MXPA04003051A

MXPA04003051A - Human tissue factor antibodies.

Info

Publication number: MXPA04003051A
Application number: MXPA04003051A
Authority: MX
Inventors: Freskgaard Per-Ola
Original assignee: Novo Nordisk As
Priority date: 2001-10-02
Filing date: 2002-09-30
Publication date: 2004-07-05
Also published as: WO2003029295A1; CZ2004454A3; HUP0401658A2; IL160998A0; PL368989A1; EP1434802A1; JP2005512970A; ZA200402303B; CA2460917A1; BR0213046A; CN1575302A; KR20040045478A; NO20041802L; RU2004113373A

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

TISSUE FACTOR ANTIBODIES HOG ÑO FIELD OF THE INVENTION The present invention relates to isolated antibodies that do not react with tissue factor (FT) to inhibit the binding of coagulation factor VIla (FVIIa) and thus an immunotherapeutic method that uses human antibodies against FT to inhibit the formation of thrombi associated with surgery, microsurgery, angioplasty or trauma or to inhibit the formation of thrombi and other functions of TF in abnormal haemostatic conditions associated such as diseases such as deep vein thrombosis, disseminated intravascular coagulation (DIC), artery disease coronary, sepsis, inflammation, arteriesclerosis or cancer. It is also described in a method for the preparation of antibodies as well as cell lines for the preparation of human monoclonal antibodies (Acsm). BACKGROUND OF THE INVENTION Blood coagulation is a process consisting of a complex interaction of several components, or factors, of the blood, which eventually results in a fibrin picture. Generally, the components of the blood that participate in what has been referred to as the "cascade" of coagulation are proenzymes or zymogens, enzymatically inactive proteins which are converted to Reis 15S 17 proteolytic enzymes by the action of an activator, itself a Activated coagulation factor. Coagulation Factors that have undergone that conversion and that are generally referred to as "Active Factors", and designated by the addition of an "a" muscle suffix (for example, Factor Vlla). Activated Factor X ("Xa") is required to convert prothrombin to thrombin, which then converts fibrinogen to fibrin as a final step in the formation of a fibrin box. There are two systems, or pathways, that promote the activation of Factor X. The "intrinsic pathway" refers to those reactions that lead to the formation of thrombin through the use of factors present only in the plasma. A series of activations mediated by proteasis finally generate Factor IXa which, in conjunction with Factor Villa, cleaves Factor X in Xa. An identical proteolysis is carried out by FVIIA and its cofactor, FT, in the "intrinsic pathway" of blood coagulation. FT is a protein bound to the membrane and normally does not circulate actively in plasma. After disruption of a spleen, however, FT can be complexed with FVIIa to catalyze the activation of Factor X or the activation of Factor IX in the presence of Ca2 + and phospholipid. Although the relative importance of the two coagulation pathways in hemostasis is unclear, it has been found that Factor VII and TF play a central role in the initiation of blood coagulation. It is often necessary to selectively block the coagulation cascade in a patient. Anticoagulants such as heparin, coumarin, coumarin derivatives, indandione derivatives, or other agents that may be used, for example, during kidney dialysis, or to treat deep vein thrombosis, disseminated intravascular coagulation (DIC), and a host of other medical disorders. For example, treatment with heparin or extracorporeal treatment with citrate ion can be used in dialysis to prevent coagulation during the course of treatment. Heparin is also used to prevent thrombosis of deep veins in patients undergoing surgery. Treatment with heparin and other anticoagulants may, however, have undesirable side effects. The available anticoagulants generally act through the body, rather than acting especially at the site of injury or damage. Heparin, for example, can cause profuse bleeding. In addition, with a half-life of approximately 80 minutes, heparin is rapidly eliminated from the blood, requiring frequent administration. Because heparin acts as a cofactor for antithrombin III (ATIII), and ATIII is rapidly depleted in the treatment of DIC, it is often difficult to maintain the appropriate dose of heparin, requiring continuous verification of the levels of ATIII and heparin. Heparin is also not effective if the exhaustion of ATIII is extreme. In addition, prolonged use of heparin may also increase platelet aggregation and reduce platelet count, and has been implicated in the development of heparin-induced thrombocytopenia. The indandione derivatives can also have toxic side effects. In addition to the anticoagulants briefly described above, it has been found that several natural proteins have anticoagulant activity. ATIII has also been proposed as a therapeutic anticoagulant. International Application No. O 92/15686 is related to inactive factor Vlla to inhibit blood coagulation. Antibodies are specific immunoglobulin (Ig) polypeptides produced by the immune system of vertebrates in response to challenges by proteins, glycoproteins, cells, or other foreign antigenic substances. The sequence of events that allows the organism to overcome the invention by foreign cells or to rid the system of foreign substances is at least partially understood. An important part of this process is the manufacture of antibodies which bind specifically to a particular foreign substance. The binding specificity of those polypeptides to a particular antigen is highly r &tinááá and the multitude of specificities capable of being generated by individual vertebrates is remarkable in its complexity and variability. Millions of antigens are capable of producing antibody responses, each antibody almost exclusively directed towards the particular antigen that produced it. Two main sources of antibodies in vertebrates are currently used, in vitro generation by mammalian B lymphocytes, and generation in cell culture by B cell hybrids. The antibodies are generated in themselves as a result of the differentiation of immature B lymphocytes into cells plasma levels, which occurs in response to stimulation by specific antigens. In undifferentiated B cells, the portions of DNA that code for the different regions of the immunoglobulin chains are separated into the genomic DNA. The sequences are sequentially mounted before the expression. The resulting rearranged gene is capable of expressing itself in the mature B lymphocyte to produce the desired antibody. However, even when a particular mammal is exposed to only one antigen, a uniform population of antibodies does not result. The immune response in situ to a particular antigen is defined by the mosaic of responses to the different determinants that are present on the antigen. Each subset of homologous antibodies is a contribution of a single population of B cells, consequently the in situ generation of antibodies is "polyclonal". This limited but inherent heterogeneity has been overcome in numerous particular cases by the use of hybridoma technology to create "monoclonal" antibodies in cell cultures by B-cell hybridomas. In this process, the splenocytes or lymphocytes of relatively short life, or fatal , of a mammal that has been injected with antigen are fused with an immortal tumor cell line, thus producing hybrid cells are "hybridomas" which are immortal or unable to produce the genetically encoded antibodies of the B cell. The hybrids thus formed they are segregated into unique genetic strains by selection, dilution, and regrowth, and each strain thus represents a single genetic line. They therefore produce antibodies which are, safely, homogeneous against a desired antigen. These antibodies, in reference to their pure genetic origin, are called "monoclonal". Monoclonal antibodies with monospecificity have greatly influenced immunology, and its usefulness and has been demonstrated in sciences such as biology, pharmacology, biochemistry and others. These monoclonal antibodies have found widespread use not only as diagnostic reagents, but also therapeutically (see, for example, Ritz and Schlossman, Blood, 59: 1-11, (1982)). The monoclonal antibodies produced by hybridomas, although theoretically effective as discussed above and clearly preferable to polyclonal antibodies due to their specificity, suffer from a significant disadvantage. In many applications, the use of monoclonal antibodies produced in non-human animals is severely restricted, where monoclonal antibodies are to be used in humans. Repeated injections of a "foreign" antibody in humans, such as a mouse antibody, can lead to dangerous hypersensitivity reactions. That non-human derived monoclonal antibody, when injected into humans, produces an anti-non-human antibody response. The therapeutic use of ACSM against TF is known from US Patent Nos. 6,001,978 and 5,223,427. International Application No. WO 99/51743 relates to human / mouse chimeric monoclonal antibodies directed against human TF. European patent application No. 833911 relates to antibodies grafted with CDR against human TF. Presta L. et al., Thrombosis and Hae ostasis, Vd. 85 (3) pp 379-389 (2001) is related to an antibody immunized against TF. There is still a need in the art for improved compositions that have air-coagulant activity that can be administered at relatively low doses and do not produce the undesirable side effects associated with traditional anticoagulant compositions. The invention satisfies this need by providing anticoagulants that do not have the side effects associated with traditional antibodies with non-human sequences, they act specifically at the sites of injury or damage, and also provide other related advantages. Furthermore, the present invention provides compounds, which act to inhibit the cellular functions of TF, which are involved in conditions such as sepsis, atherosclerosis inflammation, restenosis or cancer. BRIEF DESCRIPTION OF THE INVENTION The present invention relates to high affinity non-immunogenic human antibodies against human TF, which inhibits the binding of coagulation factor VII / VIIa and methods for the selection of therapeutically effective human antibodies against human TF. In a first aspect, the present invention relates to an isolated human antibody, which immunoreacts with an epitope present on human TF. The terms "human tissue factor" or "human FT", as used herein, refer to the full-length polypeptide receptor comprising the amino acid sequence 1-263 of the native human tissue factor.The term "antibody", as used here, it is intended to refer to immunoglobulin molecules and fragments thereof, which have the ability to specifically bind to an antigen (e.g., human TF). The full-length antibodies comprise four polypeptide chains, two heavy (P) chains and two light chains (L) interconnected by disulfide bonds Each heavy chain is comprised of a variable region of the heavy chain (abbreviated as VCP or PV) and a constant region of the heavy chain The constant region of the heavy chain is comprised of three domains, CH1, CH2 and CH3 Each light chain is comprised of a variable region of the light chain (abbreviated here as RVCL or LV) and a constant region of the chain light The constant region of the light chain is comprised of a domain, CL. The PV and LV regions can be further subdivided with regions of hypervariability, called complementarity determining regions (CDR), interdispersed with regions that are more conserved, called structural regions (FR). Each PV and LV is composed of 3 CDRs and 4 FRs, arranged from the aminoterminal to the carboxyterminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Thus, within the definition of an antibody is also one or more fragments of an antibody that retains the ability to specifically bind to an antigen. { for example, the human PT). It has been shown that the antigen-binding function of an antibody can be effected by fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include (i) a Fab fragment, a monovalent fragment consisting of the LV, PV, CL and CP I domains; (ii) F (ab) 2 and F (ab ') 2 fragments, a bivalent fragment comprising two fragments of Fab linked by a disulfide bridge in the region of the hinge; (iii) a fragment of Fd consisting of two domains PV and CPl; (iv) a fragment of Fv consisting of the LV and PV domains of a single arm of an antibody; (v) a dAc fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a PV domain; and (vi) an isolated complementarity determinant region (CDR). In addition, although the two domains of the Fv, LV and PV fragment are encoded by separate genes, they can be linked, using recombinant methods, by means of a synthetic binder that allows them to be produced as a single protein chain in which the regions LV and PV pair to form monovalent molecules (known as a single Fv chain (scFv), see for example, Bird et al (1988) Science 242: 423-426 and Huston et al (1988) Proc. Nati. Acad. Sci. USA 85: 5879-5883). It is intended that those single chain antibodies also be encompassed within the term "antibody". Other forms of single chain antibody, such as antibodies, are also encompassed. Diantibodies are bivalent, specific antibodies in which the PV and LV domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementarity domains of another chain and creating two antigen binding sites (see for example, Holliger, P., et al., (1993) Proc. Nati, Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). It should be understood that human TF can have one or more antigenic determinants comprising (1) antigenic peptide determinants which consist of unique peptide chains within human TF, (2) conformational antigenic determinants which consist of more than one especially contiguous peptide chain whose respective amino acid sequences are located disjointly along the polypeptide sequence of human TF; and (3) postranslational antigenic determinants which consist, all or in part, of molecular structures covalently linked to human TF after translation, such as carbohydrate groups or the like. The terms "human antibody", "human antibodies", "human TF antibody" and "human FT antibodies", as used herein, are intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention can include residual amino acids not encoded by the human germ cell globulin sequence (for example, mutations introduced by site-specific random mutagenesis in vi tro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as the mouse, have been grafted onto human structural sequences, by example the so-called humanized antibodies or human / mouse chimeric antibodies. An "isolated human antibody", as used herein, Is intended to refer to a human antibody that is substantially free of other antibodies having different antigenic specificities (eg, an isolated antibody that specifically binds to human TF) that is substantially free of antibodies that specifically bind to antigens other than human TF) . An isolated antibody that binds specifically to human TF can, however, have cross-reactivity with other antigens, such as FT molecules from other species (as discussed in more detail below). In addition, an isolated antibody can be substantially free of other cellular material and / or chemical compounds. The term "epitope" as used herein means any antigenic determinant on an antigen to which the antibody binds. Epitope determinants usually consist of groups of chemically active surface molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. The term "intnunorreaction" or "immunoreaction", as used herein, means any binding of an antibody to its epitope with a dissociation constant ¾ of less than 10"4 M. The terms" immunoreaction "or" immunoreaction "are used where appropriate. interchangeably with the term "specific binding." The term "inhibits," as used herein, means any reduction compared to a reference, as an example, an antibody, which inhibits the binding of human coagulation factor Vlla to FT. "human" means any antibody, which reduces the ability of the human coagulation factor VIla to bind to human FT compared to the ability of the human coagulation factor Vlla to bind to human FT in the absence of the antibody.The term "affinity", as used here, it means the strength of the binding of an antibody to an epitope.The affinity of an antibody is measured by the dissociation constant ¾, defined as [Ac] x [Ag ] / [Ac-Ag] where tAc-Ag] is the concentration of the antibody-antigen complex, [Ac] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by I / Ka. Preferred methods for determining the specificity of Acsm and the affinity for competitive inhibition can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, MY, 1988), Colligan et al. , eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y. , (1992, 1993) and Muller, Meth. Enzymol. 92: 589-601 (1983), references, which are incorporated herein by reference in their entirety. In a second aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a human antibody, which does not react with an epitope present on human TF. The term "therapeutically effective amount" is the effective dose to be determined by a qualified physician;, who can titrate the dose to achieve the desired response. Factors for dose consideration will include the potency, bioavailability, desired pharmacokinetic / pharmacodynamic profiles, treatment conditions (eg, trauma, inflammation, septic shock), patient-related factors (eg, weight, health, age, etc.). ), presence of medication coadministered administration time, or other factors known to a medical practitioner. The dose of a human antibody against FT administered to a patient will vary with the type and severity of the condition to be treated, but is generally in the range of 0.1-5.0 mg / kg of body weight. The term "subject" as used herein is intended to mean any animal, in particular mammals, as humans, and may, where appropriate, be used interchangeably with the term "patient". In a third aspect, the invention relates to a composition comprising a human antibody, which immunoreacts with an epitope present on human TF. In a further aspect, the invention relates to a method for the treatment of a disorder related to FVIIa / FT in a human, which method comprises administering to human a therapeutically effective amount of a human antibody, which immunoreacts with an epitope. present about human TF "Treatment" means the administration of an effective amount of a therapeutically active compound of the invention for the purpose of preventing any symptoms or disease state from developing for the purpose of curing or facilitating its already developed symptoms or disease states. The meaning of the term "treatment" thus includes prophylactic treatment. The term "FVIIa / FT-related disorder" as used herein means a disease or disorder, where FT and FVIIa are involved. Included are related thrombotic or coagulopathic diseases or disorders, including the inflammatory response and chronic thromboembolic diseases or disorders associated with fibrin formation, including vascular disorders such as deep vein thrombosis, arterial thrombosis, postoperative thrombosis, coronary artery bypass grafting (IDAC), percutaneous transdermal coronary angioplasty (PTCA), stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, arteriosclerosis and restenosis after angioplasty, acute and chronic indications such as inflammation, septic shock, septicemia, hypotension, adult respiratory distress (ARDS), disseminated intravascular coagulopathy (IAT), pulmonary embolism, platelet deposition, myocardial infarction, or prophylactic treatment of mammals with atherosclerotic spleens at risk for thrombosis, and other diseases or disorders. The disorders related to FVIIa / FT are not limited to in vivo coagulopathic disorders such as those mentioned above but include processes related to FVIIa / FT ex vivo with coagulation that may result from extracorporeal circulation of blood, including blood removed in line of a patient in processes such as dialysis procedures, blood filtration, or other blood diversion during surgery. The term "factor Vlla" or "FVIIa" means coagulation factor VII activated "two-chain" cleaved by specific cleavage at the Argl52-Ilel53 peptide linkage. FVIIa can be purified from the blood or purified by recombinant means. It is evident that the practice of the methods described herein is independent of how the purified VIla factor is derived, and therefore, the present invention contemplated covering the use of any suitable factor Vlla preparation for use herein. Human FVIIa are preferred. The term "FVII" means coagulation factor VII "single chain". In a further aspect, the invention relates to a method for the preparation of a humán antibody, which method comprises a) Preparing human antibodies against human TF, b) Testing antibodies in an FT-induced coagulation assay and selecting the human antibody which inhibits clot formation in this assay with a CIS0 value of less than 1 nM, less than 500 pM, preferably lower, 200 pM, preferably less than 100 pM, preferably »less than 50 pM, preferably less than 10 pM, more preferably less than 5 pM, or test antibodies in a generation assay FXa and selecting the human antibody that inhibits generation of Fxa with an IC50 value of less than 100 nm (in a test with a FVIIa concentration of 0.1 nM), lower than 10 nM, preferably lower than 5 nM, preferably less than 1 nM, more preferably less than 0.1 nM, or test antibodies in an amidolytic assay of FVIIa / FT and select the human antibody that inhibits the amidolytic activity of FVIIa induced by FT, with an IC50 value of less than 100 nM (in an assay with a FVIIa concentration of 10 nM), less than 40 nM, preferably less than 20 nM, more preferably less than 10 nM, or test antibodies in a competition assay of FVIIa and select the human antibody that competes with binding of FVIIa, or testing antibodies in an FT ELISA assay comprising FT and selecting the human antibody that binds to human FT. It should be understood that the specific IC 50 values referred to the TF-induced clot assay is when normal human plasma is used. In a further aspect, the invention relates to a method for the preparation of a human antibody, which method comprises a) Preparation of human antibodies against human TF, b) testing antibodies in an FT-induced coagulation assay and selecting the human antibody that inhibits the formation of clots in this assay with a Cg0 value lower than the CIS0 value of FFR-rFVIIa + 1 nM, lower than the Cl50 value of FFR-rFVIIa + 500 pM, preferably lower than the value of IC50 of FFR-rFVIIa + 200 pM, preferably lower than the IC50 value of FFR-rFVIIa + 100 pM, preferably lower than the IC50 value of FFR-rFVIIa + 50 pM, preferably lower than the CIS0 value of FFR-rFVIIa + 10 pM, more preferably less than the IC50 value of FFR-rFVIIa + 5 pM, more preferably less than the IC50 value of FFR-rFVIIa, or test antibodies in an FXa generation assay and select the human antibody that inhibits the generation of FXa with an IC50 value lower than the IC50 value of FFR-rFVIIa + 100 nm (using 0.1 nM FVITa in the assay), lower than the IC50 value of FFR-rFVIIa + 10 nm, preferably lower than the CIS0 value of FFR-rFVIIa + 5 nM, preferably less than the IC50 value of FFR-rFVIIa + 1 nM, more preferably than the CI5o value of FFR-rFVIIa + 0.1 nM, more preferably less than the value of IC50 of FFR-rFVIIa, or test antibodies in an amidolx co assay of FVII / FT and select the human antibody that inhibits the amidolytic activity of FVIIa induced by TF, with an IC50 value lower than the CIS0 value of FFR-rFVIIa + 100 nm (using 10 nM FVIIa in the assay), less than the value? of IC50 of FFR-rFVIIa + 40 nm, preferably less than the IC50 value of FFR-rFVIIa + 20 nM, preferably lower than the IC50 value of FFR-rFVIIa + 10 nM, more preferably less than the IC50 value of FFR-rFVIIa, or test antibodies in a FVIIa competition assay and select the human antibody that competes with FVIIa binding, or test antibodies in a FT ELISA assay comprising FT and select the human antibody that does not react with the human FT. It should be understood that the specific IC 50 values reported in the CT-induced coagulation assay is when normal human plasma is used. The term "FT-induced coagulation assay" as used herein is intended to mean any assay where the coagulation time is measured in a sample comprising blood plasma and FT. An example of FT induced coagulation assay is described in Example 1, assay 7. The term "Fxa generation assay" as used herein is intended to mean any assay where the activation of the FX is measured in a sample comprising FT, FVIIa, FX, calcium and phospholipids. An example of an FXa generation assay is described in Example 1, assay 5. The term "FVIIa / FT amidolytic assay" as used herein is intended to mean any assay where the amidolytic activity, ie the cleavage of a small peptide substrate, of FVIIa is measured in the presence of FT. An example of an amidolytic assay of FVIIa / FT is described in example 1, assay 4. The term "assay by FT ELISA" as used herein is intended to mean any assay by ELISA comprising FT and antibodies against FT. Examples of FT ELISA assays are the direct and indirect FT ELISA assays described in Example 1, assays 1 and 2. The term "direct FT ELISA assay" as used herein is intended to mean any assay by ELISA of the FT. FT that includes immobilized FT. Examples of direct FT ELISA assays are described in Example 1, Assay 1. The term "indirect FT ELISA assay" as used herein is intended to mean any FT ELISA assay, where the FT is in solution. Examples of direct FT ELISA assays are described in Examples 1, e, 2. In a further aspect, the invention relates to a human antibody which immunoreacts with an epitope present on human TF and inhibits the binding of Fact of human coagulation VIla to human FT obtainable by a method comprising: a) Preparing human antibodies against human FT, b) Testing antibodies in an FT-induced coagulation assay and selecting the human antibody that inhibits clot formation in this assay with a CIso value of less than 1 nM, less than 500 pM, preferably 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, or test antibodies in an FXa generation assay and select the human antibody that inhibits the generation of Fxa with an IC50 value below 100 nm (in an assay with a concentration of F VIIa of 0.1 nM), lower than 10 nM, preferably lower than 5 nM, preferably lower than 1 nM, more preferably lower than 0.1 nM, or test antibodies in an amidolytic assay of FVIIa / FT and select the human antibody that inhibits the amidolytic activity of FVIIa induced by FT, with a lower IC50 value of 100 nM (in a test with a FVIIa concentration of 10 nM), lower than 40 nM, preferably lower than 2Q :. ffc so as to preferably 'f' less than 10 nM, or '' test antibodies in a Fome-Fome assay and select the human antibody that competes with FVIIa binding, or test antibodies in an FT ELISA assay that understand FT and select the human antibody that binds to human FT It should be understood that the specific IC50 values referred to the FT-induced coagulation assay is when normal human plasma is used In a further aspect, the invention relates to a human antibody which immunoreacts with an epitope present in the human TF and inhibits binding of the human coagulation factor VIla to a human FT obtainable by a method comprising: a) Preparation of human antibodies against human, b) testing antibodies in a test of FT-induced coagulation and select the human antibody that inhibits clot formation in this assay with a CIso value lower than the IC50 value of FFR-rFVIIa + 1 nM, less r that the IC50 value of FFR-rFVIIa + 500 pM, preferably lower than the IC50 value of FFR-rFVIIa + 200 pM, preferably lower than the IC50 value of FFR-rFVIIa + 100 pM, preferably lower than the value of IC50 of FFR-rFVTIa + 50 pM, preferably less than the CISo R-rFVII + 10 pM value, more preferably less than the ICS * value of FFR-rFVIIa + 5 pM, more preferably-less than the IC50 value of FFR-rFVIIa, or test antibodies in an FXa generation assay and select the human antibody that inhibits the generation of FXa with an IC50 value lower than the CIso value of FFR-rFVIIa + 100 nm (using FVIIa 0.1 nM in the assay), lower than the IC50 value of FFR-rFVIIa + 10 ttm, preferably lower than the CI5o value of FFR-rFVIIa + "5 nM, preferably lower than the CISo value of FFR-rFVIIa + 1 nM, more preferably than the CIFR value of FFR-rFVIIa + 0.1 nM, more preferably less than the IC50 value of FFR-rFVIIa, or test antibodies in an amideolytic FVII / Ft assay and select the human antibody that inhibits the amidolytic activity of FVIIa induced by TF, with an IC50 value lower than the CIS0 value of FFR-rFVII + 100 nm (using 10 nM FVIIa in the assay), less than the CIFR value of FFR-rFVIIa + 40 nm, preferably less than the CI5o value of FFR-rFVIIa + 20 nM, preferably lower than the IC50 value of FFR-rFVIIa + 10 nM, more preferably less than the IC50 value of FFR-rFVIIa, or test antibodies in a FVIIa competition assay and select the human antibody that competes with the binding of FVIIa, or test antibodies in an FT ELISA assay that includes FT and select the human antibody that immunoreacts with human TF. It should be understood that the specific CI values referred to in the CT-induced coagulation assay is when normal human plasma is used. In one embodiment of the invention, the method, wherein human antibodies against human TF are produced, comprises immunization of a mammal with TF, and isolation of the antibodies produced by the immunized mammal. In a preferred embodiment, the mammal is a mouse. It should be understood that the immunized mammal or mouse is capable of producing human antibodies. In a further aspect, the invention relates to a method for the preparation of a human antibody, which method comprises: a) immunization of a mouse with human FT, b) isolation of a humanized mouse antibody producing cell and preparation of immortal cells for secreting human antibodies, c) isolation of culture medium from immortal cells comprising antibodies produced, d) testing antibodies in an indirect FT ELISA assay comprising FT in solution and selecting human antibodies which bind to human TF in solution, e) test antibodies in a competition assay of FVIIa and select human antibodies that compete for binding with FVIIa, f) test antibodies in an amidolytic FVIIa / FT assay and select human antibodies that inhibit amidolytic activity of FVIIa induced by FT, with an IC50 value of less than 100 nM (in a test with a FVIIa concentration of 10 nM), less than 40 nM, preferably less than 20 nM, preferably less than 10 nM, g) test antibodies in an FXa generation assay and select human antibodies that inhibit the generation of FXa with an IC50 value of less than 100 nM (in an assay with a FVIIa concentration of 0.1 nm), less than 10 nM, preferably less than 5 nM, preferably less than 1 nM, more preferably 0.1 nM, h) test antibodies in an induced coagulation assay by FT and select the human antibody that inhibits the formation of clots in this assay with an IC50 value of less than 1 nM, less than 500 pM, preferably 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, i) selection and culture of a suitable culture medium of immortal cells; j¾¾ @ produce the selected antibody after pa ¾ d-h, j) isolation of the selected antibody from the culture medium of selected immortal cells. It should be understood that the specific IC50 values referenced in the TF-induced coagulation assay is when normal human plasma is used. In a further aspect, the invention relates to a method for the preparation of a human antibody, which method comprises a) immunization of a mouse with human FT, b) isolation of an antibody-producing cell from a humanized mouse and preparation of immortal cells for secreting human antibodies, c) isolation of culture medium from immortal cells comprising antibodies produced, d) testing antibodies in an indirect FT ELISA assay comprising FT in solution and selecting human antibodies that immunoreact with human FT in solution, e) test antibodies in a competition test of FVIIa and select the human antibodies that compete for binding with FVIIa, f) test antibodies in an amidolytic assay of FVIIa / FT and select the human antibody that inhibits the amidolytic activity of the FVIIa induced by FT, with an IC50 value lower than that,: valofSÉ CISo FFR-rFVIIa + 100 nra (using FVIIa 10 nM in the in $ I), lower GI50 of FFR-rFVIIa + 40 nm, preferably less than valbidé IC50 of FFR-rFVIIa + 20 nM, preferibl lower ¾üCe the IC50 value of FFR-rFVIIa + 10 nM , more preferably lower than the IC50 value of FFR-rFVIIa, g) testing antibodies in an FXa generation assay and selecting the human antibody that inhibits the generation of FXa with a CIS0 value lower than the IC50 FFR value -rFVIIa + 100 nm (using 0.1 nM FVIIa in the assay), lower than the IC50 value of FFR-rFVIIa + 10 nm, preferably lower than the CIS0 value of PFR-rFVIIa + 50 nM, preferably lower than the IC50 value of FFR-rFVIIa + 0.1 nM, more preferably lower than the IC50 value of FFR-rFVIIa, h) testing antibodies in an FT-induced coagulation assay and selecting the human antibodies that inhibit clot formation in this assay with a CI50 value less than the IC50 value of FFR-rFVIIa + 1 nM, men or the value of CIS0 of FFR-rFVIIa + 500 pM, pref erably lower than the IC50 value of FFR-rFVIIa + 200 pM, preferably lower than the IC50 value of FFR-rFVIIa + 100 pM, preferably lower than the value of IC50 of FFR-rFVIIa + 50 pM, preferably lower than the IC50 value of FFR-rFVIIa + 10 pM, more preferably less than the IC50 value of FFR-rFVIIa + 5 pM, more preferably less than the IC50 value of FFR-rFVIIa, i) selection and culture of a suitable culture medium of the immortal cell that produces the selected antibody after steps dh, j) isolation of the selected antibody from the culture medium of the selected immortal cell. It should be understood that the specific IC50 values referenced in the TF-induced coagulation assay is when normal human plasma is used. The term "antibody producing cell" as used herein means any cell capable of producing an antibody. Hybridomas, transfected cell lines and splenocytes and lymphocytes of relatively short life, or deadly, of a mammal that has been injected with an antigen are included. In a further aspect, the invention relates to a human antibody which immunoreacts with an epitope present on human FT and which inhibits the binding of human coagulation factor VIla to human FT obtainable by a method comprising: a) immunization of a mouse with human FT, b) isolation of a humanized mouse antibody-producing cell and preparation of immortal cells to secrete human antibodies, c) isolation of the culture medium from immortal cells comprising antibodies produced, d) testing of antibodies in a test by Indirect FT ELISA comprising FT in solution and selects human antibodies which bind to human FT. in solution, e) test antibodies in a FVIIa competition assay and select human antibodies that compete for binding with FVIIa, f) test antibodies in an amidolytic FVIIa / FT assay and select human antibodies that inhibit the amidolytic activity of FVIIa induced by FT, with an IC50 value less than 100 nM (in a test with a FVIIa concentration of 10 nM), less than 40 nM, preferably less than 20 nM, preferably less than 10 nM, g) test antibodies in a generation essay FXa and selecting human antibodies that inhibit the generation of FXa with a CIS0 value of less than 100 nM (in an assay with a FVIIa concentration of 0.1 nm), less than 10 nM, preferably less than 5 nM, preferably less than 1 nM, more preferably 0.1 nM, h) test antibodies in an FT-induced coagulation assay and select the human antibody that inhibits clots in this assay with a CIEQ value of less than 1 nM, less than 500 pM , preferably less than 200 pM, preferably less than 100 pM, preferably less than 50 pM, preferably less than 10 M, more preferably less than 5 pM, i) selection and culture of a suitable culture medium of immortal cells that produce the antibody selected after steps dh, j) isolation of the selected antibody from the culture medium of selected immortal cells. It should be understood that the specific CI values referred to in the TF-induced coagulation assay is when normal human plasma is used. In a further aspect, the invention relates to a human antibody which immunoreacts with an epitope present in human TF and inhibits binding of human coagulation factor VIla to human TF obtainable by a method comprising: a) immunization of a mouse with human FT, b) isolation of an antibody-producing cell from a humanized mouse and preparation of immortal cells to secrete human antibodies, c) isolation of culture medium from immortal cells comprising antibodies produced, d) testing of antibodies in an assay by Indirect FT ELISA comprising FT in solution and select human antibodies that immunoreact with human FT in solution, e) test antibodies in a dk cornuta FVIIa assay and select human antibodies that compile for binding with FVIIa, f) test antibodies in an amidolytic FVIIa / FT assay and select the human antibody that inhibits amidolytic activity FVIIa induced by FT, with an IC50 value lower than the IC50 FFR-rFVIIa + lO0? íim value (using 10 nM FVIIa in the assay), lower than the GI50 value of FFR-rFVIIa + 40 nm, preferably lower than the IC50 value of FFR-rFVIIa + 20 nM, preferably lower than the IC50 value of FFR-rFVIIa + 10 nM, more preferably lower than the IC50 value of FFR-rFVIIa, g) test antibodies in a FXa generation assay and select the human antibody that inhibits the generation of FXa with an IC50 value lower than the IC50 value of FFR-rFVIIa + 100 nm (using 0.1 nM FVIIa in the assay), lower than the CFR value of FFR -rFVIIa + 10 nm, preferably lower than the IC50 value of FFR-rFVIIa + 50 nM, preferably lower than the CIS0 value of FFR-rFVIIa + 0.1 nM, more preferably lower than the IC50 value of FFR-rFVIIa , h) test antibodies in an FT-induced coagulation assay and select human antibodies which inhibit n the formation of clots in this assay with a value of IC50 lower than the CIS0 value of FFR-rFVIIa + 1 nM, lower than the IC50 value of FFR-rFVIIa + 500. pM, preferably lower than the IC50 value of FFR-rFVIIa + 200 pM, preferably lower than the CIS0 value of FFR-rFVIIa + 100 pM, preferably lower than the CIS0 value of FFR-rFVIIa + 50 pM, preferably lower than the CIFR value of FFR-rFVIIa + 10 pM, more preferably less than the IC50 value of FFR-rFVIIa + 5 pM, more preferably less than the IC50 value of FFR-rFVIIa, or i) selection and culture of a suitable immortal cell culture medium which produces the selected antibody after steps dh, j) isolation of the selected antibody from the culture medium of the selected immortal cell. It should be understood that the specific CI values referred to in the TF-induced coagulation assay is when normal human plasma is used. In one embodiment of the invention, the immortal cell is a hybridoma cell. In a further aspect, the invention relates to a human antibody producing cell < They immunoreact with an epitope present on human TF and that inhibit the binding of human coagulation factor Vlla to human TF. In another embodiment, the cell is an isolated lymphoid cell.

In another embodiment, the cell is isolated from a mouse. In a further embodiment, the cell is a hybridoma cell. In one embodiment, the hybridoma cell is obtained by fusion of an antibody-producing lymphoid cell with an immortal cell to provide an antibody-producing hybridoma cell. In a further embodiment of the invention, the isolated human antibody inhibits the binding of human coagulation factor VIla to human TF. In a further embodiment of the invention, the human isolated antibody is a monoclonal antibody. The term "monoclonal antibody" as used herein, refers to a homogeneous population of immunoglobulins, ie, that the individual molecules of the antibody population are identical except for natural mutations. The antibodies are normally synthesized by lymphoid cells derived from bone marrow B lymphocytes. Lymphocytes derived from the same clone produce immoglobulin from a single amino acid sequence. The lymphocytes can not be cultured directly for prolonged periods of time to produce substantial amounts of their specific antibody. However, Kohler et al., 1975, 256: 495, it was shown that a somatic cell fusion process, specifically between a lymphocyte and a myeloma cell, could produce hybridoma cells which grow in culture and produce a specific antibody. called "monoclonal antibody". The. Myeloma cells are lymphocytic tumor cells which, depending on the strain cells, often produce antibodies by themselves, although "non-producing" strains are known. In a further embodiment of the invention, the isolated human antibody is a recombinant antibody. The term "recombinant antibody" as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a vector of recombinant expression transfected into a host cell (as best described in Section II, below), antibodies isolated from a recombinant human antibody library, combined (as best described in Section III, below), antibodies isolated from an animal (e.g., a mouse) that is gene transgenic of human immunoglobulin (see, for example, Taylor, LD, et al., (1992) Nucí Acids Res. 20: 6287-6295) or antibodies prepared, expressed, created or isolated by other means involving the splicing of gene sequences. human immunoglobulin to other DNA sequences. These recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, those recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal is used for human Ig sequences, somatic mutagenesis in vivo) and thus the amino acid sequences of the PV and LV of the recombinant antibodies are sequences that, although they are derived from and are related to the human terminal PV and LV sequences, can not exist naturally within the germline repertoire of human antibodies in vivo. In a further embodiment of the invention, the isolated human antibody is a Fac fragment In a further embodiment of the invention, the isolated human antibody is a F (ac) 2 fragment. In a further embodiment of the invention, the isolated human antibody is an F (ac ') 2 fragment. In a further embodiment of the invention, the isolated human antibody is a single chain Fv fragment. In a further embodiment of the invention, the isolated human antibody has a Ka for binding to human TF within the range of 10"15-10" 8M. It should be understood that the Ka for the binding of the human antibody to the referred human TF is determined in an assay, where the human antibody is immobilized (see assay 6).

In a modality more of the invention is the human antibody isolated from a human FT-binding Ka within the range of 10"15-10" 10M. In a further embodiment of the invention, the isolated human antibody has a human FT binding ¾ of less than 10"8 M. In a further embodiment of the invention, the isolated human antibody has a lower human FT binding K: of 10" 9M. In a further embodiment of the invention, the isolated human antibody has a ¾ for binding to human FT less than 10"10 M. In a further embodiment of the invention, the isolated human antibody has a Ka for binding to human FT less than 10_11. In a further embodiment of the invention, the isolated human antibody has a K¿ for binding to human TF less than 10"12M. In a further embodiment of the invention, the isolated human antibody has a ¾ for binding to human FT less than 10"13. In a further embodiment of the invention, the isolated human antibody has a K¿ for binding to human FT less than 10. "14M. In a further embodiment of the invention the isolated human antibody has a Ka for binding to human TF less than 10"15 M. In a further embodiment of the invention, the method for the preparation of a human antibody comprises testing antibodies in a coagulation assay induced by FT and select the human antibody that inhibits the formation of clots in this test with an IC50 value of less than 1 n.In one embodiment, the IC 50 value is lower than 500 pM. is lower than 200 pM In one embodiment the IC50 value is less than 100 pM In one embodiment the IC50 value is less than 50 pM In one embodiment the IC50 value is less than 10 pM. modality plus the CI5o value is less than 5 pM In a further embodiment of the invention the method of preparing a human antibody comprises testing antibodies in an FT-induced coagulation assay and selecting the human antibody that inhibited the formation of clots in this assay with an IC50 value lower than the IC50 value of FFR-rFVIIa + 1 nM, lower than the GI5o value of IC50 of FFR-rFVIIa + 500 pM, preferably lower than the IC50 IC50 value of FFR-rFVIIa + 200 pM, preferably lower than the IC50 CIS0 value of FFR-rFVIIa + 100 pM , preferably lower than the CIS0 CIS0 value of FFR-rFVIIa + 100 pM, preferably lower than the IC50 IC50 value of FFR-rFVIIa + 50 pM, preferably lower than the IC50 value of IC50 of FFR-rFVIIa + 10 pM, more preferably less than the IC50 value of IC50 of FFR-rFVIIa + 5 pM, more preferably less than the CI50 value of FFR-rFVIIa. It should be understood that the specific CIS0 values referenced in the coagulation assay as determined by FT is when normal human plasma is used.

In a further embodiment of the invention, the method of preparing a human antibody comprises testing antibodies in an FXa generation assay and selecting the human antibody that inhibits generation of FXa with an IC50 value of less than 100 n (in an assay with a concentration of FVIIa of 0.1 nM). In one embodiment, the IC50 value is less than 10 mM. In one embodiment, the IC50 value is less than 5 nM. In one embodiment, the IC50 value is less than 1 nM. In one embodiment, the IC 50 value is less than 0.1 nM. In a further embodiment of the invention the method for the preparation of a human antibody comprises testing antibodies in an FXa generation assay and selecting the human antibody that inhibits the generation of FXa with an IC50 value lower than the IC50 value of FFR. -rFVIIa + 100 nM (using 0.1 nM FVIIa in the assay), less than the CIFR value of FFR-rFVIIa + 10 nM, preferably lower than the IC50 value of FFR-rFVIIa + 5 nM, preferably lower than the value of IC50 of FFR-rFVIIa + 1 nM, more preferably less than the IC50 value of FFR-rFVIIa + 0.1 nM, more preferably less than the IC50 value of FFR-rFVIIa. In a further embodiment of the invention, the method for the preparation of a human antibody comprises testing antibodies in an amidolytic assay of FVIIa / FT and selecting the human antibody that inhibits the amidolytic activity of FVIIa induced by FT with a CIS value of less than 100 nM (in an assay with a FVIIa concentration of 10 nM). In one embodiment, the IC50 value is less than 40 nM. In one embodiment, the IC50 value is less than 20 nM. In one embodiment, the value of CIS0 is less than 10 nM. In one embodiment, the IC50 value is less than 5 nM. In a further embodiment of the invention, the method for the preparation of a human antibody comprises testing antibodies in an amido-lytic FVIIa / FT assay and selecting the human antibody that inhibits the amidolytic activity of FVIIa induced by FT, with a lower IC50 value that the IC50 value of FFR-rFVIIa + 100 nM (using 10 nM FVIIa in the assay), lower than the CIS0 value of FFR-rFVIIa + 40 nM, preferably lower than the CI5 value < > of FFR-rFVIIa + 20 nM, preferably lower than the IC50 value of FFR-rFVIIa + 10 nM, more preferably less than the IC50 value of FFR-rFVIIa. In a further embodiment of the invention the method of preparing a human antibody comprises testing antibodies in a FVIIa competition assay and selecting the human antibody that competes for binding with FVIIa. In a further embodiment of the invention the method for the preparation of a human antibody comprises testing antibodies in an FT ELISA assay comprising FT and selecting the human antibody that binds to human TF.

In a further embodiment of the invention the method for the preparation of a human antibody comprises testing antibodies in a FT ELISA assay comprising immobilized FT and selecting human antibodies that bind to immobilized human FT. In a further embodiment of the invention the method for the preparation of a human antibody comprises testing antibodies in an assay and by direct FT ELISA comprising FT in solution and selecting human antibodies that bind to human FT in solution. In a further embodiment of the invention, the method of preparing a human antibody comprises testing antibodies in an FXa generation assay on cells that express FT and selecting human antibodies that inhibit the generation of FXa on the cell that expresses FT with a value of IC 50 less than 500 nM (in a test with a concentration of 1 nM). In one embodiment, the IC50 value is less than 100 nM. In one embodiment, the IC 50 value is less than 50 nM. In one embodiment, the IC50 value is less than 10 nM. In one embodiment, the IC50 value is less than 5 nM. In a further embodiment of the invention, the method for the preparation of a human antibody comprises testing antibodies in an FXa generation assay on a cell expressing FT and selecting the human antibodies that inhibit the generation of FXa on the cell expressing FT with an IC 50 value lower than the GI 50 value of FF-rFVIIa + 500 nM (using 1 nM FVIIa in the assay), lower than the IC50 value of FFR-rFVIIa + 100 nM, preferably lower than the IC50 value of FFR-rFVIIa + 50 nM, preferably lower than the IC50 value of FFR-rFVIIa + 10 nM, more preferably lower that the CISo value of FFR-rFVIIa + 5 nM, more preferably less than the CISo value of FFR-rFVIIa. The term "cells expressing TF" means any mammalian cell that expresses human TF. In a further embodiment of the invention, the method of preparing a human antibody comprises testing antibodies in whole cell FT binding assays and selecting human antibodies competing for binding * with FVIIa to human FT expressed on the cell surface of whole cells. In a further embodiment of the invention, the method for the preparation of a human antibody comprises testing antibodies in a biosensor assay and selecting human antibodies with a value of ¾ for binding to human F less than 100 nm. In one embodiment, the value of Ka for binding to human TF is less than 10 nM. In one embodiment, the value of ¾ for binding to human TF is less than 5 nM. In one modality plus the value of K <; i for 1 the binding to human TF is less than 1 nM. In one embodiment, the value of ¾ for binding to human TF is less than 0.5 nM. In a further modality, the value of ¾ for the binding to human TF is less than 10"10 M. In one modality plus the value of Ka for the binding to the human TF is less than 10" 11 M. In one modality plus the value of ¾ for binding to human FT is less than FT 10"12 M. In a further mode the value of ¾ for binding to human FT is less than 10" 13 M. In one modality plus the value of Ka for the union Human FT is less than 10"14 M. In one embodiment, the Kd value for binding to human TF is less than 10" 15 M. In a further embodiment of the invention, the method for the preparation of a human antibody comprises test antibodies in a MAPK signaling assay and select human antibodies that inhibit FVIIa-induced activation of MAPK signaling. In one embodiment, the human antibody inhibits FVIIa-induced activation of 98% MAPK signaling. In one embodiment, the human antibody inhibits the activation induced by FVIIa of signaling with MAPK with 90%. In one embodiment, the human antibody inhibits FVIIa-induced activation of MAPK signaling by 70%. In one embodiment, the human antibody inhibits FVIIa-induced activation of MAPK signaling by 50%. In one embodiment, the human antibody inhibits FVIIa-induced activation of 30% MAPK signaling.

The term "signaling by MAPK" is intended to mean a cascade of ultracellular events mediating the activation of Protein Kinase Activated by Mitogen (MAPK) or its homologs in response to various extracellular stimuli. Three distinct groups of MAP kinases identified in mammalian cells have been identified: 1) extracellularly regulated kinase (Erkl / 2 or p44 / 42). 2) N-terminal kinase c-Jun (J K) and 3) p38 kinase. The Erkl / 2 pathway involves the phosphorylation of Erk 1 (p 44) and / or Erk 2 (p 4.2). Activated MAP kinases, for example MPAK p44 / 42 can be translocated to the nucleus where they can phosphorylate and activate transcription factors including (Elk 1) and signal transducers and transcription activators (Stat). Erk 1/2 can also phosphorylate the p90RSK kinase in the cytoplasm or nucleus, and p900RSK can then activate several transcription factors. The term "protein kinase" is intended to indicate an enzyme that is capable of phosphorylating serine and / or threonine and / or tyrosine in peptides and / or proteins. The term "FVIIa-induced activation of MAPK signaling" is intended to indicate that FVIIa binds to TF in a mammalian cell and therefore induces signaling by MAPK. In a further embodiment of the invention the method for the preparation of a human antibody comprises proving antibodies in a gene expression analysis assay (assay 15) and selecting human antibodies that inhibit FVIIa-induced upregulation of genes selected independently from the list comprising Fra-1, Id2, and Cyr61. It should be understood that antibodies against FT, which inhibit FT activity, can bind to different epitopes present on FT and can inhibit both binding of FVIIa or binding of FXa to human FT. An object of the present invention is to select antibodies; that inhibit the binding of FVIIa to human TF and therefore the intracellular signaling induced by FVIIa. In a further embodiment of the invention the method for the preparation of a human antibody comprises testing antibodies in a human cancer assay (assay 13) and selecting human antibodies that inhibit the growth or metastasis of human cancers. In a further embodiment of the invention, the isolated human antibody inhibits the upregulation induced by FVIIa of genes selected independently from the list comprising Fra-1, Id2, and Cyr61. In a further embodiment of the invention, the isolated human antibody does not inhibit the binding of FX or FXa to human TF. In a further embodiment of the invention, the isolated human antibody inhibits the intercellular activity of the TF.

In a further embodiment of the invention the method for the preparation of a human antibody comprises testing antibodies in an epitope map trace test and selecting the human antibodies that bind to preferred epitopes on the TF. In one embodiment the preferred epitope comprises one or more of the residues Trp45, Lys46 and Tyr94. In one embodiment, the preferred epitope comprises the Trp45 residue. In one embodiment the preferred epitope comprises the residue Lys46. In a preferred embodiment the preferred epitope comprises the residue Tyr94. In a further embodiment of the invention the isolated human antibody binds to an epitope within the interface between FT and FVlIa. The residues in the TF that are responsible for the interaction between the protease domain of FVIIa and the TF determined from the X-ray structure (Banner et al., L996 Nature, 380: 41-46) are: Ser39, Gly43, Trp45, Ser47, Phe50, Arg74, Phe76, Tyr94, Pro92. This interface between the protease domain of FVIIa and FT is characterized by a complete interface region containing many intermolecular hydrogen bonds that allow many fine contacts between FT and FVIIa to obtain a specificity in the binding process of FVIIa . The present invention also relates to human monoclonal antibodies of high affinity with FT.

The surface of the FT that contains the contact interface for the protease domain of FVIIa contains a specific topology that is prone to react to create protein-protein interactions, where another type of protein-protein interaction is the formation of complexes between an antibody and a protein ligand. In this way, monoclonal antibodies directed against the epitope on human TF give high affinity Acsm. One aspect of the present invention is high affinity human monoclonal antibodies, which are immunoreactive with the contact interface of the protease domain of FVIIa. The human FT antibodies of the present invention act as antagonists for the induction of FT-mediated coagulation, thereby inhibiting the binding of coagulation FVIIa to FT and thus blocking the production of thrombin and the subsequent deposition of fibrin. . Human FT antibodies are particularly useful for administering to humans to treat a variety of conditions involving intravascular coagulation. Therefore, human TF antibodies can be used to inhibit TF activity which results, for example, in the inhibition of blood coagulation, thrombosis or platelet deposition. In addition, human TF antibodies according to the present invention, which act to inhibit the cellular functions of TF, the TF signaling function, may be useful in conditions such as sepsis, inflammation, arteriosclerosis, restenosis or cancer. Human FT antibodies can be useful in a variety of diseases. Included are thrombotic or coagulopathic diseases or disorders including the inflammatory response and chronic thromboembolic diseases or disorders associated with fibrin formation, including vascular disorders such as deep vein thrombosis, arterial thrombosis, postoperative thrombosis, coronary artery bypass graft (CABG) ), percutaneous transdermal coronary angioplasty (PTCA), stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, atherosclerosis and restenosis followed by angioplasty, acute and coronary locations such as inflammation, septic shock, septicemia, hypotension, adult respiratory distress syndrome (ADRA), systemic inflammatory response syndrome (SIRS), disseminated intravascular coagulopathy (DIC), pulmonary embolism, pathological platelet deposition, myocardial infarction, or prophylactic treatment of mammals with atherosclerotic spleens at risk of thrombosis bosis, veno-occlusive disease after transplantation of peripheral blood progenitor cells (CPSP), hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP) and other diseases or disorders. Human FT antibodies can be used to prevent the occurrence of thromboembolic complications in high-risk patients identified, such as those undergoing surgery or those with congestive heart failure. Human FT antibodies may be particularly useful in the treatment of intimal hyperplasia or restenosis due to acute vascular damage. Acute vascular injuries are those that occur rapidly (ie, for a few hours to months), in contrast to the chronic vascular damage (eg, atherosclerosis) that develops during life.Acute vascular injuries often result from surgical procedures such as vascular reconstruction, where the techniques of angioplasty, endarterectomy, atherectomy, vascular graft replacement or the like are used.Hyperplasia can also occur as a delayed response in response to, for example, graft replacement or organ transplantation. of human FT are more selective than heparin, binding only the FT that has been exposed at the sites of damage, and because human FT antibodies do not destroy or inhibit other coagulation proteins, they will be more effective and probably cause less Heparin complications when used prophylactically for the prevention of deep vein thrombosis.

As shown in the following examples, the human FT antibodies of the present invention are capable of selectively binding to the FT of the cell surface and inhibiting its functional activity by inhibiting the binding of FVIIA from coagulation to FT. Human FT antibodies that maintain FT binding inhibit platelet accumulation at the site of vascular damage by blocking the production of thrombin and the subsequent deposition of fibrin. Due to the ability of human FT antibodies to block thrombin deposition and limit platelet deposition at sites of acute vascular damage, human FT antibodies that maintain FT binding thus inhibit the binding of FVIIA can be used to inhibit vascular restenosis. Compositions comprising human TF antibodies are particularly useful in methods of treating patients when formulated in pharmaceutical compositions, where they can be given to individuals suffering from a variety of disease states to treat conditions related to coagulation. These human FT antibodies, capable of binding to TF and inhibiting the binding of FVIIa to TF, may have a prolonged plasma half-life and thus a correspondingly greater period of clotting activity when compared to other anticoagulants. Among the medical indications for the target compositions are those commonly treated, with anticoagulants, such as, for example, deep vein thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIC), fibrin deposition in the lungs and kidneys associated with sepsis, antiphospholipid (SAF), atherosclerosis and myocardial infarction. The compositions that can be used to inhibit vascular restenosis that occurs after mechanical vascular damage, such as damage caused by surgery, microsurgery, balloon angioplasty, endarterectomy, reductive atherectomy, placement of a stent device, laser therapy or rotoablation , like the one that occurs secondary to vascular grafts, stent devices, diversion grafts or organ transplants. The compositions can thus be used to inhibit platelet deposition and associated disorders. Thus, a method for inhibiting coagulation, vascular restenosis or platelet deposition, for example, comprises administering to a patient a composition comprising human PT antibodies in an amount sufficient to effectively inhibit coagulation, vascular restenosis or platelet deposition, methods also find use in the treatment of acute closure of a coronary artery in an individual (for example acute myocardial infarction), which comprises administering human TF antibodies, in conjunction with tissue plasminogen activator or streptokinase, and can accelerate the thrombolysis induced by tPA. Human FT antibodies are given before, in conjunction with, or immediately after administration of a thrombolytic agent, as a tissue plasminogen activator. According to the invention, human monoclonal antibodies directed against FT humstno can be produced by immunizing transgenic mice. { Obtained from Medarex) that contain parts of the human immune system in place of the mouse system with human TF. Splenocytes from those transgenic mice are used to produce hybridomas secreting human monoclonal antibodies as described (see, for example, Wood et al International Application WO 91/00906, Kucherlapati et al PCT Publication WO 91/10741; Lonberg et al. International Application WO 92/03918, Kay et al, International Application 92/03917, Lonberg, N. et al., 1994, Nature 368: 856-859, Green, LL, et al., 1994, Nature Genet, 7: 13-21, Morrison. , SL et al., 1994 Proc. Nati, Acad. Sci. USA 81: 6851-6855; Bruggeman et al., 1993 Year Immunol 7: 33-40; Tuaillon et al., 1993 PNAS 90: 3720-3724; Bruggeman et al. 1991 Eur J Immunol 21: 1323-1326). Human monoclonal antibodies directed against human TF can also be produced by phage display. Libraries of human antibodies can be constructed from persons immunized and presented on the surface of filamentous phages. Fragments of high-affinity human single chain Fv antibodies (ScFv) and Pac, in many cases, they have been isolated from these libraries using a sieve technique in which the antigen of interest immobilized on a solid surface, ie microtitre plates or beads (Barbas CF, III and Burton, DR Trends, Biotechrol, 1996, 14: 230-234; Aujame L. et al, Hum Antibodies 1997, 8: 155-68). The presentation of phage from large candid libraries has also been possible to isolate human antibodies directly without immunization (DeHaard H. J. et al J. Biol. Chem. 1999, 18218-18230). BRIEF DESCRIPTION OF THE DRAWINGS The present invention is described in greater detail in the examples with reference to the accompanying figures in which Figure 1. Schematic presentation of a selection and assay assay for the selection of high affinity human monoclonal antibodies against FT. Figure 2. Detailed schematic representation of the selection tests 1-3 as described in example 1. Figure 3. Detailed schematic representation of the selection trials 4-7 as described in example 1. Figure 4. Detailed schematic representation of selection tests 8-10 as described in example 1. Figure 5. An example of selection antibodies by assay no. 4. Inhibition of the amidolytic activity of sFT / FVIIa by FFR-rFVIIa (closed circles) and the anti-human FT monoclonal antibody HuTF-31F2 (open circles). Figure 6. An example of selection antibodies by assay no. 5. Inhibition of factor Xa generation by FFR-rFVIIa (closed circles) and the anti-human FT monoclonal antibody HuTF-31F2 (open circles). Figure 7. An example of selection antibodies by assay no. 7. Inhibition of human TF-induced coagulation by FFR-rFVIIa (closed circles) and the anti-human FT monoclonal antibody HuTF-31F2 (open circles). Figure 8. An example of selection antibodies by assay no. 10. Only anti-FT monoclonal antibodies that prevent the binding of FVIIa to inhibit FT / FVIIa-mediated signaling. Figure 9. Human anti-FT mAb inhibits FVIIa-induced phosphorylation of MAPK p44 / 42 (assay # 10). BHK cells transfected with TF were deprived of nutrients for 2 hours to produce inactive cells. Antibodies HuMab 30F5 (500 nM) and Hu ab 31F2 (500 nM) were added to the cells 15 min before the addition of FVIIa (15 nM). The cells were lysed and the proteins were separated on SDS-PAGE and transferred to nitrocellulose by electrospinning. Western electroblot analysis was performed using phospho-specific polyclonal antibodies for M > K p44 / 42. The secondary antibodies were anti-rabbit IgG conjugated to Radish Peroxidase. The chemiluminescence detection was performed using a cold CCD camera. The bands on the digitized image were quantified and the band obtained with FVIIa was fixed as 100%. When the cells were pre-incubated with HuMab 30F5 (500 nM), a 50% reduction in the phosphorylated band was observed when the cells were preincubated with HuMab 31F2 (500 nM) a reduction of 25% was observed. In conclusion, this experiment shows that human antibodies against TF (30F5 and 31F2) partially inhibited the FVIIa-induced phosphorylation of MAPK p44 / 42. Similar results were obtained using 50 nM FVIIa. Figure 10. An example of selection antibodies by in assay no. 16. The figure demonstrates the inhibition of the intracellular activity of TF in cells expressing TF by monoclonal antibodies against TF. Anti-FT B mAb inhibits the intracellular activity of TF, whereas anti-TF A mAb does not. Figure 11. An example of antibody d selection by in assay no. 12. Velocity profile of thromboelastograms obtained with 0.5 nM of FFR-rFVIIa and the anti-human TF antibody HuFT-31F2.

DETAILED DESCRIPTION OF THE INVENTION The present invention is best illustrated by the following examples, which, however, should not be construed as limiting the scope of protection. The features described in the following description and in the following examples may, separately and in any combination thereof, be material for realizing the invention in various forms thereof. EXAMPLES Example 1 Preparation of immunospecific Acsm for human TF. Reagents FT can be isolated from the human brain according to that described by Rao, L.V.M., Thrombosis Research, 51: 373-384 1988. The lipidated recombinant human FT (Dade Innovin, Baxter) can also be used as a human thromboplastin reagent. The thromboplastin of rat, rabbit, baboon and pig is prepared from brain tissue. Two volumes of 0.9% NaCl are added at 45 ° C to the brain tissue, and the tissue is homogenized with a manu l glass homogenizer. After 30 minutes of incubation at 45 ° C with occasional agitation, the samples are centrifuged for 20 minutes at 2000 x g. The precipitates are discarded, and the supernatant is stored at -80 ° C until use.

The relipidated TF can be obtained by the reconstitution of the recombinant human full-length TF (American Diagnostica # 4500) in phospholipid vesicles (PC / PS 75/25). The biotinylated human FT is produced as follows: Biotin-NHS (n-succinimido biotin, Sigma H-1759) is dissolved in DMF (dimethylformamide) at a concentration of 1.7 mg / ml. At 1 mg / ml of human FT in NaHCC buffer > 3 0.1 M do you add 60 μ? of the biotin-NHS solution and allowed to react for 4 hours at room temperature. The reaction solution containing the biotinylated TF is dialyzed against PBS buffer overnight. The FVIIa used is recombinant human FVIIa prepared according to that described by Thim, K. et al. Biochem 27: 7785-7793, 1988. FTs: Recombinant human soluble FTi-209 expressed in E. coli and purified essentially as described by Freskgard, P.-O. Et al. Protein Sci. 5, 1531-1540 (1996). S2288: reconstituted in H20 at 17.24 mg / ml (Chromogenix) FX: FX of purified human plasma (HFX, Enzyme Research Laboratories Ltd.) FXa: Activated FX of purified human plasma Oon Russel Snake Venom (HFXa, Enzyme Research Laboratories Ltd .) Chromozyme Z: Dissolved in H20 at 1.25 mg / ml. (Boehringer Mannheim) 125I-FVIIa is obtained by standard radioactive labeling procedures. FFR-rFVIIa: FVIIa blocked at the active site with D-Phe-L-Phe-L-Arg-chloromethyl ketone. Prepared according to what was described by Sorensen B.B. et al. J. Biol. Chem. 272: 11863-11868, 1997. Immunization Human FT is emulsified in Freund's Complete Adjuvant. 40 g of AcmHu are provided to mice or hybrids thereof (Medarex) by subcutaneous injection. 14 and 28 days later, and eventually more times with intervals of 14 days, the mice are reinforced with a similar injection of 20 μ9 of FT in Incomplete Freund's Adjuvant. Ten days after the last injection a blood sample is taken and the sera are tested for specific antibodies to human TF by FT ELISA (Tests 1 and 2). Fusion Mice with positive serum test 1-3 are reinforced with 20 μg of human FT by intravenous injection and sacrificed 3 days later. The spleen is removed aseptically and dispersed to a simple cell suspension. I¾ Fox myeloma cells are grown in hybridoma CD medium (Gibco 11279-023). The fusion of spleen cells and myeloma cells (P3x63 Ag8.653, ATCC CRL-1580) and Sp2 / 0 myeloma cell lines (ATCC CRL.1581) for our fusions are carried out by the PEG methods (Kohler , G &Milstein C. (1976), European J. Immunology, 6: 511-19). The cells are seeded in microtiter plates and incubated at 37 ° C. The medium is changed three times during the next two weeks. 100 μ? of supernatant of the hybridoma cells from each well and tested for FT-specific antibodies in FT ELISA (Tests 1 and 2). Example 2: Selection. The different assays used in the selection of serum and culture supernatants for specific selected antibodies are described below. Direct FT ELISA assay (Assay 1): Nunc immunoplates are coated during the noch at 4 ° C with 1 μg / ml of human FTs in PBS. The plates are blocked with blocking buffer (T3S with CaCl2 5 ti and 2% BSA) and washed in TBS + 0.05% Tween 20, and the supernatants of the hybridoma cells are added. After incubation at room temperature for 1 hour, the plates are washed and anti-human IgG is added with horseradish peroxidase (POR). After another hour of incubation, the plates are washed and developed with TMB substrate (Kem-EN-Tec) as described by the manufacturers. The absorbance at 450 nm is measured in an ELISA reader. Indirect FT ELISA assay (Assay 2): Nunc immunoplate coated with 0.5 μg / ml goat anti-human IgG (Southern Biotechnology Associates, Cat- # 2040-l) in PBS and incubated overnight at 4 ° C. . The plates are blocked with block buffer (TBS with 5 mM CaCl 2 and 2% BSA) and washed in TBS + 5 mM CaCl 2 + 0.05% Tween 20. The culture supernatants of the hybridoma cells and the plates are incubated for one hour at room temperature. After another wash, biotinylated human FTs are added at a concentration of 1 9 / p ?, and 'Be incubated for 1 hour. After washing, 100 μ? of a solution of Streptavidin_POR and incubated for 1 hour. The plates are developed with TMB substrate as described for test 1. FVIIa competition assay (Test 3): Nunc immunoplates are incubated with human FTs (concentration of 5 μg / ml in PBS) overnight, 4 ° C . Plates are washed and blocked in TBS buffer with 5mM CaCl2 and 2% BSA. Human anti-FT Acsm is added and the plates are incubated for 2 hours. The plates are washed before the bidphylyphilated human FVIIa (1 μ? / P ?? in TBS buffer with 5 mM CaCl2 and 2% BSA) is added and the plates are incubated for 1 hour. The plates are washed before the addition of Streptavidin labeled with HRPO and incubated for 45 minutes. The plates are washed again before revealing with TMB substrate (Kera-EN-Tec) according to what is described by the manufacturers. Inhibition of the amidolytic activity of FVIIa / FTs (Test 4): The inhibition of the amidolytic activity catalyzed by FVIIa-FT by human anti-FT mAbs was tested using soluble human FT (10 nM), recombinant human FVIIa (10 nM) and increasing the concentrations of the Acsm (0.0122-50 nM). Several concentrations of human anti-FT Acsm or FFR-rFVIIa are preincubated with 10 nM FTs and 10 nM FVIIa in BSA buffer (50 nM Hepes, pH 7.4, 100 mM NaCl, 5 mM CaCl 2 and 1 mg / ml BSA) during 60 minutes at room temperature before the addition of the S2288 substrate (1.2 mM, Chromogenix). The color development is continuously measured for 30 minutes at 405 nm. The amidolytic activity is presented as ODm / min. The IC50 values for the inhibition of the amidolytic activity of FVIIa / TF by the Acsm can be calculated. The values of Cls0 for FFR-rFVIIa is 7 +/- 3 nM in this test. of FXa (Essay 5). FT lipidated (10 M), FVIIa (100 pM) and anti-FT Acsm or FFR-rFVIIa (0-50 nM) are incubated in BSA buffer (see assay 4) for 60 minutes at room temperature before it is added. the FX (50 nM). The reaction is stopped after a further 10 minutes by the addition of ¾ volume of the interruption buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 20 mM EDTA). The amount of FXa generated is determined by the addition of the substrate S2765 (0.6 mM, Chromogenix, and measuring the absorbance at 405 nm continuously for 10 minutes.) The IC 50 values for the inhibition of the mAb of the activation mediated by FVIIa / FT lipidated F can be calculated The IC 50 value for FFR-rFVIIa is 51 +/- 26 pM in this assay Biodetector assay (Test 6) Antibodies are tested in the Biacore instrumentation by passing a standard solution of human anti-FT mAb over a microcircuit integrated with immobilized antibody to human IgG.This is followed by different concentrations of FTs in 10 mM hepes, pH 7.4 with a content of 150 mM NaCl, 10 mM CaCl2, and polysorbate 20 at 0.0003% .The values of ¾ are calculated of the detectograms using the integrated Biacore evaluation programming programs and systems.

FT (Test 7): an ACL300 Research coagulation apparatus (ILS Laboratories). Dilutions of human anti-FT ACSM are mixed in imidazole SO mM, pH 7.4, 100 mM NaCl, 0.1% BSA with 25 mM CaCl 2 in the ratio of 2 to 5 and added to sample cups in the coagulation apparatus. The thromboplastin of human, rat, rabbit, baboon or pig diluted with the imidazole buffer to give a coagulation time of approximately 30 sec. in samples without antibody is placed in the reservoir of reagents 2, and the plasma of human, rat, rabbit, baboon or pig, in the reservoir of reagent 3. During the analysis are transferred 70 μ? of a mixture of CaCl2 antibody at 25 μ? of thromboplastin reagent and preincubated for 900 sec. before the addition of 60 μ? of plasma and measured the clotting time. The maximum coagulation time is set at 400 sec. A thromboplastin dilution was used as the standard curve to convert the clotting times into the activity of FT in relation to the control if anti-FT or ACSM. FFR-rFVIIa aggregates. The IC50 value for FFR-rFVIIa is 4.4 +/- 0.4 pM in this assay. Inhibition of FVIIa / FT catalyzed activation of the FX cell surface by Acsm (Assay 8): Monolayers of cells expressing human TF, for example human lung fibroblasts WI-38 (ATTC No. CCL-75), cell line of human bladder carcinoma J82 (ATTC No. HTB-1), human keratinocyte cell line CC 1102KerTr (ATTC No. CRL-2310), human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231, are employed, source of FT in the activation catalyzed by FVIIa / FT of the FX. Monolayers of confluent cells in a plate of 24, 48 or 96 wells are washed once in buffer A (10 mM Hepes, pH 7.45, 150 mM NaCl, 4 mM Cl, and 11 mM glucose) and once in B buffer ( buffer A supplemented with 1 mg / ml of BSA and 5 mM Ca2 +). FVIIa (1 nM) FX (135 nM) and several concentrations of Acm (or FFR-rFVIIa) in buffer B are added to the cells simultaneously. Alternatively, the cells are preincubated 15 min. with anti-FT ACSM or FFR-rFVIIa before the addition of rFVIIa and FX. The formation of FXa is allowed for 15 min. at 37 ° C. Aliquots of 50 μ? Are removed? and added to 50 μ? of interruption buffer (shock absorber supplemented with 10 mM EDTA and 1 mg / ml BSA). The amount of FXa generated is determined by transferring 50 μ? of the previous mixture to a well of a microtiter plate and adding 25 μ? of chromozyme X (final concentration of 0.6 mM) to the wells. The absorbance at 405 nM is measured continuously and the initial rates of color development are converted to FXa concentrations using a standard FXa curve. The IC50 value for FFR-rFVIIa is 1.5 nM in this assay. Inhibition of binding of 12SI-FVIIa to FT of the cell surface by mAb (Assay 9): Binding studies were used using cells expressing human TF, for example human lung fibroblasts I-38 (ATTC No. CCL-75), human bladder carcinoma cell line J82 (ATTC No. HTB-1), human keratinocyte cell line CCD 1102KerTr (ATTC No. CRL-2310 ), U87 human glioblastoma cell line, or human breast cancer cell line DA-MB231. The confluent monolayers in 24-well tissue culture plates are washed once with buffer A (see test 8) and once with buffer B (see test 8). The monolayers are pre-incubated 2 min. with 100 μ? of shock absorber B cold. Variable concentrations of Acsm (or FFR-rFVIIa) and radiolabeled FVIIa (125 I-FVIIa 0.5 nM) are added simultaneously to the cells (final volume of 200 μ?). The plates are incubated for 2 hours at 4 ° C. At the end of the incubation, the unbound material is removed, the cells are washed 4 times with buffer B cooled with ice and washed with 300 μ? of lysis buffer (200 m NaOH, 1% SDS and 10 m EDTA). Radioactivity is measured in a gamma counter (Cobra, Packard Instruments). The binding data are analyzed and the curve adjusted using GraFit4 (Erithacus Software, Ltd., (R.U.)). The IC50 value for FFR-rFVIIa is 4 nM in this assay.

Inhibition p44 / 42 induced by FVIIa / FT by the MCA (Test 10): The amount of phosphorylated MAP44 p42 / 42 and / or Akt, and / or p90RSK is determined by the quantitative detection of chemiluminescence (Fujifilm LAS-1000) of the analysis of western electromagnet transfer. Cells expressing human TF, for example CCD1102KerTr, HEK P166, human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231, are cultured in medium with 0.1% FSC for 24 or 48 hours before the experiment to inactivate the cells. On the day of the experiment the cells should have a confluence of 70-80%. The experiment is carried out by preincubating the cells with excess of Acm or FFR-rFVIIa in medium without serum for 30 min. at 37 ° C before the addition of 10-100 nM FVIIa and incubating for 10 minutes. As a positive control of cell signaling, the cells are treated with 10% FSC for 10 minutes. The cells are washed twice in ice-cold PBS before the cells are lysed in lysis buffer (20 mM Tris, 0.1% Triton X-100, ImM EDTA, lmM EGTA, 50 mM sodium fluoride, β-glycerophosphate of sodium 10 mM, 5 mM sodium pyrophosphate, 150 mM NaCl, pH 7.5 with a content of 4- (2-aminoethyl) benzenesulfonyl fluoride (FAEBS) 0.1 mM and 1 mM benzamidine, added just before use: orthovanadate 1 mM sodium, 5 μg / ml leupeptin, 10 μg / ml aprotinin). The lysates were SDS-sampler buffer and loaded onto polyacrylamide. A standard biotinylated protein marker is loaded into each gel. The separated proteins on SDS-polyacrylamide gel were transferred to nitrocellulose by electrospinning, and MAPK kinases p44 / 42, Akt and p90RSK were visualized by immunostaining by phospho-specific antibodies, and the chemiluminescence is quantified by Fujifilm LASIOO. Epitope map trace assay (Assay 11): Preparation of soluble TF variants (FXs). The variants of FTs (I22C, W45C, K46C, Y94C, F140C, W158C, K201C) are constructed using inverse PCR (QuikChange, Stratagene, La Jolla, CA, USA) using natural plasmid (Freskgard et al., Protein Sci. 5, 1531). -1540, 1996) as a model. The wild type and variants are expressed and purified in E. coli according to what is described elsewhere (Freskgard et al., Protein Sci. 5, 1531-1540, 1996) with some modifications. Cell lysis is performed by the X-press technique (Biox, Sweden) in 10 mM Tris-HCl buffer, pH 7.5 and subsequently suspended in the same buffer with the addition of 1 mg of DNase. The solution is centrifuged at 11OOOxg for 20 min. at 4 ° C, and the inclusion bodies are denatured in 75 ml of 6 M GuHCl, 0.5 M NaCl, 20 mM Tris-HCl, pH 8.0. Refolding is achieved after 1 hour of incubation at room temperature by dripping by diluting the denatured protein in 1 L of solution containing 50 mM Tris-HCl, 0.25 mM NaCl, pH 8.0 with gentle agitation for about 2 hours. The purification is effected using Q-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden) and affinity chromatography of PVIIa according to what is described by Freskgard et al. (nineteen ninety six). The homogeneity of the protein is verified by SDS-PAGE. The concentration is measured at A2B0nm and determined using a calculated extension coefficient of 37440 M "1cm * 1 (Gilí and von Hippel, 1989) .AxiSorp plates (Nunc-Immuno) are coated with natural FTs and variants (10 μg / ml ) in TBS and blocked with blocking buffer (TBS with Tween 20 al, 0.1% and 0.5% BSA) .The plates are washed with ford buffer (TBS and TWEEN 20 to 0.1%) .The human anti-TFs Acsm are applied at a concentration of 1 ng / ml of blocking buffer and incubated for 1 hour.The plates are then washed (6x) using the washing buffer.The antibody binding is subsequently detected using ati-human IgG labeled with POR ( Helica Biosystems, Inc.) to a 1: 2000 dilution in blocking buffer using the TMBpiug substrate (Kem Tech Cat. 4390A) The final ELISA signal (D04so-62o) is used as a measure of the binding of each antibody to all the variants of FTs.

Human thromboplastin (for example, Innovin, Dabe Behring, final dilution 50,000 x) was mixed with CaCl 2 (final concentration of 16.7 mM) and anti-Ft ACSM and incubated for 15 min. at room temperature. Citrate-stabilized human whole blood (280 μ?) Was added to RoTEG sampling cups (Pentapharm) and preheated for 5 min. at 37 ° C, before the addition of 40 μ? of thromboplastin / CaCl2 / ACM anti-FT mixture. Thromboelastography is followed for one hour in a Ro-TEG device (Penthapharm). The velocity profiles are obtained from the programs using the programs and programming systems CoagProMR (MedScience, Árhus, Denmark). Example 3 Human cancer assay. Investigation of the effects of treatment with human anti-TF mAbs on the growth and metastasis of human cancers in mouse models (Assay 13) Treatment: ACSM anti-human FT given by injection i.v. of bolus; 10 mg / kg = 0.1 mg / lOg; the injection volume is 0.1 ml per 10 g of mouse of any of three treatment solutions: A. Control vehicle B. 1 mg / ml of human FFR-rFVIIa C. 1 mg / ml of anti-FT ACSM Description of 3 «Portholes J. Primary growth and hepatic metastasis of colon cancer Healthy female mice (nu imj5 7-8 weeks old) were used to destroy the residual immunoresistance of nude mice at the impiatiation of human cells. mice are routinely irradiated, at 5 Gy 2 days before human tumor grafting (Vogel et al., 1997) .The mice are challenged by grafting tumor from human colon carcinoma cell LS174T (ATCC CCL 188) cultured in RPMI 1640 with 15 % fetal sheep serum (FSC) according to those described (Li et al., Human Gene Therapy 10: 3045-3053, 1999.) In brief, cells are harvested with trypsin-EDTA, washed twice, and resuspended in serum-free RPMI supplemented with heparinate solution of sodium (1 U / ml). A small left subcostal incision is then made in the mice under anesthesia and 33 106 LS174T cells are injected in 50 m phosphate buffered saline (PBS) in the spleen. After 3 to 5 minutes, the spleens of the spleen are ligated and the spleen is surgically removed. This procedure will lead to a stable incidence of liver metastases (more than 95%). Treatment with anti-TF Acsm will be initiated immediately after implantation and will last for the remainder of the study period. On days 15 and 30 after the inoculation of the tumor cells the mice are sacrificed, the livers are removed and weighed, and the number of tumor nodules visible on the surfaces of the livers is counted. Liver samples are fixed overnight in AFA (5% acetic acid, 75% ethyl alcohol, 2% formalin, 18% water), transferred to 100% ethanol, and embedded in paraffin, and prepared 5 mM sections for histological quantification of metastatic nodules, by immunohistochemistry and quantification of apoptosis. Study I-li Purpose: To examine the effects of microscopic growth and hepatic metastasis of colon tumours LS174T in nude mice of anti-FT Acsm given in a bolus by i.v injection; 10 mg / kg Mice: 60 NRMI females of 6 weeks of age nu / nu homozygous. Groups The mice are assigned randomly in 4 groups of 15 and treated with solutions A, B or C.

Term: At a weight loss of > 20% or other objective signs of severe toxicity the animal is finished. Parameters: The size of the tumor in two orthogonal diameters is recorded daily during the growth phase. Body weight is recorded 5% S - 3 times per week. Postmortem determination of metastasis formation in the liver. II. Primary growth and me ii * t3¾áá; to% monar & Breast cancer Human breast carcinoma cells MDA-MB-231 (ATCC HTB26) are cultured in Dulbeco's modified Eagle's medium (DMEM) supplemented with 10% fetal sheep serum (FSC). MDA-MB-231 (3 3 106) cells are injected subcutaneously in nude mice (female mice 7 to 8 weeks old). Primary growth of the tumor and metastasis is evaluated as described previously (LI et al., Human Gene Therapy 12: 515-526, 2001) Study II-1 Purpose: To examine the effect on macroscopic growth and lung metastasis of mammary tumors MB-231 in nude mice of anti-FT Acsm given in a bolus by i.v.; 10 mg / kg Mice: 60 NRMI females of 6 weeks of age nu / nu homozygous. Groups The mice are assigned randomly in 4 groups of 15 and treated with solutions A, B or C.

Term: At a weight loss of > 20% or other objective signs of severe toxicity the animal is finished.

Parameters: in two diameters daily during the growth phase. The oral body weight is initially registered 2-3 times per week. Postmortem determination of metastasis formation in the lung. JJJ Primary growth of glioma tumor xenograft The MG U373 tumor cell line is a human glioblastoma multiforme cell line, with high angiogenic activity, high vascular density and rapid growth in nude mice. Flank tumors are inoculated, following standard procedures (see attached protocols for the experimental plan). The mice are observed twice a day for signs of toxicity and the mice are measured daily in two perpendicular diameters. Tumors are transplanted to the flanks of homozygous nu / nu nude mice with a history of NMRI. The mice are 7-week-old males obtained from M &B (Ry, Denmark). The animals are obtained in a gnobiotic environment and receive croquettes of sterile food and drinking water ad üjbifcum. Three different studies are conducted with the glioma tumor model: Purpose: Examine Ifc * effects on the macroscopic growth of tumors. U373 e nude anti-f mAbs given in a bolus by i.v injection; 10 mg / kg Ra on 60 NRMI males 6 weeks old nu / nü homozygous. Groups The mice are assigned randomly in 4 groups of 15 and treated with solutions A, B or C.

Term: At a weight loss of > 20% or other objective signs of severe toxicity the animal is finished. Parameters The size of the tumor in two orthogonal diameters is recorded daily during the growth phase. Body weight is registered initially and 2-3 times per week.

Study III-2 Purpose: To examine the effects of macroscopic growth of U373 tumors in nude mice of anti-FT Acsm given in a bolus by i.v injection; 10 mg / kg. after the pre-therapeutic growth of the tumor has been established. Mice 60 NRMI males 6 weeks old nu / nu homozygous. Groups The mice are assigned randomly in 4 groups 5 and 5, with solutions A, B or C. Treatment begins when 6 consecutive measurements (daily) show Qompert-zynia growth. This corresponds to 100-200 mm3. Term: The treatment lasts until the tumors' has grown beyond the maximum size of approximately 1.0 cm3, ie with a tumor diameter no greater than 15 mm or until there is sodium stabilized a Gompertzian regrowth by 6 consecutive measurements. At the end of the term, the tumors of each group are excised for histological and immunochemical evaluation. At a weight loss of > 20% or other objective signs of severe toxicity the animal is finished. Parameters: The size of the tumor in two orthogonal diameters is recorded daily during the growth phase. Body weight is registered initially and twice a week.

Study III -3 Purpose: To examine the effect of anti-FT mAbs on the growth of intracranial U373 tumors in nude mice. Mice: 60 NRMI males 6 weeks old nu / nu Tumor: U373 implanted orthotopically in the right hemisphere following standard procedures. Groups The mice soii: Randomly assigned in 4 groups of 15 and treated with solutions A, B or C. Term: Mice are signs of chronic neurological damage are subjected to euthanasia. Data: Survival (ie time to neurological damage) is quantified by Kaplan-Meyer statistics. Example 4 (Test 14) In a mouse where the FT gene is blocked and the FT gene is inserted (mFT-KO / hTF-KI mice) a 0.5 ml matrigel plug will be placed under the abdominal skin. In the matrigel, b-FGF (5 ng) will be incorporated and one week later the formation of new patent spleens in the gel will be quantified by measuring the hemoglobin content (angiogenesis). The inhibitory capacity (% inhibition of the hemoglobin content) of the human anti-FT mAbs can be evaluated after single or repeated parenteral administrations of the proteins. Example 5 (Test 15) Analysis assay of gene expression to discriminate antibodies, which prevents the binding of FVIIa to FT and antibodies, which prevent the binding of FX to FT. In cDNA microarray analysis, a gene has been observed in BHK-TF cells treated with 'Aflia. These include: Fra-1, a gene that codes for an antigen relacün¾ £ k >; with Fos 1, Id2, a gene encoding a member of the helix-loop-helix class of proteins, and Cyr61 encoding a signaling protein from the extracellular matrix. The following assay was designed to select anti-FT mAbs that avoid FVIIa-induced upregulation of Fra-1, Id2 or Cyr61. Cell culture Reagents were purchased from GIBCO-BRL unless otherwise indicated. The BHK-FTr cells created according to what was described by Poulsen L.K. et al., J Biol. Chem. 273, 6228-6232, 1998, is grown in Dulbecco's modified Eagle's medium with a content of 10% FSC, 100 IU / ml penicillin, and 100 μg / ml streptomycin to obtain a confluence of 95-100 %, washed and grown for an additional 16-18 hours in medium are FSC. The cells are washed again and exposed to FSC-free medium with a 100 mM FVIIa content. For the cloning of fragments for Northern electro-Western blot analysis the cells are treated as follows. BHK-FT are grown in Dulbeco modified Eagle medium with an FSC content of 10%, 100 IU / ml penicillin, and 100 g / ml streptomycin to obtain a confluence of 95-100%, washed and grown for 16 days. -18 additional hours in media without FSC. The cells are washed again and exposed to the FSC free medium with a content of 100 nM FVIIa for 1 h. The CRL2091 cells (ATCC) are grown in Iscove modified Dulbeco medium with a content of 10% FBS, 100 U / ml of penicillin, and 100 μg / such of streptomycin to a confluence of 95-100%. Subsequently, the cells are deprived of serum for 16-18 hours and treated with FBS-free medium with a content of 100 nM FVIIa for 6 hours. Murine 3T3-L1 cells (ATCC) are maintained in modified Dulbeco's Eagle medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μ9 /? T? 1 streptomycin. The cells are grown to confluence and are induced with medium containing 1 μm dexamethasone (Sigma), 10 pg / ml human insulin (Novo Nordisk A / S), and 1 μ. of BRL 9653 (Novo Nordisk A / S) for 1 hour. Cloning of fragments for Northern blot analysis Fra-1 is cloned by reverse transcription PCR from RNA isolated from 3T3-L1 cells treated for 1 hour with dexamethasone, insulin, and BRL49653 using the Superscript II (Life Technologies) kit. according to the manufacturer's instructions. Id2 and Cyr61 are cloned by reverse transcription PCR from isolated RNA respectively. The primers upstream and current CAGCATGAAAGCCTTCAGTC-3 'and 5' -CTCTGGTGATGCAGGéÍÍAG-3 'for Id2, 5' -CGTCACCCTTCTCCACTTGA-3 'and 5'CTTGGTCTTGCTGCATTTCT-3' for Cyr61. The parameters for the PCR are a cycle of denaturation at 94 ° C for 10 s, annealing at 6 ° C for 15 s, and extension at 72 ° C for 1.5 min, a denaturation cycle at 94 ° C for 10 s, annealing at 6 ° C for 15 s, and extension at 72 ° C for 1.5 min, a denaturation cycle at 94 ° C for 10 s, annealing at 63 ° C for 15 s, and extension at 72 ° C for 1.5 min, a denaturation cycle at 94 ° C for 10 s, annealing at 62 ° C for 15 s, and extension at 72 ° C for 1.5 min, a denaturation cycle at 94 ° C for 10 s, annealing at 61 ° C for 15 s , and extension at 72 ° C for 1.5 min, a denaturation cycle at 94 ° C for 10 s, annealing at 60 ° C for 15 s, and extension at 72 ° C for 1.5 min, 40 denaturation cycle at 94 ° C for 10 s, annealing at 55 ° C for 15 s, and extension at 72 ° C for 1.5 min. All fragments are cloned in TPO 2.1 (Invitrogen) and sequenced using a Megabase sequencer. incubated following the seller's instructions. 20 μ9 of RNA are fractionated by size in a denaturing gel with a content of 1% agarose, 20 mM MOPS, 5 tnM NaOAc, 6% formaldehyde, and 1 mM EDTA, transferred to a Hybond N + membrane (Amersham) by stained by capillarity and immobilized by UV crosslinking. The cDNAs encoding-for Fra-1, Id2 or Cyr61 are labeled with the Prime It kit (Stratagene) using [oc-32P] dATP (Amersham) and hybridized using Express Hyb (Clontech) following the manufacturer's instructions and results they are visualized by autoradiography. Example 6 (Test 16). APK trial via the Elkl transcription factor / Luciferase reporter (PathDetect) HeLa cells are seeded to a confluence of 40% in a T-80 flask before transfection. Cells are transfected with 150 ng of pFA-Elkl (Stratagene), 3 μg of pFR-Luc (Stratagene), 3 μg of human FT / pcDNA3 and 3 μg of Mouse Protease Activated Receptor 2 / pcDNA31 + using 36 μ? of FuGene (Invitrogen) according to what is described in the manual. The next day the cells are detached by means of Versene ™ (Invitrogen) and seeded in 96 well, black well plates for 16 hours. The cells are preincubated for 1 hour with 20 μ? of serum free medium (control), 20 μ? of FFR-rFVIIa 2.5 μ? (control), 20 μ? of ACM anti-FT B 2.5 μ? or 20 μ? of ACM anti-FT A 2.5 μ ?. 20 μ? of FVIIa 0.5 μ? Half the wells and half the other half. After 4 hours of incubation the cells are subjected to the luciferase gene assay. LucLite reagent (Packard) is added to the cells according to the manufacturer's description. Luciferase expression levels are read on a TopCount Microplate Scintillation (Packard). It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims

ND? < ¾ Having described the invention as above, the content is claimed as property; in the following claims: 1. An isolated human antibody, characterized in that it immunoreacts with an epitope present on human TF. 2. The isolated human antibody according to claim 1, characterized in that it inhibits the binding of the human coagulation factor VIla of human TF. 3. The human antibody isolated according to any of claims 1-2, characterized in that it is a monoclonal antibody. 4. The human antibody isolated according to any of claims 1-3, characterized in that it is a recombinant antibody. 5. The human antibody isolated according to any of claims 1-4, characterized in that said antibody is a Fac fragment 6. The human antibody isolated according to any of claims 1-4, characterized in that said antibody is a fragment F (ac) 2- 7. The human antibody isolated according to any of claims 1-4, characterized in that said antibody 8. The one according to any of claims 1-4, characterized in that said antibody is a Pyrde chain fragment. simple. 9. The human antibody isolated according to any of claims 1-8, characterized in that said antibody has a ¾ for binding to human TF within the range of 10"15-10" 8. 10. The human antibody isolated according to any of claims 1-9, characterized in that said antibody has a ¾ binding to human TF within the range 10 ~ 15-10 ~ 10M. 11. A pharmaceutical composition comprising a therapeutically effective amount of a human antibody, characterized in that it immunoreacts with an epitope present on human TF. 12. The pharmaceutical composition comprising a therapeutically effective amount of a human antibody, characterized in that it immunoreacts with an epitope present on human TF, wherein said antibody is in accordance with any of claims 1-10. 13. A composition comprising a human antibody, characterized in that it immunoreacts with an epitope present on human TF. 14. The composition comprising an antibody lower than the IC50 value of FFR-rFVIIa + 100 pM, preferably lower than the IC50 value of FFR-rFVIIa + 50 of CI5o of FFR-rFVIIa + than the CIso value of FFR-rFVIIa + 5 pM, more preferable less than the IC50 value of FFR-rFVIIa, or test antibodies in an FXa generation assay and select the huínamo antibody that inhibits the generation of FXa with an IC50 value lower than the IC50 value of FFR-rFVIIa + 100 nm (using 0.1 nM FVIIa in the assay), lower than the CI5o value of FFR-rFVIIa + 10 nm, preferably lower than the CI5t value) of FFR-rFVIIa + 5 nM, preferably lower than the CFR value of FFR -rFVIIa + 1 nM, more preferably than the CIS0 value of FFR-rFVIIa + 0.1 nM, more preferably less than the IC50 value of FFR-rFVIIa, or test antibodies in an amidolytic FVIIa / FT assay and select the human antibody that inhibits the amidolytic activity of FVIIa induced by FT, with an IC50 value lower than the CIS0 value of FFR-rFVIIa + 100 nm (using 10 nM FVIIa in the assay), less than the IC50 value of FFR-rFVIIa + 40 nm, preferably less than the IC50 value of FFR-rFVIIa + 20 nM, preferably lower than the IC50 value of FFR-rFVIIa + 10 nM, more preferably lower than the IC50 value of FFR-rFVIIa, or test antibodies in a FVIIa competition assay and select the human antibody that competes with preferable lower than the IC50 value of FFR-rFVIIa. rFVIIa + 40 nM, preferably lower than the IC50 value of FFR-rFVIIa + 20 nM, preferably lower than the IC50 value of FFR-rFVIIa + 10 nM, more preferably less than the IC50 value of FFR-rFVIIa. 2. 3 . The method according to any of claims 17-22, characterized in that it comprises testing antibodies in a competition test of FVIIa and selecting the anticue 24. The method of trust with any of claims 17-23, characterized in that it comprises testing antibodies in an FT-ELISA assay comprising FT and selecting the human antibody that immunoreacts with human TF. 25. A human antibody characterized by immunoreaction with an epitope present on the human TF and inhibits the binding of the human coagulation factor Vlla to the human FT obtainable by a method comprising a) Preparation of human antibodies against human TF, b) test antibodies in an FT-induced coagulation assay and select the human antibody that inhibits clots in this assay with a CI value less than the IC50 value of FFR-rFVlIa + 1 nM, lower than the CI5o value of FFR-rFVIIa + 500 p, preferably lower than the IC50 value of FFR-rFVIIa + 200 pM, preferably lower than the IC50 value of FFR-rFVIIa + 100 pM, preferably lower than the CIFR value of FFR-rFVIIa + 50 pM, preferably less than the IC50 value of FFR-rFVIIa + 10 pM, more preferably less than the IC50 value of FFR-rFVIIa + 5 pM, more preferably less than the IC50 value of FFR-rFVIIa, or test antibodies in a test or FXa generation select anti-human generation of FXa with IC50 of FFR-rFVIIa + 100 nm (using FVIIa 0.1 nM in the assay), lower than the S & amp;; of FFR-rFVTIa + 10 nM, preferably lower than the IC50 of FfR-rFVIIa + 5 nM, preferably lower than the CI value of FFR-rPVHa + 1 nM, more preferably than the IC50 value of FFR-rFVIIa + 0.1 nM, more preferably less than the IC50 value of FFR-rFVIIa, or test antibodies in an amidolytic assay of FVIIa / FT and select the human antibody that inhibits the amidolytic activity of FVIIa induced by TF, with a value of IC50 lower than the IC50 value of FFR-rFVIIa + 100 nM (using 10 nM FVIIa in the assay), lower than the IC50 value of FFR-rFVIIa + 40 nm, preferably lower than the IC50 value of FFR-rFVIIa + 20 nM, preferably lower than the IC50 value of FFR-rFVIIa + 10 nM, more preferably lower than the IC50 value of FFR-rFVIIa, or test antibodies in a FVIIa competition assay and select the human antibody that compete with the binding of FVIIa, or test antibodies in an FT ELISA assay that includes FT and select the human antibody that immunoreacts with human TF. 26. A method for the preparation of a human antibody, characterized 'ßf§? «ß eífl (PEodo comprises a) immunization1 mouse cou;' f * $: hu« ¾ho, b) isolation of a producing cell d antibodies of a mouse immunization and preparation of immortal cells to secrete human antivirals, c) isolation of culture medium from immortal cells comprising antibodies produced, d) testing antibodies in an indirect FT ELISA assay comprising FT in solution and selecting antibodies humans that immunoreact with human FT in solution, e) test antibodies in a FVIIa competition assay and select human antibodies that compete for binding with FVIIa, f) test antibodies in an amidolytic d FVIIa / FT assay and select the human antibody that inhibits the amytolytic activity of FVIIa induced by FT, with an IC50 value lower than the IC50 value of FFR-rFVIIa + 100 nM (using 10 nM FVIIa in the assay), lower r to the IC50 value of FFR-rFVIIa + 40 nM, preferably lower than the IC50 value of FFR-rFVIIa + 20 nM, preferably lower than the IC50 value of FFR-rFVIIa + 10 nM, more preferably lower than the IC50 value of FFR-rFVIIa, g) test antibodies in an FXa generation assay and select the human antibody that inhibits the generation of FXa with an IC50 value lower than the value of of CI5o of FFR-rFVIIa + 5 pM, more preferably less than the IC50 value of FFR-rFVIIa, or i) selection and culture of a suitable culture medium of the immortal cell that produces the selected antibody after steps dh , j) isolation of the selected antibody from the culture medium of the selected immortal cell. 27. The method of compliance with the claim further comprises testing antibodies in a generation assay of FXa on a cell expressing FT and selecting human antibodies that inhibit the generation of FXa on the cell expressing FT with a CIso value less than the IC50 value of FFR-rFVIIa + 500 nM (using 1 nM FVIIa in the assay), lower than the IC50 value of FFR-rFVIIa + 100 nM, preferably lower than the IC50 value of FFR-rFVIIa + 50 nM, preferably lower than the FFR CIso value -rFVIIa + 10 nM, more preferably less than the IC50 value of FFR-rFVIIa + 5 nM, more preferably less than the IC50 value of FFR-rFVIIa. 29. The method according to any of claims 26-28, characterized in that the method further comprises testing antibodies in an FT binding assay of whole cells and selecting human antibodies competing for binding with FVIIa to expressed human TF. on the cell surface of whole cells. 30. The method according to any of claims 26-29, characterized in that the method further comprises testing "antibodies in a foio¾iK¾ibt assay and selecting antibodies with a Ka for binding to human FT less than 100 nM, less than 10 nM, preferably less than 5 nM, preferably less than 1 nM, more preferably less than 0.5 nM. 31. The method according to any of claims 26-30, characterized in that the method further comprises testing antibodies in a MAPK signaling assay and selecting human antibodies that inhibit FVIIa-induced activation of MAPK signaling. 32. The method according to any of claims 26-31, characterized in that the method further comprises testing antibodies in a tracing test on the epitope map and selecting human antibodies that immunoreact with preferred epitopes on the TF. 33. The method according to claim 32, characterized in that the preferred epitope comprises residues Trp45, Lys46 and Tyr94. 34. The method according to any of claims 26-33, characterized in that the immortal cell is a hydridome cell. 35. A human antibody characterized in that it immunoreacts with an epitope present on human TF and that inhibits the binding of human coagulation factor Vlla to human TF, which can be obtained by a method comprising: a) mouse with FT no, b) isolation ¾e a antibody producing cell of an immunized mouse and preparation of immortal cells to secrete human antibodies, c) isolation of the culture medium from immortal cells comprising antibodies produced, d) testing antibodies in an indirect FT ELISA assay comprising FT in solution and select human antibodies that immunoreact with human FT in solution, e) test antibodies in a FVIIa competition assay and select human antibodies that compete for binding with FVIIa, f) test antibodies in an amidoolytic FVIIa / FT assay and select the human antibody that inhibits the amidolytic activity of FVIIa induced by FT, with a C value I50 lower than the CIS0 value of FFR-rFVIIa + 100 nM (using 10 nM FVIIa in the assay), lower than the IC50 value of FFR-rFVIIa + 40 nM, preferably lower than the IC50 value of FFR-rFVIIa + 20 nM , preferably lower than the IC50 value of FFR-rFVIIa + 10 nM, more preferably lower than the IC50 value of FFR-rFVIIa, g) testing antibodies in an FXa generation assay and selecting the human antibody that inhibits the generation of FXa with an IC50 value lower than the CIS0 value of FFR-rFVIIa + 100 nM (using 0.1 nM FVIIa in the assay), lower than the IC50 value of FFR-rFVIIa + 10 nM, preferably lower than the IC50 fence of FFR-rFVIIa + 5 nM, preferably lower than the IC50 value of FFR-rFVIIa + 1 nM, more preferably lower than the value of IC50 of FFR-rFVIIa + 0.1 nM, more preferably lower than the IC50 value of FFR-rFVIIa, h) test antibodies in an FT-induced coagulation assay and select human antibodies that inhibit clot formation in this assay with an IC50 value less than the IC50 value of FFR-rFVIIa + 1 nM, lower than the CIS0 value of FFR-rFVIIa + 500 pM, preferably lower than the IC50 value of FFR-rFVIIa + 200 pM, preferably lower than the IC50 value of FFR-rFVIIa + 100 pM, preferably lower than the IC50 value of FFR-rFVIIa + 50 pM, preferably lower than the IC50 value of FFR-rFVIIa + 10 pM, more preferably less than the IC 50 value of FFR-rFVIIa + 5 pM, more preferably less than the value of IC50 of FFR-rFVIIa, i) selection and culture of a suitable culture medium of the immortal cell that produces the selected antibody after steps d-h, j) isolation of the selected antibody from the culture medium of the selected immortal cell. 36. The human antibody characterized in that it immunoreacts with an epitope present in the human TF and inhibits the binding of the human coagulation factor VIla: produced by a method according to any of claims 26-34. 37. A human antibody producing cell characterized by immunoreacting with an epitope present in human TF and inhibiting the binding of human clotting factor Vlla to human TF. 38. The cell according to claim 37, characterized in that the cell is an isolated lymphoid cell. 39. The cell according to any of claims 37-38, characterized in that the cell is isolated from a mouse. 40. The cell according to claim 37, characterized in that the cell is a hybridoma cell. 41. The cell according to claim 40, characterized in that the hybridoma cell is obtained by fusion of an antibody-producing lymphoid cell with an immortal cell to provide an antibody-producing hybridoma cell. 42. The cell according to any of claims 37-41, characterized in that the antibody inhibits the binding of the human coagulation factor Vlla to the human TF. 43. The cell according to any of claims 37-42, characterized in that the antibody that immunoreacts with a 3-dimensional surface involves all and Tyr9. 44. with any of claims 37-43, characterized in that the "antibody is a Fac fragment 45. The cell according to any of claims 37-44, characterized in that the antibody is an F (ac) 2-46 fragment. The cell according to any of claims 37-44, characterized in that the antibody is an F (ac ') 2-47 fragment. The cell according to any of claims 37-44, characterized in that the antibody is a fragment. scFv 48. The cell according to any of claims 37-47, characterized in that the antibody has a Kd for binding to human TF within the range of 10"15-10'8M. 49. The cell according to any of claims 37-48, characterized in that the antibody has a ¾ for binding to human TF within the range of 10"15-10 * 10M.