysis 16) MAPK analysis via Elkl transcription factor / luciferase reporter gene (PathDetect) HeLa cells were seeded in T-80 culture flasks up to 40% confluence one day before transfection. Cells were transfected with 150 μg of pFA-Elkl (Starkin), 3 μg of human TF / pcDNA3, and 3 μg of mouse-activated receptor 2 / pcDNA3 as described in the manual using FuGene (Rocky). , L +. The next day the cells were detached with VerseneTM and seeded in black 96-slot inspection plates (Packard) at a cell density of 20,000 cells per well. After the cells had re-adsorbed the plate, the medium was replaced with 160 ml of blood-free Duchenne's Modified Inger's Medium (Invitokine) and cultured for 16 hours. Cells were treated with 20 μl of blood-free 淸 media (control group), 20 μl 59 200407427 2.5 μM FFR-rFVIIa (control group), 20 μl of 2.5 μM anti-TF Mab B or 20 μl of 2.5 μM anti-TF Mab A. Add 20 μl of 0.5 μM FVIIa to one half of the tank and medium to the other half. After 4 hours of incubation, cells were analyzed for luciferase gene. LucLlte (Packard) reagent was added to the cells as instructed by the manufacturer. Read the luciferase expression on a TopCount microplate reader (Packard). [Brief description of the drawings] The present invention will be described with reference to the accompanying drawings in each of the embodiments, where FIG. 1. illustrates the illustrated example of screening for a single human gastric affinity antibody against TF Screening analysis. Figure 2. Detailed illustration of screening analysis 1-3 as described in Example 1 ° Figure 3. Detailed illustration of screening analysis 4-7 as described in Example 1 ° Figure 4. As described in Example 1 The detailed graphic description of the screening analysis 8-10 is described in Figure 5.-Example of screening antibody by No. 4 analysis. Inhibition of sTF / FVIIa amidolytic activity by FFR_ rFVIIa (filled circle) and human anti-TF monoclonal antibody HuTF-31F2 (€ circle). Figure 6.-Example of antibody screening by assay No. 5. Inhibition of factor Xa by FFR-rFVIIa (filled circles) and human anti-TF monoclonal antibody HuTF-31F2 (filled circles). Figure]. An example of screening antibodies by analysis No. 7. Inhibition of human TF-induced clot formation by FFR-rFVIIa (filled circles) and human anti-TF monoclonal antibody HuTF-31F2 (filled circles). Figure 8. —Example of screening antibodies by analysis No. 10. Only anti-TF monoclonal antibodies that prevent 200407427 from blocking FVIIa binding can inhibit TF / FVIIa-mediated signaling. Figure 9. Human anti-TF Mab inhibits P44 / 42 MAPK phosphorylation induced by FVIIa (Analysis 10). BHK cells transfected with TF were subjected to blood starvation for 2 hours to quiescent cells. The antibodies HuMab 30F5 (500 nM) and HuMab 31F2 (500 nM) were added to the cells 15 minutes before FVIIa (15 nN) was added. The cells were lysed and the proteins were separated on SDS-PAGE and transferred to a nitrocellulose membrane by electroporation. Western blot analysis was performed using multiple phosphate-specific antibodies to P44 / 42 MAPK. The secondary antibody was an anti-rabbit IgG conjugated to horseradish peroxidase. Chemiluminescence was performed using a cooled CCD4 eyepiece. The band on the digitized film was quantified and the band measured by FVIIa was set to 100%. A 50% reduction in phosphorylated bands was observed when cells were precultured with HuMab 30F5 (500 nM), and a 25% reduction was observed when cells were precultured with HuMab 31F2 (500 nM). In conclusion, this experiment shows that anti-TF human antibodies (30F5 and 31F2) can partially inhibit the phosphorylation of P44 / 42 MAPK induced by FVIIa. Similar results were observed with 50 nM FVIIa. Figure 10 · —Example of antibody screening by No. 16 analysis. This figure demonstrates that the intracellular activity of TF in TF expressing cells can be inhibited by an anti-TF monoclonal antibody. Anti-TF Mab B inhibits TF intracellular activity, while anti-TF Mab A does not. Figure 11. An example of screening antibodies by analysis No. 12. Rate profile of hemagglutination measured with 0.5 nM of FFR-rFVIIa and human anti-TF antibody HuTF-31F2. 61

Claims (1)

200407427 Patent application patent application ^ ^ ^ 1. An isolated human antibody that reacts with an antigenic epitope on human TF. 2. The isolated human antibody according to item 1 of the scope of the patent application, which inhibits the binding of human coagulation factor Vila to human TF. 3. The isolated human antibody according to any of claims 1 to 2 of the scope of patent application, which is a monoclonal antibody. 4. The isolated human antibody according to any of claims 1 to 3 of the scope of patent application, which is a recombinant antibody. 5. The isolated human antibody according to any one of claims 1 to 4, wherein the antibody is a Fab fragment. 6. The isolated human antibody according to any one of claims 1 to 4, wherein the antibody is a F (ab) 2 fragment. 7. The isolated human antibody according to any one of items 1 to 4 of the scope of the patent application, wherein the antibody is a F (ab,) 2 fragment. 8. The isolated human antibody according to any one of claims 1 to 4, wherein the antibody is a single-chain Fv fragment. 9. The isolated human antibody according to any one of claims 1 to 8 of the scope of the patent application, wherein the antibody has a Kd 値 with human TF in the range of 10-I5-1 (r 8 M. 10. According to The isolated human antibody according to any one of the claims 1 to 9, wherein the antibody has a Kd 値 with human TF in the range of 10_15_1 (T1G M). 11. A pharmaceutical composition comprising A therapeutically effective amount of a human antibody that immunologically reacts with an antigenic epitope presented on 62 200407427 human TF. 12. A pharmaceutical composition comprising a therapeutically effective amount of an epitope presented on human TF A human antibody that undergoes an immune response, wherein the anti-system is the antibody according to any one of claims 1 to 10. 13. A composition comprising an immune response to an antigenic epitope presented on human TF 14. A composition comprising a human antibody that immunologically reacts with an antigenic epitope presented on human TF, wherein the anti-system is the antibody according to any one of claims 1 to 10 of the scope of the patent application. 15 · A method for treating humans with FVIIa / TF-related diseases, comprising administering to the human a therapeutically effective amount of a human antibody that immunologically reacts with an antigenic epitope presented on human TF. 16. A method for treating humans A method for human and FVIIa / TF-related diseases, comprising administering to the human a therapeutically effective amount of an antibody according to any one of claims 1 to 10. 17. A method for preparing a human antibody, Comprising a) preparing human antibodies against human TF, b) testing the antibodies in a TF-induced clot assay and screening for an IC5Q (lower than FFR-rFVIIa) IC5Q (+ 1 nM) in this analysis, For example, IC5Q 値 +500 pM lower than FFR-rFVIIa, preferably IC5 lower than FFR-rFVIIa.値 + 200 pM, preferably lower than the IC50 of FFR-rFVIIa 値 + 100 pM, preferably lower than the IC5 of FFR-rFVIIa.値 + 50 pM, preferably lower than IC5G of FFR-rFVIIa 値 + 10 pM, more preferably lower than IC5Q 値 of FFR-rFVIIa + 5 pM, more preferably lower than IC5Q of FFR-rFVIIa, inhibiting the formation of clots 63 200407427 Human antibody, or test the antibody in FXa production analysis and screen it out. 1 (: 5 "straight to FFR-rFVIIa's IC5Q 100 + 100 nM (using 0.1 nM FVIIa) 'For example, lower than FFR-rFVIIa's IC5G値 +10 nM, preferably lower than IC5Q 値 of FFR-rFVIIa + 5 nM, preferably lower than IC5Q 値 of FFR-rFVIIa + 1 nM, more preferably lower than IC50 of FFR-rFVIIa + 0.1 nM, more preferably IC5Q 値 below FFR-rFVIIa, human antibodies that inhibit FXa production 'or test antibodies in FVIIa / TF 醯 amine decomposition analysis and screen out IC5Q 値 whose IC5Q 値 is lower than FFR-rFVIIa + 100 nM (in this item 10 nM FVIIa) is used in the analysis, for example, IC5G 値 + 40 nM lower than FFR-rFVIIa, preferably IC5Q 値 + 20 nM lower than FFR-rFVIIa, and preferably IC5G 値 +10 nM lower than FFR-rFVIIa A human antibody that is better than the IC50 of FFR-rFVIIa, which inhibits the amine-induced degradation activity of FVIIa by TF, or competes with FVIIa Analyze test antibodies and screen for human antibodies that can compete with FVIIa, or test antibodies in TF ELISA assays containing TF and screen for human antibodies that can immunoreact with human TF. 18. According to item 17 of the scope of patent application A method in which the human anti-human TF system is prepared by: a) immunizing a mammal with human TF, and b) isolating an antibody produced by the immunized mammal. 19. According to the scope of the patent application The method according to item 18, wherein the lactating animal is a mouse. 64 200407427 20. According to the method according to any one of claims Π to 19 in the scope of application patents, which comprises testing antibodies in a TF-induced clot analysis And it can be selected in this analysis that its IC5G 値 IC5QF lower than FFR-rFVIIa + 1 ηM ', such as IC50Q + 500 ρM lower than FFR-rFVIIa, preferably IC5Q lower than FFR-rFVIIa 値+ 200 ρΜ, preferably lower than the IC50 of FFR-rFVIIa 値 + 100 ρM, preferably lower than the IC5Q 値 of FFR-rFVIIa + 50 ρM, preferably lower than the IC5G F of FFR-rFVIIa + 10 ρΜ, more preferably lower than FFR-rFVIIa IC5Q 値 + 5 ρΜ, better Of FFR-rFVIIa in IC5G Zhi, human antibody inhibition of clot formation. 21. The method according to any one of claims 17 to 20 of the scope of patent application, which comprises testing antibodies in FXa yield analysis and screening for IC5Q 値 which is lower than FFR-rFVIIa +100 nM (in the analysis Use 0.1 nM FVIIa), for example, IC5G 値 + 10 nM lower than FFR-rFVIIa, preferably IC5Q 値 + 5 nM lower than FFR-rFVIIa, and preferably IC5 lower than FFR-rFVIIa.値 + 1 nM, preferably lower than the IC5Q of FFR-rFVIIa 値 + 0.1 nM, more preferably lower than the IC5 () of FFR-rFVIIa, a human antibody that inhibits the production of FXa 22. According to claims 17 to 21 in the scope of patent application Any one of the methods, which includes testing antibodies in a FVIIa / TF amidine decomposition assay and screening for IC5Q (lower than FFR-rFVIIa) IC5Q (+100 nM) (for this analysis, 10 nM FVIIa is used), such as low IC5Q 値 + 40 nM at FFR-rFVIIa, preferably lower than IC5Q 値 + 20 nM at FFR-rFVIIa, preferably lower than IC5Q 値 +10 nM at FFR-rFVIIa, more preferably lower than FFR-rFVIIa IC5 () 値, a human 65 200407427 antibody that inhibits FVIIa amidinolytic activity induced by TF. 23. The method according to any one of claims 17 to 22 of the scope of patent application, which comprises testing antibodies in a FVIIa competition assay and screening for human antibodies that can compete with FVIIa competition. 24. The method according to any one of claims 17 to 23 of the scope of patent application, which comprises testing antibodies in a TF ELISA analysis including TF and screening for human antibodies that can immunoreact with human TF. 25. An isolated antibody that immunologically reacts with an antigenic epitope present on human TF and inhibits the binding of human coagulation factor Vila to human TF, which can be obtained by a method comprising the following steps: a) Preparation of anti-human TF Human antibodies, b) test the antibodies in a TF-induced clot assay and screen out those that can be used in this analysis with IC5G 値 lower than FFR-rFVIIa IC5G 値 1 nM, such as lower than FFR-rFVIIa IC5G 値 + 500 pM, preferably lower than IC5 of FFR-rFVIIa.値 + 200 pM, preferably lower than IC50 of FFR-rFVIIa 値 + 100 pM, preferably lower than IC5Q 値 of FFR-rFVIIa + 50 pM, preferably lower than IC5 of FFR-rFVIIa.値 + 10 pM, better than IC50 of FFR-rFVIIa + 5 pM, more preferably IC5G of FFR-rFVIIa, human antibodies that inhibit clot formation, or test antibodies and screen out in FXa yield analysis The IC5G may be lower than the IC5 of FFR-rFVIIa.値 + 100 nM (using 0.1 nM FVIIa), for example, IC5G 値 + 10 nM lower than FFR-rFVIIa, preferably IC5G 値 + 5 nM lower than FFR-rFVIIa, preferably IC5G 値 + 1 lower than FFR-rFVIIa nM, better than FFR-rFVIIa IC5G 値 + 0.1 nM, better than 66 200407427 FFR-rFVIIa IC50 値, human antibodies that inhibit FXa production, or test antibodies and screen in FVIIa / TF amine decomposition analysis Out can its IC5.値 lower than IC50 of FFR-rFVIIa + 100 nM (10 nM FVIIa used in this analysis), for example, lower than IC5Q of FFR-rFVIIa 値 + 40 nM, preferably lower than IC5Q of FFR-rFVIIa 値 + 20 nM, preferably less than IC5Q 値 +10 nM of FFR-rFVIIa, more preferably less than IC50 of FFR-rFVIIa, human antibodies that inhibit TF-induced FVIIa amidolytic activity, or compete with FVIIa Test antibodies during analysis and screen for human antibodies that can compete with FVIIa, or test antibodies in TF ELISA assays containing TF and screen for human antibodies that can immunoreact with human TF. 26. A method for preparing a human antibody, the method comprising: a) immunizing a mouse with human TF, and b) isolating an antibody-producing cell from the immunized mouse and preparing immortalized cells capable of secreting human antibody , C) separating the medium containing the produced antibodies from immortalized cells, d) testing the antibodies in an indirect TF ELISA assay containing TF in solution, and screening for human antibodies that can immunoreact with human TF in solution, e) Test antibodies in FVIIa competition analysis and screen for human antibodies that can compete with FVIIa, f) Test antibodies in FVIIa / TF scan analysis and screen for IC50 below FFR-rFVIIa IC50値 + 100 nM (use 10 nM FVIIa in this analysis 67 200407427), for example, IC5 () 低于 + 40 nM lower than FFR-rFVIIa, preferably lower than IC5Q 値 + 20 nM, lower lower than FFR-rFVIIa, preferably lower Human antibodies that inhibit IC5Q 値 + 10 nM of FFR-rFVIIa, and better than IC50 F of FFR-rFVIIa, inhibit human activity of FVIIa amine-induced degradation by TF, g) test antibodies in FXa production analysis and screen out Its IC5 () 値IC5G 値 + 100 nM below FFR-rFVIIa (0.1 nM FVIIa is used in this analysis), for example, IC5Q 値 + 10 nM below FFR-rFVIIa, and preferably IC5 below FFR-rFVIIa.値 + 5 nM, preferably lower than IC5 of FFR-rFVIIa.値 + 1 nM, better than IC5Q of FFR-rFVIIa 値 + 0 · 1 nM, better than IC5Q of FFR-rFVIIa, inhibiting human antibodies produced by FXa h) in TF-induced clot analysis Test the antibody and select an IC5G 値 + 1 nM 'whose IC50 is lower than FFR-rFVIIa in this analysis, such as IC5G 値 + 500 pM lower than FFR-r * FVIIa, preferably lower than FFR- IC5Q 値 +200 pM of rFVIIa, preferably lower than IC50 値 + 100 pM of FFR-rFVIIa, such as IC5Q 値 + 50 pM lower than FFR-rFVIIa, preferably IC5G 値 + 10 pM lower than FFR-rFVIIa, more Better than IC5Q 値 + 5 pM below FFR-rFVIIa, more preferably IC50IC below FFR-rFVIIa, human antibodies that inhibit clot formation, i) Screen and incubate in a suitable medium for the production of step dh Immortalized cells of the screened antibody, j) Isolate the screened antibody from the cultured medium of immortalized screen. 27. The method according to item 26 of the patent application scope, wherein the method further includes step 2004200427 to test antibodies in a direct TF ELISA analysis including immobilized TF and to screen out human antibodies that can immunoreact with immobilized human TF. 28. The method according to any one of claims 26 to 27 in the scope of the patent application, wherein the method further comprises testing antibodies in an FXa yield analysis performed by TF-expressing cells, and screening for IC5 () 値 lower than FFR- IC5Q 値 + 500 nM of rFVIIa (1 nM FVIIa is used in this analysis), for example, IC5 () 値 + 100 nM lower than FFR-rFVIIa, preferably IC5Q 値 + 50 nM lower than FFR-rFVIIa, preferably Lower IC5 than FFR-rFVIIa.値 + 10 nM, more preferably IC5G lower than FFR-rFVIIa 値 + 5 nM, more preferably lower than IC50 値 F of FFR-rFVIIa, inhibit human antibodies produced by FXa. 29. The method according to any one of claims 26 to 28, wherein the method further comprises testing antibodies in a complete cell TF binding assay, and screening for human TFs that can compete with FVIIa and display on the surface of intact cells Binding human antibody. 30. The method according to any one of claims 26 to 29, wherein the method further comprises testing antibodies in a biosensor analysis, and screening for Kd 値 having a binding to human TF of less than 100 nM, such as Human antibodies below 10 nM, preferably below 5 nM, preferably below 1 nM, more preferably below 0.5 nM. 31. The method according to any one of claims 26 to 30, wherein the method further comprises testing antibodies in the MAPK signal analysis and screening for human antibodies that can inhibit the activation of MAPK signals induced by FVIIa-. 32. The method according to any one of claims 26 to 31, 69 200407427, wherein the method further comprises testing the antibody in the epitope mapping analysis, and screening for an immune response with a better antigenic epitope on TF Human antibodies. 33. The method according to item 32 of the patent application, wherein the preferred antigenic epitope comprises residues Trp45, Lys46 and Tyr94. 34. The method according to any one of claims 26 to 33, wherein the immortal proliferating cells are fused with tumor cells. 35. A human antibody that immunoreacts with an antigenic epitope present on human TF and inhibits the binding of human coagulation factor Vila to human TF, which can be obtained by a method comprising the following steps; a) Immunizing mice with human TF , B) isolating antibody-producing cells from the immunized mouse and preparing immortalized cells capable of secreting human antibodies, c) separating the medium containing the produced antibodies from immortalized cells, d) in TF containing the presence solution Test antibodies in an indirect TF ELISA assay and screen human antibodies that can immunoreact with human TF in solution, e) test antibodies in a FVIIa competition assay and screen for human antibodies that can compete with FVIIa, f) Test the antibodies in the FVIIa / TF amidolysis analysis and screen out that IC5G may be less than 100 nM (in an analysis with a FVIIa concentration of 10 nM), such as less than 40 nM, preferably less than 20 nM, more Human antibodies that inhibit FVIIa amidolytic activity induced by TF, preferably below 10 nM, g) Test antibodies in FXa production analysis and screen out IC50 that is lower than F50 of FFR-rFVIIa + 100 nM (70 200407427 0.1 nM FVIIa is used in this analysis), for example, IC50 値 + i0nM lower than FFR-rFVIIa, preferably IC50 値 + 5 nM lower than FFR-rFVIIa, preferably lower than FFR-rFVIIa IC50 値 + 1 nM, better than IC50 値 + 0.1 nM, better than FFR-rFVIIa IC50 値, better than FFR-rFVIIa IC50 抑制, inhibit human antibodies produced by FXa, h) in TF-induced clot analysis Test the antibody and select an IC50 of less than FFR-rFVIIa + IC50 値 + 1 nM in this analysis, such as IC50 of FFR-rFVIIa + 500 pM, preferably less than IC50 of FFR-rFVIIa値 +200 pM, preferably lower than IC50 of FFR-rFVIIa 値 + 100 pM, for example, lower than IC50 of FFR-rFVIIa 値 + 50 pM, preferably lower than IC50 of FFR-rFVIIaF + 10 pM, more preferably lower than IC50 r + 5 pM of FFR-rFVIIa, better than IC50 F of FFR-rFVIIa, human antibodies that inhibit clot formation, i) Screen and grow in a suitable medium to produce the product obtained by screening in step dh Immortalized cells of the antibody, j) Isolate the screened antibody from the cultured medium of immortalized cells. 36. A human antibody that immunologically reacts with an antigenic epitope present on human TF and inhibits the binding of human coagulation factor Vila to human TF, which can be obtained by a method according to any one of claims 26 to 34 System 37. A cell that produces a human antibody that immunologically reacts with an antigenic epitope present on human TF and inhibits the binding of human coagulation factor Vila to human TF. 71 200407427 38. The cell according to item 37 of the application, wherein the cell is an isolated lymphocyte. 39. The cell according to any one of claims 37 to 38, wherein the cell line is isolated from a mouse. 40. The cell according to item 37 of the application, wherein the cell is a fused tumor. 41. The cell according to item 40 of the scope of patent application, wherein the fusion tumor cell line is obtained by fusing antibody-producing lymphocytes with immortalized cells to obtain antibody-producing fusion tumor cells. 42. The cell according to any one of claims 37 to 41, wherein the antibody inhibits the binding of human coagulation factor Vila to human TF. 43. The cell according to any one of claims 37 to 42 of the scope of the patent application, wherein the antibody system performs an immune response to a 3-dimensional surface including all residues Trp45, Lys46 and Tyr94. 44. The cell according to any one of claims 37 to 43, wherein the antibody is a Fab fragment. 45. The cell according to any one of claims 37 to 44 in the scope of the patent application, wherein the antibody is a F (ab) 2 fragment. 46. The cell according to any one of claims 37 to 44 in the scope of the patent application, wherein the antibody is a F (ab ') 2 fragment. 47. The cell according to any one of claims 37 to 44 in the scope of the patent application, wherein the antibody is a scFv fragment. 48. The cell according to any one of items 37 to 47 in the scope of the patent application, wherein the antibody has a Kd 値 with human TF within the range of 10_15-1 (T8 M of the range 72 200407427). 49. According to the scope of the patent application The cell of any one of items 37 to 48, wherein the antibody has a Kd 値 with human TF in the range of 1Cr15-1 (T1 () M.) 73