HUP0401658A2 - Human tissue factor antibodies - Google Patents

Abstract

The invention deals with fully human antibodies, which enter into an immunological reaction with tissue factor (TF), thus inhibiting the binding of coagulation factor VIIa (FVIIa). HE

Description

s. B. G. & *Szabadalmi Ügyvivői tpcfi'J H-1062 Budapest, András^ tft-413 Phone: 461-1000, Fax: 461-1099

Ski 1 Ail.

78.484/SZE

Human tissue factor antibodies

The invention relates to isolated antibodies that immunologically react with tissue factor (TF) to inhibit the binding of coagulation factor VIIa, thereby providing: an immunotherapeutic method, human antibodies against TF to inhibit the formation of thrombi, which occur during surgery, microsurgery, angioplasty or accidents, or inhibition of thrombus formation and other TF functions, which represent an abnormal hemostatic condition associated with diseases such as deep vein thrombosis, disseminated intravascular coagulation (DIC), coronary artery disease, sepsis, inflammation, atherosclerosis or cancer. The invention also discloses methods for producing the antibodies, as well as human monoclonal antibodies (MAbs) using cell lines.

Blood coagulation is a complex process of interaction between various blood components, or factors, which ultimately leads to the formation of a fibrin clot. Usually, the blood components involved in this process, i.e. the members of the cascade, are proenzymes or zymogens, enzymatically inactive proteins that are proteolytically converted to an active form by the action of an activator, thus themselves being converted into activating blood coagulation factors. Blood coagulation factors that participate in such conversion are usually called active factors, and are indicated by the addition of a lowercase letter (for example, factor VIIa).

Activated factor X (Xa) is required for the conversion of prothrombin to thrombin, which then converts fibrinogen to fibrin as the final step in the formation of a fibrin clot. Two systems or pathways can be distinguished that facilitate the activation of factor X. The intrinsic pathway refers to reactions that use only factors present in plasma to produce thrombin. A protease-mediated activation cascade ultimately produces factor IXa, which, together with factor Villa, cleaves factor X to Xa. Similar proteolysis is caused by FVIIa and its cofactor TF in the extrinsic pathway of blood coagulation. TF is a membrane-bound protein and is not normally found in the circulation in its active form (i.e., it is not present in plasma). However, upon vascular injury, TF can form a complex with FVIIa, thereby catalyzing the activation of factor X or factor IX in the presence of Ca ²⁺ and phospholipids. While the significance of the ratio of the two coagulation pathways in hemostasis is unclear, factor VII and TF have been found to play a pivotal role in the initiation of blood coagulation.

Selective blockade of the coagulation cascade is often required in patients. Anticoagulants, such as heparin, coumarin, coumarin derivatives, indanedione derivatives, or other agents, may be used, for example, in renal dialysis or in the treatment of deep vein thrombosis, disseminated intravascular coagulation (DIC), and other host diseases. For example, heparin therapy or extracorporeal treatment with ciprate ions may be used in dialysis to prevent blood clotting during treatment. Heparin may also be used to prevent deep vein thrombosis in patients undergoing surgery.

However, treatment with heparin and other anticoagulants can lead to unwanted side effects. In general, the anticoagulant available acts on the whole body, not on the

only at the surgical site. For example, heparin can cause severe bleeding. Furthermore, its half-life is approximately 80 minutes, so heparin is rapidly cleared from the blood, which in turn necessitates its frequent administration. Since heparin acts as a cofactor for antithrombin III (ATIII), and ATIII is rapidly depleted in the treatment of DIC, it is difficult to maintain an adequate heparin dose, requiring continuous monitoring of ATIII and heparin levels. Heparin is also ineffective when ATIII depletion is extreme. Furthermore, repeated use of heparin can also increase platelet aggregation while decreasing platelet count, which can lead to the development of heparin-induced thrombocytopenia. Indanedione derivatives are also associated with toxic side effects. In addition to the anticoagulants described above, several naturally occurring proteins have been found to have anticoagulant activity. ATIII has also been proposed as a therapeutic anticoagulant.

The use of inactivated factor VIIa in the inhibition of blood coagulation has been disclosed in the literature [Patent Application No. WO 92/15686].

Antibodies are specific immunoglobulin (Ig) polypeptides produced in the immune system of vertebrates in response to foreign proteins, glycoproteins, cells, or other antigenic substances. The sequence of steps in the body's avoidance of invasion by foreign cells or in the elimination of foreign substances is partially known. An important part of this process is the production of antibodies that can specifically bind to the given foreign substance. The binding specificity of such polypeptides for a given antigen is very sophisticated, and the system of specificities developed in a given vertebrate is complex and >

is remarkable in its variability. Millions of antigens are capable of eliciting an antibody response, each antibody being directed almost exclusively to the specific antigen that triggered its formation.

Currently, two main sources of antibodies are used in vertebrates, one is antibodies produced in situ by mammalian B lymphocytes, while the other is antibodies produced by B-cell hybrid cultures. In situ antibodies are derived from plasma cells that are formed as a result of the differentiation of immature B lymphocytes under the stimulation of specific antigens. In undifferentiated B cells, the DNAs encoding different parts of the immunoglobulin chains occur in different parts of the genomic DNA. Before expression, the sequences are assembled in a given order. The resulting ordered gene is able to be expressed as the corresponding antibody in mature B lymphocytes. However, even if the mammal is exposed to only one antigen, we do not obtain a uniform antibody population. The in situ immune response is a mosaic response to any given antigen, since different determinants are located on the antigen. Each subclass of homologous antibodies is formed by a different population of B cells, and accordingly, the in situ formation of the antibody is polyclonal.

Limited, but not innate, heterogeneity can be overcome in many cases by hybridoma technology, by producing monoclonal antibodies using cell cultures of B cell hybridomas.

In this process, relatively short-lived or dying mammalian spleen cells or lymphocytes injected with antigen are fused with immortal tumor cell lines, thus obtaining hybrid cells or hybridomas, which are both immortal and capable of producing the antibody genetically encoded by the B cell. The hybrids thus formed can be transformed into genetic lines by selection, dilution, and repeated growth, each of which produces a specific antibody. Therefore, their antibody production is homogeneous with respect to the desired antigen. These antibodies, because they are produced by pure genetic progeny, are called monoclonal antibodies.

The monospecificity of monoclonal antibodies has had a great impact on the development of immunology, which has extended to other branches of biology, as well as pharmacology, biochemistry and other fields. Such monoclonal antibodies have found their place not only as diagnostic reagents, but also have great therapeutic significance [Ritz and Schlossman: Blood, 59, 1-11 (1982)].

Monoclonal antibodies produced by hybridoma cells, although theoretically effective as discussed above and advantageous over polyclonal antibodies due to their specificity, have an important disadvantage. In many applications, the use of monoclonal antibodies that are not produced in humans is severely limited in humans. Repeated injections of a foreign antibody, such as a murine antibody, can lead to harmful hypersensitivity reactions. Such a non-human monoclonal antibody, when injected into a human, will elicit an anti-non-human antibody response.

The therapeutic use of Mabs against TF is known [U.S. Patent Nos. 6,001,978 and 5,223,427].

The literature [Patent Application No. WO 99/51743] describes a human/mouse chimeric monoclonal antibody raised against human TF.

CDR-grafted antibodies against human TF have been described in the literature [Patent Application EP 833911].

A humanized antibody against TF has been published in the literature [Presta et al.: Thrombosis and Haemostasis, 85(3), 379-389(2001)].

There is still a need for improved compositions that have anticoagulant activity, that can be administered at relatively low doses, and that do not cause the undesirable side effects that are characteristic of conventional anticoagulant compositions. The invention satisfies this need by providing anticoagulants that do not have the side effects of conventional antibodies containing non-human sequences, and that act specifically at the site of injury, and that exhibit other associated advantages. Furthermore, the invention provides compounds that inhibit the cellular functions of TF, which may occur in conditions such as sepsis, inflammation, atherosclerosis, restenosis, or cancer.

The invention relates to non-immunogenic high affinity human antibodies raised against TF that inhibit the binding of coagulation factors VII/Vila, and the disclosed methods provide for the selection of therapeutically effective human antibodies against human TF.

The first aspect of the invention relates to an isolated human antibody that immunoreacts with an epitope on human TF.

As used herein, the term human tissue factor or human TF refers to the full-length polypeptide receptor comprising amino acids 1-263 of the native human tissue factor sequence.

As used herein, the term antibody is intended to refer to immunoglobulin molecules and fragments thereof that are capable of specifically binding to antigen (e.g., human TF). Full-length antibodies consist of four polypeptide chains, two heavy chains (H) and two light chains (L), which are linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions are further divided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed with more conserved regions, otherwise called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, in the amino- and C-terminal directions, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Thus, the definition of an antibody also includes one or more fragments of the antibody that are capable of specifically binding to an antigen (e.g., human TF). It has been shown that the antigen-binding function of an antibody can also be performed as a fragment of the full-length antibody. Examples of binding fragments, as used in the term antibody, are

are: (i) the Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH I domains; (ii) F(ab)2 and F(ab')2 fragments, bivalent fragments consisting of two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fd fragment, consisting of VH and CH1 domains; (iv) Fv fragment, consisting of the VL and VH domains of one arm of the antibody; (v) dAb fragment [Ward et al.: Nature, 341, 544546(1989)], which contains a VH domain; and (vi) an isolated complementarity-determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by recombinant methods with a synthetic linker that allows the formation of a single chain in which the VL and VH regions form a pair of monovalent molecules (referred to as single-chain Fv (scFv)) [Bird et al.: Science, 242, 423-426 (1988): and Huston et al.: Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988)]. Such single-chain antibodies are also included within the scope of the invention as an antibody. Other forms of single-chain antibodies, such as dibodies, are also included. Di-bodies are bivalent, dual-specific antibodies in which the VH and VL domains are expressed from a single polypeptide chain, but are too short to connect the two domains within the same chain using a linker, so that the domains are forced to pair with a domain of the complementary chain, thus forming two antigen-binding sites [Holliger et al.: Proc. Natl. Acad. Sci. USA, 90, 6444-6448(1993); Poljak et al.: Structure, 2, 1121-1123(1994)]. It is understood that human TF may have one or more antigenic determinants, which include (1) a peptide antigenic determinant consisting of a single peptide chain within human TF, (2) a conformational

antigenic determinants consisting of more than one spatially continuous peptide chain, the corresponding amino acid sequences of which are not located in a linked manner on the human polypeptide sequence; and (3) post-translational antigenic determinants, which contain molecular structures covalently linked in whole or in part to human TF, such as carbohydrates or the like.

As used herein, the terms human antibody, human antibodies, human TF antibody, and human TF antibodies are intended to encompass antibodies that contain the variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the invention may also contain amino acids that are not found in human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), such as in the CDRs, particularly CDR3. However, as used herein, the term human antibody does not include antibodies whose CDR sequences are derived from the germline of another mammalian cell, such as murine, that are transferred to a human framework sequence, such as so-called humanized antibodies or human/mouse chimeric antibodies.

As used herein, the term isolated human antibody is intended to refer to human antibodies that are substantially free of antibodies of different antigen specificity (e.g., an isolated antibody that specifically binds to human TF is substantially free of

(i.e., antibodies that bind to antigens other than TF). However, an isolated antibody that specifically binds to human TF may cross-react with other antigens, such as TF molecules from other species (discussed below). Furthermore, the isolated antibody may be substantially free of other cellular materials and/or compounds.

As used herein, the term epitope refers to any antigenic determinant of an antigen that binds to an antibody. Epitope determinants typically include chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have a characteristic three-dimensional structure and can be characterized by specific charges.

As used herein, the term immunologically responsive refers to the binding of any antibody to an epitope where the dissociation constant Kd is less than ΙΟ ⁴ M. As used herein, the term immunologically responsive is used interchangeably with the term specifically binding.

As used herein, the term inhibit refers to a reduction in binding relative to a reference value. For example, an antibody that inhibits binding of human coagulation factor VIIa is one that reduces the binding ability of human coagulation factor VIIa to TF, compared to the binding ability of human coagulation factor VIIa to human TF in the absence of the antibody.

As used herein, the term affinity describes the strength of antibody-epitope binding. Antibody affinity is expressed as a dissociation constant (Kd), which is defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the unbound antibody, and [Ag] is the molar concentration of the unbound antigen. The affinity constant K _is equal to 1/Kd. The preferred methods for determining the specificity and affinity of Mabs are based on competitive inhibition [Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), eds. Colligan et al., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, NY, (1992, 1993); and Muller, Meth. Enzymol., 92, 589-601 (1983), which is hereby incorporated by reference in its entirety.

A second aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a human antibody that immunologically reacts with an epitope on human TF.

As used herein, the term therapeutically effective amount refers to an effective dose determined by the physician, who may titrate the dosage to achieve the desired response. Factors influencing dosages include: potency, bioavailability, desired pharmacokinetic/pharmacodynamic profiles, circumstances of treatment (e.g., accident, inflammation, septic shock), patient-related factors (e.g., weight, health status, age, etc.), concomitant medications, timing of administration, or other factors known to the skilled practitioner. The dosage of human antibody directed against TF administered to a patient may vary depending on the type and severity of the condition being treated, but will generally range from 0.1 to 5.0 mg/kg body weight.

··· ··· ·· : ···. ·*: .*·. :.

······ ·· ··

As used herein, the term subject may refer to any animal, particularly mammals such as humans, and where appropriate, may be used interchangeably with the term patient.

A third aspect of the invention relates to a composition comprising a human antibody that immunologically reacts with an epitope on human TF.

A further aspect of the invention relates to a method of treating human diseases associated with FVIIa/TF, which comprises administering to the human a therapeutically effective amount of a human antibody that immunologically reacts with an epitope on human TF.

The term treatment refers to the administration of a therapeutically effective amount of a compound of the invention with the intention of preventing any symptom or pathological condition, or of curing or eliminating such symptoms or pathological conditions once they have developed. The term treatment thus also includes preventive treatment.

As used herein, the term FVIIa/TF-related diseases refers to diseases or conditions in which TF and FVIIa play a role. These include thrombotic or coagulopathic diseases or conditions, including inflammatory responses, and chronic thromboembolic diseases and conditions associated with fibrin formation, such as vascular diseases such as deep vein thrombosis, arterial thrombosis, post-operative thromboses, coronary artery bypass grafts (CABG), percutaneous transdermal coronary angioplasty (PTCA), stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, atherosclerosis, and restenosis following angioplasty, acute and chronic indications such as inflammation, septic shock, septicemia, hypotension, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulopathy (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or prophylactic treatment of mammals for to reduce the risk of thrombosis in blood vessels prone to atherosclerosis, and other diseases or disorders. FVIIa/TF-related diseases do not only refer to in vivo coagulopathic diseases, as mentioned above, but also to ex vivo FVIIa/TF processes, such as blood clotting, which may result from extracorporeal circulation of blood, including removal of blood from the patient in-line, or in processes such as dialysis, blood filtration, or intraoperative blood bypass.

Factor VIIa, or FVIIa, is a term used to describe two chain activated coagulation factor VII that are specifically cleaved at the Arg152Ilel53 peptide bond. FVIIa can be purified from blood or produced recombinantly. It is understood that the practice of the methods described herein is independent of the purification method or origin of factor VIIa, and therefore the invention contemplates any method of producing factor VII that is suitable for the use herein. Human factor VII is preferred.

The term FVII refers to single-chain coagulation factor VII.

A further subject matter of the invention relates to methods for producing human antibodies, which method comprises: a) producing human antibodies against human TF,

b) testing the antibodies in a TF-induced blood clot formation assay and selecting the human antibody that inhibits blood clot formation in this assay, where the IC50 value is less than 1 nM, such as less than 500 pM, preferably less than 200 pM, preferably less than 100 pM, preferably less than 50 pM, preferably less than 10 pM, more preferably less than 5 pM; or testing the test antibodies in a FXa formation assay and selecting the human antibody that inhibits FXa formation with an IC50 value of less than 100 nM (where the FVIIa concentration in the assay is 0.1 nM), such as less than 10 nM, preferably less than 5 nM, preferably less than 1 nM, more preferably less than 0.1 nM; or testing the antibodies in a FVIIa/TF amidolytic system and selecting the human antibody that inhibits TF induced factor VIIa amidolytic activity at an IC50 of less than 100 nM (where the FVIIa concentration in the assay is 10 nM), such as less than 40 nM, preferably less than 20 nM, more preferably less than 10 30 nM; or testing the test antibodies in a FVIIa competition system and selecting the human antibody that competes with FVIIa binding; or testing the test antibody in a TF ELISA system and selecting the human antibody that binds human TF.

It should be understood that the specific IC50 values for the TF induced clot assay are obtained in the presence of normal human plasma.

A further aspect of the invention relates to a method for producing a human antibody, which method comprises the following:

a) production of human antibodies against human TF,

b) testing the antibodies in a TF-induced blood clot formation assay and selecting the human antibody that inhibits blood clot formation in this assay when the IC50 value is less than the FFR-rFVIIa IC50 value + 1 nM, such as less than the FFR-rFVIIa IC50 value + 500 pM, preferably less than the FFR-rFVIIa IC50 value + 200 pM, preferably less than the FFR-rFVIIa IC50 value + 100 pM, preferably less than the FFR-rFVIIa IC50 value + 50 pM, more preferably less than the FFR-rFVIIa IC50 value + 10 pM, preferably less than the FFR-rFVIIa IC50 value + 5 pM, preferably less than the FFR-rFVIIa IC50 value; or testing the test antibodies in an FXa training assay and selecting the human antibody with an IC50 value less than the FFR-rFVIIa IC50 value + 100 nM (using 0.1 nM FVIIa in the assay), such as less than the FFR-rFVIIa IC50 value + 10 nM, preferably less than the FFR-rFVIIa IC50 value + 5 nM, preferably less than the FFR-rFVIIa IC50 value + 1 nM, preferably less than the FFR-rFVIIa IC50 value + 0.1 nM, preferably less than the FFR-rFVIIa IC50 value; or testing the antibodies in a FVIIa/TF amidolytic system, and selecting the human antibody that inhibits TF-induced factor VIIa amidolytic activity at an IC50 of less than the FFR-rFVIIa IC50 +100 nM (where the FVIIa concentration in the assay is 10 nM), such as less than the FFR-rFVIIa IC50 + 40 nM, preferably less than the FFR-rFVIIa IC50 + 20 nM, more preferably less than the FFR-rFVIIa IC50 + 10 nM, preferably less than the FFR-rFVIIa IC50; or • ···· ·« ·· · ·*· · · · ·* ::·<:· ^: ·τ testing the test antibodies in a FVIIa competition system, and selecting the human antibody that competes with FVIIa binding; or testing the test antibody in a TF ELISA system and selecting the human antibody that binds human TF.

It should be understood that the specific IC50 values for the TF induced clot assay are obtained in the presence of normal human plasma.

As used herein, the term TF induced clotting assay is intended to refer to any assay in which the clotting time is measured in a sample system comprising blood plasma and TF. An example of a TF induced clotting assay is described in Assay 7 of Example 1.

As used herein, the term FXa formation assay is intended to refer to any assay in which FXa activation is measured in a sample containing TF, factor VIIa, FX, calcium, and phospholipids. An example of an FXa formation assay is described in Assay 5 of Example 1.

As used herein, the term FVIIa/TF amidolytic assay is intended to refer to any assay in which the amidolytic activity of FVIIa, i.e., the cleavage of small peptide substrates, is measured in the presence of TF. An example of a FVIIa/TF amidolytic assay is described in Assay 4 of Example 1.

As used herein, the term TF ELISA assay is intended to refer to any ELISA assay that contains antibodies directed against TF. TF • · · · · 9 · 9

J ”*· · ·**· ··<·

Examples of ELISA assays include direct and indirect TF ELISA assays, as described in assays 1 and 2 of Example 1.

As used herein, the term direct TF ELISA assay is intended to refer to any ELISA assay in which a captured TF is present. An example of a direct TF ELISA assay is described in Assay 1 of Example 1.

As used herein, the term indirect TF ELISA assay is intended to refer to any ELISA assay in which TF is in solution. An example of an indirect TF ELISA assay is described in Assay 2 of Example 1.

A further aspect of the invention relates to human antibodies that immunologically react with an epitope on human TF and inhibit the binding of human coagulation factor VIIa to human TF, which can be obtained by a process comprising:

a) producing human antibodies effective against human TF, b) testing the antibodies in a TF-induced blood clot formation assay and selecting the human antibody that inhibits blood clot formation in this assay, wherein the IC50 value is less than 1 nM, such as less than 500 pM, preferably less than 200 pM, preferably less than 100 pM, preferably less than 50 pM, preferably less than 10 pM, more preferably less than 5 pM; or testing the test antibodies in an FXa formation assay and selecting the human antibody that inhibits FXa formation with an IC50 value of less than 100 nM (where the FVIIa concentration in the assay is 0.1 nM), such as less than 10 nM, preferably less than 5 nM, preferably less than 1 nM, more preferably less than 0.1 nM; or testing the antibodies in a FVIIa/TF amidolytic system and selecting the human antibody that inhibits TF induced factor VIIa amidolytic activity at an IC50 of less than 100 nM (where the concentration of FVIIa in the assay is 10 nM), such as less than 40 nM, preferably less than 20 nM, more preferably less than 10 nM; or testing the test antibodies in a FVIIa competition system and selecting the human antibody that competes with FVIIa binding; or testing the test antibody in a TF ELISA system and selecting the human antibody that binds human TF.

It should be understood that the specific IC50 values for the TF induced clot assay are obtained in the presence of normal human plasma.

A further aspect of the invention relates to a human antibody that immunologically reacts with an epitope on human TF and inhibits the binding of human coagulation factor VIIa to human TF, obtainable by a process comprising:

a) production of human antibodies effective against human TF,

b) testing the antibodies in a TF-induced blood clot formation assay and selecting the human antibody that inhibits blood clot formation in this assay when the IC50 value is less than the FFR-rFVIIa IC50 value + 1 nM, such as less than the FFR-rFVIIa IC50 value + 500 pM, preferably less than the FFR-rFVIIa IC50 value + 200 pM, preferably less than the FFR-rFVIIa IC50 value + 100 pM, preferably less than »·

FFR-rFVIIa IC50 value + 50 pM, more preferably less than FFR-rFVIIa IC50 value + 10 pM, preferably less than FFRrFVIIa IC50 value + 5 pM, preferably less than FFR-rFVIIa IC50 value; or testing the test antibodies in an FXa formation assay and selecting the human antibody that inhibits FXa formation at an IC50 value less than the FFR-rFVIIa IC50 value + 100 nM (using 0.1 nM FVIIa in the assay), such as less than the FFR-rFVIIa IC50 value + 10 nM, preferably less than the FFR-rFVIIa IC50 value + 5 nM, preferably less than the FFR-rFVIIa IC50 value + 1 nM, preferably less than the FFR-rFVIIa IC50 value + 0.1 nM, preferably less than the FFR-rFVIIa IC50 value; or testing the antibodies in a FVIIa/TF amidolytic system and selecting the human antibody that inhibits TF induced factor VIIa amidolytic activity with an IC50 value less than the FFR-rFVIIa IC50 +100 nM (where the FVIIa concentration in the assay is 10 nM), such as less than the FFR-rFVIIa IC50 +40 nM, preferably less than the FFR-rFVIIa IC50 +20 nM, more preferably less than the FFR-rFVIIa IC50 +10 nM, preferably less than the FFR-rFVIIa IC50; or testing the test antibodies in a FVIIa competition system and selecting the human antibody that competes with FVIIa binding; or testing the test antibody in a TF ELISA system and selecting the human antibody that binds human TF.

It should be understood that the specific IC50 values for the TF induced clot assay are obtained in the presence of normal human plasma.

.·. !·’· ·ζ\ 1

7· ·**' ··»·

In one embodiment of the method of the invention, a human antibody against human TF is produced, which comprises immunizing a mammal with human TF and isolating the antibodies produced by the immunized animal. In a preferred embodiment, the mammal is a mouse. It is to be understood that the immunized mammal or mouse is capable of producing human antibodies.

A further aspect of the invention relates to a method for producing a human antibody, comprising the following steps: a) immunizing a mouse with human TF, b) isolating antibody-producing cells from the immunized mouse and producing immortalized cells secreting human antibodies, c) isolating the antibody produced from the culture medium of the immortalized cells, d) testing the antibody by indirect TF ELISA in a TF solution and selecting human antibodies binding to human TF from the solution, e) testing the antibodies in a FVIIa competition assay and selecting a human antibody capable of competing with FVIIa binding, f) testing the antibody in a FVIIa/TF amidolytic system and selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity, having an IC50 value of less than 100 nM (where the FVIIa concentration in the assay is 10 nM), such as less than 40 nM, preferably less than 20 nM, more preferably less than 10 nM;

g) testing the antibody in an FXa formation assay and selecting the human antibody that inhibits FXa formation with an IC50 of less than 100 nM (wherein the assay

J. ,’P ·'.*

FVIIa concentration (0.1 nM), such as less than 10 nM, preferably less than 5 nM, preferably less than 1 nM, more preferably less than 0.1 nM;

h) testing the antibodies in TF induced blood clotting and selecting those human antibodies which inhibit blood clotting in this assay, where the IC50 value is less than 1 nM, such as less than 500 pM, preferably less than 200 pM, preferably less than 100 pM, preferably less than 50 pM, preferably less than 10 pM, more preferably less than 5 pM;

i) selecting and culturing the immortalized cells in a suitable culture medium, thereby ensuring the production of the antibodies selected in points d-h, j) isolating the antibodies released from the selected immortalized cells from the culture medium.

It should be understood that the specific IC50 values for the TF induced clot assay are obtained in the presence of normal human plasma.

A further aspect of the invention relates to a method for producing human antibodies, which method comprises the following steps: a) immunizing a mouse with human TF, b) isolating antibody-producing cells from the immunized mouse and producing immortal cells secreting human antibodies, c) isolating the produced antibody from the culture medium of the immortalized cells, d) testing the antibody by indirect TF ELISA, which is performed in a solution of TF, and selecting human antibodies binding to human TF from the solution,

e) testing antibodies in the FVIIa competition assay and selecting a human antibody capable of competing with FVIIa binding,

f) testing the antibody in a FVIIa/TF amidolytic system and selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity, having an IC50 value less than the FFRrFVIIa IC50 + 100 nM (where the FVIIa concentration in the assay is 10 nM), such as less than the FFR-rFVIIa IC50 + 40 nM, preferably less than the FFR-rFVIIa IC50 + 20 nM, more preferably less than the FFR-rFVIIa IC50 + 10 nM, preferably less than the FFR-rFVIIa IC50;

g) testing the antibodies in an FXa formation system and selecting a human antibody that inhibits FXa formation with an IC50 value less than the FFR-rFVIIa IC50 value +100 nM (using 0.1 nM FVIIa in the assay), such as less than the FFR-rFVIIa IC50 value +10 nM, preferably less than the FFR-rFVIIa IC50 value +5 nM, preferably less than the FFRrFVIIa IC50 value +1 nM, preferably less than the FFR-rFVIIa IC50 value +0.1 nM, preferably less than the FFR-rFVIIa IC50 value;

h) testing the antibodies in a TF-induced blood clot formation system, and selecting those human antibodies that inhibit blood clot formation in this assay, where the IC50 value is less than the FFR-rFVIIa IC50 value + 1 nM, such as less than the FFR-rFVIIa IC50 value + 500 pM, preferably less than the FFR-rFVIIa IC50 value + 200 pM, preferably less than the FFR-rFVIIa IC50 value + 100 pM, preferably less than the FFR-rFVIIa IC50 value + 50 pM, more preferably less than the FFR-rFVIIa IC50 value + 10 pM, preferably less than the FFR-rFVIIa IC50 value + 5 pM, preferably less than the FFR-rFVIIa IC50 value;

i) selection and cultivation of immortalized cells in appropriate culture medium, thereby ensuring the production of antibodies selected in points d-h.,

j) isolating antibodies released from the selected immortalized cells from the culture medium.

It should be understood that the specific IC50 values for the TF induced clot assay are obtained in the presence of normal human plasma.

As used herein, the term antibody-producing cell refers to any cell capable of producing antibody. Including hybridomas, transfected cell lines, and relatively short-lived or dying spleen cells or lymphocytes from mammals that have been injected with antigen.

A further aspect of the invention relates to human antibodies that immunologically react with epitopes on human TF and inhibit the binding of human coagulation factor VIIa to human TF, which can be obtained by a process comprising:

a) immunizing the mouse with human TF, b) isolating antibody-producing cells from the immunized mouse and producing immortal cells secreting human antibodies, c) isolating the produced antibody from the culture medium of the immortalized cells,

d) testing the antibody with indirect TF ELISA, which is performed in the TF solution, and selecting human antibodies binding to human TF from the solution,

e) testing antibodies in the FVIIa competition assay and selecting a human antibody capable of competing with FVIIa binding,

f) testing the antibody in a FVIIa/TF amidolytic system and selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity and has an IC50 value of less than 100 nM (where the FVIIa concentration in the assay is 10 nM), such as less than 40 nM, preferably less than 20 nM, more preferably less than 10 nM;

g) testing the antibody in an FXa formation assay and selecting the antibody that inhibits FXa formation at an IC50 of less than 100 nM (where the FVIIa concentration in the assay is 0.1 nM), such as less than 10 nM, preferably less than 5 nM, preferably less than 1 nM, more preferably less than 0.1 nM;

h) testing the antibodies in TF induced blood clotting and selecting those human antibodies which inhibit blood clotting in this assay, where the IC50 value is less than 1 nM, such as less than 500 pM, preferably less than 200 pM, preferably less than 100 pM, preferably less than 50 pM, preferably less than 10 pM, more preferably less than 5 pM;

i) selection and cultivation of immortalized cells in appropriate culture medium, thereby ensuring the production of antibodies selected in points d-h.,

j) isolating antibodies released from the selected immortalized cells from the culture medium.

It should be understood that the specific IC50 values for the TF induced clot assay are obtained in the presence of normal human plasma.

A further aspect of the invention relates to a human antibody that immunologically reacts with epitopes on human TF and inhibits the binding of human coagulation factor VIIa to human TF, obtainable by a process comprising:

a) immunization of mice with human TF,

b) isolation of antibody-producing cells from the immunized mouse and production of immortal cells secreting human antibodies, c) isolation of the produced antibody from the culture medium of the immortalized cells,

d) testing the antibody with indirect TF ELISA, which is performed in the TF solution, and selecting human antibodies binding to human TF from the solution,

e) testing antibodies in the FVIIa competition assay and selecting a human antibody capable of competing with FVIIa binding,

f) testing the antibody in a FVIIa/TF amidolytic system and selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity, having an IC50 value less than the FFRrFVIIa IC50 +100 nM (where the FVIIa concentration in the assay is 10 nM), such as less than the FFR-rFVIIa IC50 +40 nM, preferably less than the FFR-rFVIIa IC50 +20 nM, more preferably less than the FFR-rFVIIa IC50 +10 nM, preferably less than the FFR-rFVIIa IC50;

g) testing the antibodies in an FXa formation system and selecting a human antibody that inhibits FXa formation with an IC50 value less than the FFR-rFVIIa IC50 value + 100 nM (using 0.1 nM FVIIa in the assay), such as less than the FFR-rFVIIa IC50 value + 10 nM, preferably less than the FFR-rFVIIa IC50 value + 5 nM, preferably less than the FFR-rFVIIa IC50 value + 1 nM, preferably less than the FFR-rFVIIa IC50 value + 0.1 nM, preferably less than the FFR-rFVIIa IC50 value;

h) testing the antibodies in a TF-induced blood clot formation system, and selecting those human antibodies that inhibit blood clot formation in this assay, where the IC50 value is less than the FFR-rFVIIa IC50 value + 1 nM, such as less than the FFR-rFVIIa IC50 value + 500 pM, preferably less than the FFR-rFVIIa IC50 value + 200 pM, preferably less than the FFR-rFVIIa IC50 value + 100 pM, preferably less than the FFR-rFVIIa IC50 value + 50 pM, more preferably less than the FFR-rFVIIa IC50 value + 10 pM, preferably less than the FFR-rFVIIa IC50 value + 5 pM, preferably less than the FFR-rFVIIa IC50 value;

i) selection and cultivation of immortalized cells in appropriate culture medium, thereby ensuring the production of antibodies selected in points d-h.,

j) isolating antibodies released from the selected immortalized cells from the culture medium.

It should be understood that the specific IC50 values for the TF induced clot assay are obtained in the presence of normal human plasma.

In one embodiment of the invention, the immortal cell is a hybridoma.

A further aspect of the invention relates to cells producing human antibodies that immunologically react with epitopes on human TF and inhibit the binding of human coagulation factor VIIa to human TF.

In one embodiment, the cell is an isolated lymphoid cell.

In a further embodiment, the isolated cell is derived from a mouse.

In a further embodiment, the cell is a hybridoma.

In one embodiment, the hybridoma is obtained by fusion of the antibody-producing lymphoid cell and the immortal cell, thereby providing antibody-producing hybridoma cells.

In a further embodiment of the invention, the isolated human antibody inhibits the binding of human coagulation factor VIIa to human TF.

In a further embodiment, the isolated human antibody is monoclonal.

As used herein, the term monoclonal antibody refers to a homogeneous population of immunoglobulins, i.e., the individual molecules of the antibody population are identical except for natural mutations. Antibodies are normally synthesized by B lymphocytes derived from the bone marrow. Lymphocytes derived from the same clone produce immunoglobulins from a single amino acid sequence. Lymphocytes cannot be cultured directly for long periods of time to produce their specific antibodies in sufficient quantities.

However, others in the literature have shown [Kohler et al.: Nature, 256, 495(1975)] that fusion of somatic cells, specifically lymphocytes and myeloma cells, can result in hybridoma cells that grow in culture and produce a specific antibody called a monoclonal antibody. Myeloma cells are lymphocytic tumor cells that, depending on the cell line, often produce antibodies themselves, although they are known as non-producing strains.

In a further embodiment of the invention, the isolated human antibody is recombinant.

As used herein, the term recombinant antibody is intended to encompass all recombinantly produced, expressed, generated, or isolated antibodies, such as those produced in host cells transfected with a recombinant expression vector (described in more detail in Section II below), antibodies obtained from recombinant, combinatorially produced human antibody libraries (described in more detail in Section III below), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes [Taylor et al. Nucl. Acids Res., 20, 6287-6295 (1992)], or antibodies produced, expressed, generated, or isolated by any other means in which the human immunoglobulin gene is spliced to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or when the animal is transgenic for the human Ig sequences used) and thus represent the amino acid sequence of the VH and VL regions of the recombinant antibodies, while being derived from and related to the human germline VH and VL sequences, and therefore, of course, cannot be included in the human germline pool in vivo.

In a further embodiment of the invention, the isolated human antibody is a Fab fragment.

In a further embodiment of the invention, the isolated human antibody is an F(ab)2 fragment.

In a further embodiment of the invention, the isolated human antibody is an F(ab')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 binds to human TF in the range of ΙΟ ¹⁵ - ΙΟ ⁸ M. It is to be understood that the Kd value of the binding of the human antibody to human TF is determined in an assay in which the human antibody is immobilized (see Assay 6).

In a further embodiment of the invention, the isolated human antibody has a Kd value for human TF binding of 10 ¹⁵ -10 ¹⁰ M.

In a further embodiment of the invention, the isolated human antibody has a Kd value of less than ΙΟ- ⁸ M when binding to human TF. In a further embodiment of the invention, the Kd value of less than ΙΟ- ⁹ M when binding to human TF. In a further embodiment of the invention, the Kd value of less than 10 ¹⁰ M when binding to human TF. In a further embodiment of the invention, the Kd value of less than 10- ¹¹ M when binding to human TF. In a further embodiment of the invention, the Kd value of less than 10- ¹² M when binding to human TF. In a further embodiment of the invention, the Kd value of less than

than ΙΟ ¹³ Μ. In a further embodiment of the invention, the Kd value when the human antibody binds to human TF is less than ΙΟ ¹⁴ M. In a further embodiment of the invention, the Kd value when the human antibody binds to human TF is less than ΙΟ ¹⁵ M.

In a further embodiment of the method of the invention, the preparation of the human antibody further comprises the step of testing the antibody in a TF-induced blood clotting assay and selecting human antibodies that inhibit blood clotting in the blood clotting assay with an IC50 value of less than 1 nM. In a further embodiment, the IC50 value is less than 500 pM. In a further embodiment, the IC50 value is less than 200 pM. In a further embodiment, the IC50 value is less than 100 pM. In a further embodiment, the IC50 value is less than 50 pM. In a further embodiment, the IC50 value is less than 10 pM. In a further embodiment, the IC50 value is less than 5 pM.

In a further embodiment of the method of the invention, the preparation of the human antibody also comprises the step of testing the antibody in a TF-induced blood clotting assay and selecting those human antibodies which have an IC50 inhibition of blood clotting in the blood clotting assay that is less than the FFR-rFVIIa IC50 + 1 nM, such as less than the FFR-rFVIIa IC50 + 500 pM, preferably less than the FFR-rFVIIa IC50 + 200 pM, preferably less than the FFR-rFVIIa IC50 + 100 pM, preferably less than the FFR-rFVIIa IC50 + 50 pM, more preferably less than the FFR-rFVIIa IC50 + 10 pM, preferably less than the FFR-rFVIIa IC50 + 5 pM, preferably less than the FFR-rFVIIa IC50.

It should be understood that the specific IC50 values for the TF induced clot assay are obtained in the presence of normal human plasma.

In a further embodiment of the method of the invention, the preparation of the human antibody further comprises the step of testing the antibody in an FXa formation assay and selecting antibodies that inhibit FXa formation at an IC50 value of less than 100 nM (where the concentration of FVIIa in the assay is 0.1 nM). In a further embodiment, the IC50 value is less than 10 nM. In a further embodiment, the IC50 value is less than 5 nM. In a further embodiment, the IC50 value is less than 1 nM. In a further embodiment, the IC50 value is less than 0.1 nM.

In a further embodiment of the method of the invention, the preparation of the human antibody also comprises the step of testing the antibody in an FXa formation assay and selecting antibodies that inhibit FXa formation with an IC50 value less than the FFR-rFVIIa IC50 value + 100 nM (using 0.1 nM FVIIa in the assay), such as less than the FFR-rFVIIa IC50 value + 10 nM, preferably less than the FFR-rFVIIa IC50 value + 5 nM, preferably less than the FFR-rFVIIa IC50 value + 1 nM, preferably less than the FFR-rFVIIa IC50 value + 0.1 nM, preferably less than the FFR-rFVIIa IC50 value.

In a further embodiment of the method of the invention, the preparation of the human antibody further comprises the step of testing the antibody in a FVIIa/TF amidolytic assay and selecting those human antibodies which exhibit amidolytic activity with an IC ₅₀ value of less than 100 nM.

(where the concentration of FVIIa in the assay is 0.1 nM). In a further embodiment, the IC50 value is less than 40 nM. In a further embodiment, the IC50 value is less than 20 nM. In a further embodiment, the IC50 value is less than 10 nM. In a further embodiment, the IC50 value is less than 5 nM.

In a further embodiment of the method of the invention, the preparation of the human antibody further comprises the step of testing the antibody in a FVIIa/TF amidolytic assay and selecting antibodies that inhibit the amidolytic activity with an IC50 value less than the FFR-rFVIIa IC50 +100 mM (where the FVIIa concentration in the assay is 0.1 nM), such as less than the FFR-rFVIIa IC50 +40 nM, preferably less than the FFR-rFVIIa IC50 +20 nM, more preferably less than the FFR-rFVIIa IC50 +10 nM, preferably less than the FFR-rFVIIa IC50.

In a further embodiment of the method of the invention, the production of the human antibody also comprises the step of testing the antibody in a FVIIa competition assay and selecting human antibodies that compete with human FVIIa.

In a further embodiment of the method of the invention, the production of the human antibody also comprises the step of testing the antibody in a TF ELISA assay, and selecting those human antibodies, and selecting those antibodies that bind human TF.

In a further embodiment of the method of the invention, the production of the human antibody also comprises the step of testing the antibody in a TF ELISA assay using bound TF and selecting antibodies that interact with the bound human TF.

In a further embodiment of the method of the invention, the production of the human antibody also comprises the step of testing the antibody in a TF ELISA assay using TF in solution and selecting those human antibodies that bind to human TF in solution.

In a further embodiment of the method of the invention, the preparation of the human antibody further comprises the step of testing the antibody in an FXa production assay and selecting antibodies that inhibit FXa production on TF expressing cells at an IC50 value of less than 500 nM (where the concentration of FVIIa in the assay is 1 nM). In a further embodiment, the IC50 value is less than 100 nM. In a further embodiment, the IC50 value is less than 50 nM. In a further embodiment, the IC50 value is less than 10 nM. In a further embodiment, the IC50 value is less than 5 nM.

In a further embodiment of the method of the invention, the preparation of the human antibody also comprises the step of testing the antibody in an FXa formation assay and selecting antibodies that inhibit FXa formation on TF-expressing cells with an IC50 value less than the FFR-rFVIIa IC50 + 500 nM (using 1 nM FVIIa in the assay), such as less than the FFR-rFVIIa IC50 value +100 nM, preferably less than the FFR-rFVIIa IC50 value + 50 nM, preferably less than the FFR-rFVIIa IC50 value + 10 nM, preferably less than the FFRrFVIIa IC50 value + 5 nM, preferably less than the FFR-rFVIIa IC50 value.

The term TF-expressing cell may refer to any human TF-expressing cell.

In a further embodiment of the method of the present invention, the production of the human antibody also comprises testing the antibodies in a whole cell TF binding assay and selecting those antibodies that compete with FVIIa for binding to human TF expressed on the surface of whole cells.

In a further embodiment of the method of the invention, the preparation of the human antibody also comprises testing the antibodies in a biosensor assay and selecting those human antibodies that compete for FVIIa binding with a Kd value of less than 100 nM. In a further embodiment, the Kd value is less than 10 nM. In a further embodiment, the Kd value is less than 5 nM. In a further embodiment, the Kd value is less than 1 nM. In a further embodiment, the Kd value is less than 0.5 nM. In a further embodiment, the Kd for binding to human TF is less than 10 ¹⁰ M. In a further embodiment, the Kd for binding to human TF is less than 10 ¹¹ M. In a further embodiment, the Kd for binding to human TF is less than ΙΟ ¹² M. In a further embodiment, the Kd for binding to human TF is less than 10- ¹³ M. In a further embodiment, the Kd for binding to human TF is less than 10 ¹⁴ M. In a further embodiment, the Kd for binding to human TF is less than ΙΟ ¹⁵ M.

In a further embodiment of the method of the invention, the preparation of the human antibody also comprises testing the antibodies in a MAPK signaling assay and selecting those human antibodies that inhibit FVIIa-induced activation of MAPK signaling. In one embodiment, the human antibody inhibits FVIIa-induced MAPK signaling by 98%. In one embodiment, the human antibody inhibits FVIIa-induced MAPK signaling by 90%. In one embodiment, the human antibody inhibits FVIIa-induced MAPK signaling by 70%. In one embodiment, the human antibody inhibits FVIIa-induced MAPK signaling by 50%. In one embodiment, the human antibody inhibits FVIIa-induced MAPK signaling by 30%.

For our purposes, the term MAPK signaling refers to the sequence of intracellular events that mediate the activation of mitogen-activated protein kinase (MAPK) or its homologues in response to various extracellular stimuli. Three distinct groups of MAP kinases have been identified in mammalian cells: 1) extracellularly regulated kinase (Erk1/2 or p44/42), 2) c-Jun N-terminal kinase (JNK), and 3) p38 kinase. The Erk1/2 pathway involves the phosphorylation of Erk 1 (p44) and/or Erk 2 (p42). Activated MAP kinases, such as p44/42 MAPK, can translocate to the nucleus, where they phosphorylate and activate transcription factors, including Elk 1, as well as signal transducers and activators of transcription (Stat). In the cytoplasm or nucleus, Erk1/2 is also capable of phosphorylating p90RSK kinase, which may then enable p90RSK to activate multiple transcription factors.

For our purposes, the term protein kinase refers to an enzyme capable of phosphorylating the amino acids serine and/or threonine and/or tyrosine of peptides and/or proteins.

As used herein, the term FVIIa-induced activation of MAPK signaling refers to the binding of FVIIa to TF in mammalian cells and thus initiating MAPK signaling.

In a further embodiment of the method of the invention, the human antibody assay is combined with gene expression analysis (assay 15) and the selection of human antibodies that inhibit FVIIa-induced up-regulation of genes, independently of the substance selected from the list, which genes are: Fra-1, Id2 and Cyr11.

It should be understood that antibodies raised against TF that inhibit TF activity may bind to different epitopes on TF and may inhibit both FVIIa binding and FX or FXa binding to human TF. The present invention relates to the selection of antibodies that inhibit the binding of FVIIa to human TF and thereby inhibit FVIIa-induced intracellular signaling.

In a further embodiment of the method of the invention, the production of the human antibody also includes testing the antibody on human cancers (Test 13) and selecting those human antibodies that inhibit the growth or metastasis of human cancers.

In a further embodiment of the invention, the isolated human antibodies inhibit FVIIa-induced up-regulation of genes, independently selected from the list, which genes are: Fra-1, Id2 and Cyrol.

In a further embodiment of the invention, the isolated human antibody does not inhibit the binding of FX or FXa to human TF.

• ··· *· · · · · ·

J. ...· .·· ~· · :·

In a further embodiment of the invention, the isolated human antibody inhibits intracellular TF activity.

In a further embodiment of the method of the invention, the antibody is screened by epitope mapping to produce a human antibody and a human antibody is selected that binds to a preferred epitope of TF. In one embodiment, the preferred epitope comprises one or more of the amino acids Trp45, Lys46 and Tyr94. In one preferred embodiment, the epitope is Trp45. In one preferred embodiment, the epitope is Lys46. In one preferred embodiment, the epitope is Tyr94.

In one preferred embodiment, the isolated human antibody binds to an epitope located at the interaction of TF and FVIIa.

The amino acids of TF are responsible for the interaction between the FVIIa protease domain and TF, the structure of which was determined by X-ray analysis [Banner et al.: Nature, 380, 41-46(1996)], the following amino acids participate in its structure: Ser39, Gly43, Trp45, Ser47, Phe50, Arg74, Phe76, Tyr94, Pro92. The interaction area between the FVIIa protease domain and TF has a complex structure, as it contains numerous intermolecular hydrogen bonds, which form the fine structure of the interaction between TF and FVIIa, so the FVIIa binding process is very specific.

The invention also relates to high affinity human monoclonal TF antibodies. The surface of TF, which interacts with the protease domain of FVIIa, has a specific topology that is prone to interfere with the formation of protein-protein interactions in which other types of protein-protein interactions form a complex between the antibody and the protein ligand, and thus monoclonal antibodies raised against such epitopes on human TF are high affinity Mabs.

One aspect of the invention relates to high affinity human monoclonal antibodies that immunologically interact with the surface of the FVIIa protease domain.

The human TF antibodies of the invention act as antagonists in TF-mediated induction of blood coagulation, thereby inhibiting the binding of FVIIa to TF, thereby blocking thrombin generation and subsequent fibrin deposition. The human TF antibodies are particularly useful when administered to humans for the treatment of various conditions, including intravascular coagulation, such that the human TF antibodies are useful in inhibiting TF activity, resulting in, for example, inhibition of blood coagulation, thrombosis or platelet deposition. Furthermore, the human TF antibodies of the invention, which inhibit the cellular functions of TF, through the signaling function of TF, may be useful in the following conditions: sepsis, inflammation, atherosclerosis, restenosis or neoplastic diseases.

Human TF antibodies can be used in a variety of diseases. Including: thrombotic or coagulopathic diseases or conditions, such as inflammatory response, and chronic thromboembolic diseases or conditions, which are associated with fibrin formation, such as vascular diseases, such as deep vein thrombosis, arterial thrombosis, post-operative thromboses, coronary artery bypass grafts (CABG), percutaneous transdermal coronary angioplasty (PTCA), stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, atherosclerosis, and restenosis following angioplasty, acute and chronic indications, such as inflammation, septic shock, septicemia, hypotension, adult respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), disseminated intravascular coagulopathy (DIC), pulmonary embolism, pathological platelet deposition, myocardial infarction, or the preventive treatment of mammals to reduce the risk of thrombosis in atherosclerotic vessels, veno-occlusive disease after peripheral blood progenitor cell transplantation (PBPC), hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP) and other diseases and conditions. Human TF antibodies can also be used to prevent thromboembolic complications in patients in identified high-risk groups, such as those who have undergone surgery or who have developed congestive heart failure. Human TF antibodies are particularly useful in the treatment of intimal hyperplasia or restenosis due to acute vascular injury. Acute vascular injury is of a rapid course (lasting from a few hours to months) in contrast to chronic vascular injury (e.g. atherosclerosis), which can last a lifetime. Acute vascular injuries often occur as a result of surgical procedures such as vascular remodeling using angioplasty, endarterectomy, atherectomy, vascular grafts, or the like. Hyperplasia can also occur as a result of these procedures, such as graft placement or organ transplantation. Because human TF antibodies are more selective than heparin, binding only to TF at the site of injury, and because human TF antibodies do not destroy or inhibit other coagulation proteins, they are more effective and have a lower risk of bleeding _{complications} than heparin when used for the prevention of deep vein thrombosis.

As demonstrated in the following examples, the human TF antibodies of the invention are capable of selectively binding to cell surface TF and inhibiting its activity by preventing FVIIa from binding to TF. By blocking thrombin generation at the site of injury, the human TF antibodies inhibit platelet aggregation by binding to TF and thus the subsequent fibrin deposition.

Since human TF antibodies are able to block thrombin generation and limit platelet deposition at the site of acute vascular injury, human TF antibodies that maintain FVIIa binding by inhibiting it can be used to inhibit vascular restenosis.

Compositions comprising human TF antibodies are particularly well suited for the treatment of patients when formulated as pharmaceutical compositions for administration to individuals suffering from various diseases for the treatment of coagulation-related conditions. Such human TF antibodies, which are capable of binding TF and inhibiting the binding of FVIIa and TF, have a longer plasma half-life and thus are able to exert their anticoagulant activity for a longer period of time compared to other anticoagulants. The medical indications of the compositions of the invention are the same as those of commonly used anticoagulants, such as deep vein thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIC), fibrin deposition in the lungs and kidneys with septicemia, antiphospholipid syndrome (APS), atherosclerosis and myocardial infarction. The compositions are useful for inhibiting vascular restenosis, which conditions occur after mechanical injury, such as surgery, microsurgery, balloon angioplasty, endarterectomy, reductive atherectomy, stent placement, laser therapy or rotablation, or may occur secondary to vascular grafts, stents, bypass grafts or organ transplantation. Thus, the compositions are useful for inhibiting platelet deposition and the diseases associated therewith. Thus, methods for inhibiting blood coagulation, vascular restenosis or platelet deposition, for example, comprise administering to the patient a composition comprising human TF antibodies in an amount effective to inhibit blood coagulation, vascular restenosis or platelet deposition. Methods find their place in the treatment of acute coronary artery disease (e.g., acute myocardial infarction) that involve the administration of human TF antibodies in combination with tissue plasminogen activator or streptokinase, which are capable of accelerating tPA-induced thrombolysis. Human TF antibodies have previously been administered before or shortly after thrombolytic agents, such as tissue plasminogen activator.

The human monoclonal antibodies of the invention, which are raised against human TF, can be produced by immunizing transgenic mice (available from Medarex) that carry components of the human immune system rather than the mouse system, together with human TF. Spleen cells obtained from such transgenic mice can be used to form hybridomas.

J. ·-* ‘Μ* are used for the production of human monoclonal antibodies that are secreted by the human monoclonal antibodies described in the literature [Wood et al.: WO 91/00906, Kucherlapati et al.: WO 91/10741; Lonberg et al.: WO 92/03918; Kay et al.: 92/03917; Lonberg et al.: Nature, 368, 856-859(1994); Green et al.: Nature Genet., 7, 13-21(1994); Morrison et al.: Proceedings of the National Academy of Sciences, USA 81, 6851-6855(1994); Bruggeman et al.: Year Immunol., 7, 33-40(1993); Tuaillon et al.: Proceedings of the National Academy of Sciences, USA, 90, 3720-3724(1993); Bruggeman et al.: Eur. J. Immunol., 21, 1323-1326(1991)].

Human monoclonal antibodies against human TF can also be produced by phage display. Human antibody libraries can be generated from immunized individuals and displayed on the surface of filamentous phages. High-affinity human single-chain Fv (ScFv) and Fab antibody fragments have been isolated from such libraries several times using the panning technique, in which the antigen of interest is immobilized on a solid surface, i.e. microtiter plates or beads [Barbas and Burton: Trends. Biotechnoi., 14, 230-234 (1996); Aujame et al.: Hum. Antibodies, 8, 155-168 (1997)]. Large-scale naive phage display has also made it possible to isolate human antibodies without direct immunization [DeHaard et al.: J. Biol. Chem., 18218-18230 (1999)].

The invention is illustrated in more detail through the attached figures.

Figure 1 is a schematic diagram of an exemplary screening procedure for selecting human monoclonal antibodies that exhibit high affinity for TF.

Figure 2 is a detailed, schematic representation of screening tests 1-3 described in Example 1.

Figure 3 is a detailed, schematic representation of screening tests 4-7 described in Example 1.

Figure 4 is a detailed, schematic representation of screening tests 8-10 described in Example 1.

Figure 5 is an example of screening with assay 4, showing the inhibition of sTF/FVIIa amidolytic activity by FFR-rFVIIa (closed circles) and the human anti TF monoclonal antibody HuTF-31 F2 (open circles).

Figure 6 is an example of screening with assay 5, showing the inhibition of FXa formation with FFR-rFVIIa (closed circles) and the human monoclonal antibody huTF31 F2 (open circles).

Figure 7 is an example of screening with Assay 7, showing inhibition of human TF-induced clot formation with FFR-rFVIIa (closed circles) and the human monoclonal antibody huTF-31 F2 (open circles).

Figure 8 is an example of screening with assay 10. Only anti-TF monoclonal antibodies prevent FVIIa binding in inhibitory TF/FVIIa-mediated signaling.

Figure 9 shows that anti TF Mab inhibits FVIIa-induced p44/42 MAPK phosphorylation (assay 10). BHK cells were transfected with TF, after which the cells were serum-starved for 2 h to reach a quiescent state. The HuMab 30F5 (500 nM) and HuMab 31F2 (500 nM) antibodies were added to the cells 15 min before FVIIa treatment (15 nM). The cells were lysed and the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes for electroblotting. Western blotting was performed with phospho-specific polyclonal antibodies to p44/42 MAPK. The secondary antibody was anti-rabbit IgG conjugated to horseradish peroxidase. Chemiluminescence detection was performed with a cooled CCD camera. The bands were quantified in the digitized image and the band obtained with FVIIa was considered as 100%. When the cells were preincubated with HuMab 30F5 (500 nM), a 50% decrease in the phosphorylated band was observed, and when the cells were preincubated with HuMab 31F2 (500 nM), a 25% decrease was observed. In conclusion, these experiments show that human TF antibodies (30F5 and 31F2) partially inhibit FVIIa-induced phosphorylation of p44/42 MAPK. Similar results were obtained with 50 nM FVIIa.

Figure 10 shows an example of antibody screening assay 16. The figure shows that inhibition of TF intracellular activity in TF expressing cells is possible with monoclonal antibody TF antibody. Anti-TF Mab B inhibits TF intracellular activity, while anti-TF Mab A does not.

Figure 12 shows an example of antibody screening assay 12. The acceleration profile of thromboelastograms was examined with 0.5 nM FFR-rFVIIa and human anti-TF antibody HuTF-31 F2.

The invention is further illustrated by way of examples, which are designed not to limit the scope of the invention. The features disclosed in the following description and the following examples are each intended to be used individually, as any .t ;·’* .··. .··. t ,. 7 e . ··* ·· > »

K ...» 'w ·’«’ in combination represent different embodiments of the invention.

Examples

Example 1

Production of Mabs specific for human TF Reagents:

Human TF can be isolated from human brain as described in the literature [Rao: Thrombosis Research, 51, 373-384 (1988)].

Lipid-labeled recombinant human TF (Dade Innovin, Baxter) can also be used as a human thromboplastin reagent. Rat, rabbit, baboon, and porcine thromboplastin are prepared from brain tissue. Two volumes of 45°C 0.9% NaCl are added to the brain tissue and the tissue is homogenized using a hand-held glass homogenizer. After incubation at 45°C for thirty minutes with a few shakes, the samples are centrifuged at 2000 g for 20 minutes. The precipitate is discarded and the supernatant is stored in equal aliquots at -80°C until use.

Re-lipidated TF can be obtained by forming recombinant full-length TF (American Diagnostica #4500) in phospholipid vesicles (PC/PS 75/25).

Biotinylated human TF is prepared as follows: Biotin-NHS (n-succinimido biotin, Sigma Η-1759) is dissolved in DMF (dimethylformamide) at a concentration of 1.7 mg/ml. 1 mg/ml of human TF in 0.1 M NaHCO ₃ buffer is added to 60 μΐ of biotin-NHS solution and allowed to react for 4 hours at room temperature. The biotinylated TF in the reacted solution is dialyzed against PBS buffer overnight.

The FVIIa used is recombinant human FVIIa, which is prepared as described in the literature [Thim et al.: Biochem, 27, 7785-7793 (1988)].

sTF: Recombinant human soluble TF1-209 expressed in E. coli and purified essentially as described in the literature [Freskgard et al.: Protein Sci., 5, 1531-1540 (1996)].

S2288: solution in water at a concentration of 17.24 mg/ml (Chromogenix).

FX: Purified human plasma FX (HFX, Enzyme Research Laboratories Ltd.).

FXa: Purified human plasma FX activated with Russell's viper venom (HFXa, Enzyme Research Laboratories Ltd.). Chromozyme X: A solution of 1.25 mg/ml in water is used (Boehringer Mannheim).

¹²⁵ I-FVIIa is prepared by a standard isotope method.

FFR-rFVIIa: This is FVIIa blocked in its active site with D-Phe-L-Phe-LArg-chloromethyl ketone. Its preparation has been described in the literature [Sorensen et al.: J. Biol. Chem., 272, 1186311868 (1997)].

Immunization:

Human TF is emulsified in Freund's complete adjuvant. 40 pg of HuMab mice or hybrids (Medarex) are injected subcutaneously. After 14 and 28 days, and finally at several 14-day intervals, the mice are immunologically boosted with a similar injection of 20 pg of TF in incomplete Freund's adjuvant. 10 days after the last injection, blood samples are drawn and serum is tested for human TF-specific antibody by ELISA (Studies 1 and 2).

Fusion:

Mice showing positive sera in studies 1-3 are boosted with 20 pg human TF by intravenous injection and sacrificed after three days. The spleen is removed under sterile conditions and a single cell suspension is prepared.

Fox myeloma cells were grown in CD Hybridoma Medium (Gibco 11279-023).

Spleen cells and myeloma cells (P3x63 Ag8.653, ATCC CRL-1580) and myeloma cells Sp210 (ATCC CRL-1581) were fused by the PEG method [Köhler and Milstein: European J. Immunology, 6, 511-519(1976)]. The cells were plated on microtiter plates and incubated at 37°C. The medium was changed three times over the next two weeks. 100 μΐ of the supernatant from the hybridoma cells were removed from each well and assayed for TF specific antibodies by TF ELISA (assays 1 and 2).

Example 2

Filtration

Screening of specifically selected antibodies from serum and culture supernatants was performed using the following procedures: Direct TF ELISA assay (assay 1):

Nunc immunoplates were coated with 1 pg/ml human sTF (in PBS) overnight at 4 °C. The plates were blocked with blocking buffer (TBS supplemented with 5 mM CaCl and 2% BSA) and washed with TBS + 0.05% Tween 20, and the supernatants of the hybridoma cells were added. After 1 hour of incubation at room temperature, the plates were washed and horseradish peroxidase (HRPO)-labeled anti-human IgG was added. After an additional hour of incubation, the plates were washed and developed with TMB substrate (KemEN-Tec) according to the manufacturer's instructions. Absorbance was measured at 450 nm using an ELISA reader.

Indirect TF ELISA test (2 tests)

Nunc immunoslides were coated overnight at 4°C with 0.5 pg/ml goat anti-human IgG (in PBS) (Southern Biotechnology Associates, Cat-#2040-l). The slides were blocked with blocking buffer (TBS supplemented with 5 mM CaCl ₂ and 2% BSA) and washed with TBS + 5 mM CaCl ₂ + 0.05% Tween 20. The supernatants of the hybridoma cultures were added and incubated for 1 hour at room temperature. After further washing, biotinylated human sTF was added at a concentration of 1 pg/ml and incubated for 1 hour. After washing, 100 μΐ Streptavidin-HRPO solution was added and incubated for 1 hour. The slides were developed as described in Experiment 1.

FVIIa competition assay (assay 3):

Nunc immunoplates were incubated overnight at 4°C with 5 pg/ml human sTF (in PBS). Plates were blocked with blocking buffer (TBS supplemented with 5 mM CaCl ₂ and 2% BSA) and incubated for 2 hours. Before adding biotinylated human FVIIa, plates were washed (1 pg/ml in TBS buffer supplemented with 5 mM CaCl ₂ and 2% BSA) and incubated for 1 hour. Before adding HRPO-labeled streptavidin, plates were washed and incubated for 45 minutes. Plates were washed again and developed with TMB substrate (Kem-EN-Tec) according to the manufacturer's instructions.

Inhibition of FVIIa/sTF amidolytic activity (study 4)

Inhibition of FVIIa-TF-catalyzed amidolytic activity was investigated with anti-human TF Mabs using soluble human TF (10 nM), recombinant human FVIIa (10 nM) and increasing concentrations of Mabs (0.0122 - 50 nM). Varying concentrations of anti-human TF Mabs, or FFRrFVIIa-1, were preincubated with 10 nM sTF and 10 nM FVIIa in BSA buffer (50 mM Hepes, pH = 7.4, 100 mM NaCl, 5 mM CaCl 2 and 1 mg/ml BSA) for 60 min at room temperature, followed by the addition of S2288 substrate (1.2 mM, Chromogenix). Color development was measured continuously at 405 nm for 30 min. Amidolytic activity was expressed as mOD/min. The IC50 values of the Mabs for inhibition of FVIIa/TF amidolytic activity can be calculated as follows. In this assay, the IC50 value of FFR-rFVIIa is 7 +/- 3 nM.

Inhibition of FXa formation (study 5).

Lipid-labeled TF (10 pM), FVIIa (100 pM) and anti-TF Mabs or FFR-rFVIIa-1 (0 - 50 nM) in BSA buffer (see study 4) were incubated for 60 min at room temperature, then FX (50 nM) was added. The reaction was stopped after 10 min with 1/2 volume of stop buffer (50 mM Hepes, pH = 7.4, 100 mM NaCl, 20 mM EDTA). The amount of FXa formed was determined with S2765 substrate (0.6 mM, Chromogenix) and the absorbance was measured continuously at 405 nm for 10 min. The FX

IC50 values for Mab-mediated inhibition of activation by FVIIa/lipid-loaded TF were calculated. The IC50 value for FFR-rFVIIa was 51 +/26 pM in this assay.

Biosensor test (test 6)

The antibodies were tested using a Biacore instrument by passing the anti-human TF Mab solution over the chip, where the immobilized antibody was human IgG. Subsequently, different concentrations of sTF were added in 10 mM Hepes (pH = 7.4) containing 150 mM NaCl, 10 mM CaCl, and 0.0003% polysorbate 20. Kd values were calculated from the sensorgrams using the integrated Biacore evaluation program.

The TF-dependent clotting assay (assay 7)

The assay was performed using an ACL300 Research clot formation device (ILS Laboratories). Dilutions of anti-human TF Mabs were made up in 50 mM imidazole, pH = 7.4, 100 mM NaCl, 0.1% BSA, mixed with 25 mM CaCl in a ratio of 2 to 5, and placed in the sample caps of the clot formation device. Human, rat, rabbit, baboon or porcine thromboplastin was diluted in imidazole buffer such that the clot formation time in the samples without antibody was approximately 30 seconds and placed in the 2nd tank, while human, rat, rabbit, baboon or porcine plasma was placed in the 3rd tank. During the analysis, 70 μΐ of antibody and CaCh mixture was transferred to 25 μΐ of thromboplastin reagent and preincubated for 900 seconds, then 60 μΐ of plasma was added and the clotting time was measured. The maximum clotting time was 400 seconds. The thromboplastin dilutions were used as a standard curve to calculate the clotting time from the TF relative to the control.

for calculating activity, which did not include anti-TF Mabs or to which FFR-rFVIIa was added. In this study, the IC50 value of FFR-rFVIIa was 4.4 +/- 0.4 pM.

Inhibition of FVIIa/cell surface TF catalyzed FX activation with Mabs: (study 8)

For the FVIIa/TF catalyzed activation of FX, monolayer cultures of cells expressing human TF were used as a source of TF, such as human lung fibroblast WI-38 (ATTC No. CCL-75), human bladder carcinoma cell line J82 (ATTC No. HTB-1), human keratinocyte cell line CCD 1102KerTr (ATCC no. CRL-2310), human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231. Monolayer cultures that reached confluent growth were washed in 24-, 48- or 96-well plates with buffer A (10 mM Hepes, pH = 7.45, 150 mM NaCl, 4 mM KCl and 11 mM glucose) and buffer B (buffer A supplemented with 1 mg/ml BSA and 5 mM Ca ²⁺ ). FVIIa (1 nM), FX (135 nM) and various concentrations of Mab (or FFR-rFVIIat) in buffer B were added simultaneously to the cells. Alternatively, cells were preincubated with anti-TF Mabs or FFR-rFVIIa for 15 min, followed by the addition of rFVIIa and FX. FXa formation was allowed to proceed for 15 min at 37 °C. 50 μΐ were removed from each well and 50 μΐ of stop buffer (in this case buffer A was supplemented with 10 mM EDTA and 1 mg/ml BSA) were added. The amount of FXa formed was determined by pipetting 50 μΐ of the above mixture into the wells of the microtiter plates and adding 25 μΐ of Chromozym X (final concentration 0.6 mM). The absorbance was continuously measured at 405 nm and the initial rate of color development was expressed as FXa .t :··· .··. .··.: .

- ··. ··· · · ♦ ·

J, ...· ..· ”1 concentrations were converted using an FXa standard curve. In this study, the IC50 of FFR-rFVIIa was 1.5 nM.

Inhibition of ¹²⁵ I-FVIIa binding to cell surface TF by Mab (study 9)

Binding assays were performed on human TF-expressing cells, such as human lung fibroblast WI-38 (ATTC No. CCL-75), human bladder carcinoma cell line J82 (ATTC No. HTB-1), human keratinocyte cell line CCD 1102KerTr (ATCC no. CRL-2310), human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231. Monolayer cultures that reached saturating growth were washed in 24-well plates with buffer A (see study 8) and buffer B (see study 8). Monolayer cultures were preincubated for 2 min with 100 μΐ ice-cold buffer B. Different concentrations of Mabs (or FFR-rFVIIa) and labeled FVIIa (0.5 nM ^sj-pvila) were added to the cells simultaneously (final volume 200 μΐ). The plates were incubated for 2 h at 4 °C. At the end of the incubation, unbound material was removed, the cells were washed four times with ice-cold buffer B and lysed with 300 μΐ lysis buffer (200 mM NaOH, 1 % SDS and 10 mM EDTA). Radioactivity was measured using a gamma counter (Cobra, Packard Instruments). The binding data were analyzed and fitted to the curve using GraFit4 program (Erithacus Software, Ltd., (U.K.). The IC50 value of FFR-rFVIIa in this assay was 4 nM.

Inhibition of FVIIa/TF-induced p44/42 MAPK activation with Mab (study 10)

The phosphorylated amount of p44/42 MAPK, and/or Akt, and/or p90RSK was quantified from Western blotting by chemiluminescence (Fujifilm LAS-1000). Cells expressing human TF, such as CCD1102KerTr, NHEK P166, human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231, were cultured in medium supplemented with 0-0.1% FCS for 24 or 48 hours, so that experiments could be performed on quiescent cells. On the day of the experiment, the cells should reach 70-80% growth saturation. The experiment was performed by preincubating cells in excess Mab or FFR-rFVIIa in serum-free medium for 30 min at 37 °C, then adding 10 - 100 nM FVIIa and incubating for 10 min. As a positive control for signal transduction, cells were treated with 10% FCS for 10 min. Cells were washed twice with ice-cold PBS and then lysed in lysis buffer (20 mM Tris, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 10 mM sodium (3-glycerophosphate, 5 mM sodium pyrophosphate, 150 mM NaCl, pH = 7.5, containing 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) and 1 mM benzamidine. Immediately before use, the following were added: 1 mM sodium or orthovanadate, 5 pg/ml leupeptin, 10 pg/ml tiny tinin. The lysates were mixed with SDS sample buffer and loaded onto SDS-polyacrylamide gels. A marker consisting of standard biotinylated proteins was loaded onto each gel. Proteins separated from the SDS-polyacrylamide gels were electroblotted were transferred to nitrocellulose and the p44/42 MAPK, Akt and p90RSK kinases were visualized by immunoblotting using phosphospecific antibodies and chemiluminescence was quantified on a Fujifilm LA51000.

Epitope mapping (study 11)

Production of soluble TF (sTF) variants

The sTF variants (I22C, W45C, K46C, Y94C, F140C, W158C, K201C) were generated by inverse PCR (QuikChange, Stratagene, La Jolla, CA, USA) using a wild-type plasmid as a template [Freskgard et al.: Protein Sci., 5, 1531-1540 (1996)]. The wild-type and variants were expressed and purified from E. coli as described in the literature [Freskgard et al.: Protein Sci., 5, 1531-1540 (1996) with some modifications. Cell lysis was performed with X-press (Biox, Sweden) in 10 mM Tris-HCl buffer, pH = 7.5, and then taken up in the same buffer to which 1 mg DNase was added. The solution was centrifuged at 11,000 x g for 20 min at 4 °C and the inclusion bodies were denatured in 75 ml of a solution of the following composition: 6 M GuHCl, 0.5 M NaCl, 20 mM Tris-HCl, pH = 8.0. Refolding was achieved after 1 h of incubation at room temperature by adding the denatured protein dropwise to 1 liter of a solution of 50 mM Tris-HCl, 0.25 M NaCl, pH = 8.0, under gentle stirring, over a period of approximately 2 h. Purification was performed on a Q-Sepharose Fast Flow column (Pharmacia, Uppsala, Sweden) and FVIIa affinity chromatography as described in the literature [Freskgard et al. (1996)]. Protein homogeneity was confirmed by SDS-PAGE. The concentration was determined at A280 nm and the extinction coefficient was calculated to be 37440 M^cm ¹ [Gill and von Hippel, (1989)].

MaxiSorp plates (Nunc-Immuno) were coated with wild-type sTF and its variants (10 µg/ml) (dissolved in TBS) and blocked with buffer (TBS supplemented with 0.1% Tween 20 and 0.5% BSA). The plates were washed with wash buffer (TBS containing 0.1% Tween 20). Anti-human TF Mabs were used at a concentration of 1 ng/ml in blocking buffer and incubated for 1 hour. After this, the plates were washed 6x with wash buffer. Antibody binding was then detected with HRP-labeled anti-human IgG (Helica Biosystems, Inc) at a 1:2000 dilution prepared in blocking buffer using TMB _{plus substrate} (Kern Tech Cat. 4390A). The final ELISA signal (OD450-620) was used to determine the binding of each antibody to each sTF variant.

Thromboelastography (study 12)

Human thromboplastin (e.g. Innovin, Dade Behring, final dilution 50,000-fold) was mixed with CaCl2 (final concentration 16.7 mM) and anti-TF Mabs and incubated at room temperature for 15 min. Citrate-stabilized human whole blood (280 μΐ) was added to RoTEG sample caps (Pentapharm) and pre-warmed at 37 °C for 5 min before adding 40 μΐ of thromboplastin/ _CaCl2 /anti-TF Mab. Thromboelastography was monitored for 1 h in a RoTEG instrument (Pentapharm). Velocity profiles were obtained from the thrombograms using CoagPro Software™ (MedScience, Arhus, Denmark).

Example 3

Human Cancer Assay. Study with human antiTF Mabs to investigate treatment effects in mouse models that target the growth and metastasis of human cancers (Study 13)

Treatment:

Human anti-TF Mabs were administered as a bolus injection i.v.; 10 mg/kg = 0.1 mg/10 g. Injection volume per 10 g of mouse was 0.1 ml for any of the three treatment solutions, which were as follows:

A. vehicle control,

B. 1 mg/ml human FFR-rFVIIa,

C. 1 mg/ml anti-TF Mabs.

Description of the models

I. Primary growth and growth of liver metastases of colon cancer:

Healthy female athymic mice (nu/nu) aged 7-8 weeks were used for the experiments. To eliminate the residual resistance of nude mice to human cell implants, the mice were routinely irradiated with 5 Gy for 2 days and then human tumor grafts were introduced [Vogel et al.: (1997)]. The mice were immunologically challenged with LS174T human colon carcinoma tumor cell grafts (ATCC CCL 188) cultured in RPMI 1640 in the presence of 15% fetal calf serum (FCS) as described in the literature [Li et al.: Human Gene Therapy, 10, 3045-3053(1999)]. Briefly, cells were harvested with trypsin-EDTA, washed twice, and resuspended in serum-free RPMI supplemented with sodium heparinate (1 unit/ml). Mice were anesthetized by making a small left subcostal incision and 3 × 106 LS174T cells in 50 μΐ phosphate-buffered saline (PBS) were injected into the spleen. Splenic vessels were ligated after 3–5 min, and spleens were surgically removed. This procedure leads to a stable incidence of liver metastases (>95%). Treatment with anti-TF Mabs was initiated immediately after implantation and continued throughout the remaining period. Mice were sacrificed 15 and 30 days after inoculation with tumor cells, livers were removed and weighed, and the number of visible tumor foci on the liver surface was counted. Liver samples were fixed overnight with AFA (5% acetic acid, 75% ethyl alcohol, 2% formalin, 18% water), transferred to 100% ethanol, and embedded in paraffin, and 5-μm sections were prepared for histological evaluation of metastatic foci, immunochemistry, and apoptosis quantification.

The I-1 study

Objective: To investigate the liver metastases and macroscopic growth of LS174T colon tumors in nude mice given iv bolus injection of 10 ^m g/kg anti-TF Mabs.

Mice: 60 homozygous nu/nu 6-week-old NMRI males.

Groups: Mice were randomly divided into groups of 15 and treated with solution A, B or C.

Termination of the experiment: Upon weight loss of more than 20% or upon the appearance of other signs of severe toxicity.

Parameters: During the growth phase, the two orthogonal diameters of the tumor were recorded daily. Body weight was recorded 2-3 times per week. Postmortem, liver metastases were determined.

II. Primary growth and lung metastasis of human breast carcinoma MDA-MB-231 cells (ATCC HT826) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). MDA-MB-231 cells (3 × 106) were injected subcutaneously into 7-8-week-old female nude mice. Primary tumor growth and metastasis were evaluated as previously described [Li et al.: Human Gene Therapy, 12, 515-526(2001)].

Study II -1

Objective: To investigate lung metastases and macroscopic growth of MDA-MB-231 breast tumors in nude mice given iv bolus injection of 10 mg/kg anti-TF Mabs.

Mice: 60 homozygous nu/nu 6-week-old NMRI males.

Groups: Mice were randomly divided into groups of 15 and treated with solution A, B or C.

Termination of the experiment: Upon weight loss of more than 20% or upon the appearance of other signs of severe toxicity.

Parameters: During the growth phase, the two orthogonal diameters of the tumor were recorded daily. Body weight was recorded 2-3 times per week. Postmortem, liver metastases were determined.

Study III: Primary growth of glioma xenografts

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. Tumors were inoculated into the flank according to standard procedures described in the literature (see attached experimental design). Mice were examined twice daily for signs of toxicity, and tumor diameters were measured twice daily in perpendicular directions. Tumors were transplanted into the flanks of nu/nu homozygous mice (NMRI background). 7-week-old male mice were purchased from M&B (Ry, Denmark). Animals were maintained in a gnotobiotic environment and received sterile food and water ad libitum.

We performed three different experiments on the glioma tumor model:

Study III-1

Objective: To investigate the macroscopic growth of U373 tumors in nude mice given iv bolus injection of 10 ^m §/kg anti-TF Mabs.

Mice: 60 homozygous nu/nu 6-week-old NMRI males.

Groups: Mice were randomly divided into groups of 15 and treated with solution A, B or C.

Termination of the experiment: Upon weight loss of more than 20% or upon the appearance of other signs of severe toxicity.

Parameters: During the growth phase, the two orthogonal diameters of the tumor were recorded daily. Body weight was recorded 2-3 times per week.

Study III-2

Objective: To investigate the macroscopic growth of U373 tumors in nude mice given iv bolus injections of 10 ^μg /kg anti-TF Mabs after pre-therapeutic tumor growth was established.

Mice: 60 homozygous nu/nu 6-week-old NMRI males.

Groups: Mice were randomly divided into groups of 15 and treated with solution A, B or C.

Treatments were started when 6 consecutive measurements (daily) showed Gompertzian growth. This corresponds to 100200 mm ³

End of the experiment: Treatments were continued until the tumor reached a maximum size of approximately 1.0 cm ³ , i.e. a tumor diameter greater than 15 mm. Gompertzian regrowth was demonstrated by 6 consecutive measurements. At the end of the experiment, tumors were evaluated histologically and immunologically in each group. The experiment was terminated when there was a weight loss of more than 20% or when there was any other sign of severe toxicity.

Parameters: During the growth phase, the two orthogonal diameters of the tumor were recorded daily. Body weight was recorded 2-3 times per week.

Study III-3

Objective: To investigate the intracranial growth of U373 tumors in nude mice in the presence of anti-TF Mabs.

Mice: 60 homozygous nu/nu 6-week-old NMRI males.

Tumor: U373, which was implanted orthotopically into the right hemisphere using a standard procedure.

Groups: Mice were randomly divided into groups of 15 and treated with solution A, B or C.

End of the experiment: Upon chronic neurological injury, the mice were euthanized.

Data: Survival (i.e. time to neurological injury) was determined using the Kaplan-Meyer statistical method.

Example 4 (Test 14)

In mice in which the TF gene was knocked out and into which the human TF gene was constructed (mTF-KO/hTF-KI mice), a 0.5 ml matrigel plug was placed subcutaneously in the abdominal skin. The matrigel b-FGF (5 ng) was incorporated, and after one week the new blood vessels grown into the gel were quantified by measuring the hemoglobin content (angiogenesis). The inhibitory capacity (% inhibition of hemoglobin content) of human anti-TF Mabs can be evaluated after single or repeated parenteral administration of the protein.

Example 5 (Test 15)

Gene expression analysis to distinguish between antibodies that prevent TF binding to antibodies and those that prevent FX binding to TF.

By cDNA microarray analysis, we observed upregulation of three genes in BHK-TF cells treated with FVIIa. These genes are: Fra-1, which encodes Fos-related antigen 1; Id2, which encodes the helix-loop-helix members of proteins; and Cyról, which encodes an extracellular matrix signaling protein. To screen anti-TF Mabs, we developed the following assay that prevents FVIIa-induced upregulation of Fra-1, Id2, or Cyról.

Cell culture:

Reagents were purchased from GIBCO-BRL Life Technologies unless otherwise stated.

BHK-TF cells, which were established as described in the literature [Poulsen et al.: Journal of Biological Chemistry 273, 6228-6232 (1998)], were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS, 100 international units/ml penicillin and 100 pg/ml streptomycin. Cultures were grown to 95-100% confluence, the cells were washed and grown for an additional 16-18 hours in FCS-free medium. The cells were washed again and 100 nM FVIIa in FCS-free medium was added to the cultures. For Northern blotting and fragment cloning, the cells were treated as follows. BHK-TF cells were grown in Dulbecco's modified Eagle's medium containing 10% FCS, 100 international units/ml penicillin, and 100 μg/ml streptomycin, and when 95-100% confluence was reached, the cells were washed and grown for an additional 16-18 hours in FCS-free medium. The cells were washed again and 100 nM FVIIa was added to the cultures in FCS-free medium for 1 hour. CRL2091 cells (ATCC) were grown in Iscove's modified Eagle's medium containing 10% FBS, 100 international units/ml penicillin, and 100 μg/ml streptomycin, and the cells were grown to 95-100% confluence. Afterwards, the cells were washed and grown for another 16-18 hours in FCS-free medium, and 100 nM FVIIa-1 was added to the cultures in FCS-free medium for 6 hours.

Mouse 3T3-L1 cells (ATCC) were grown in Dulbecco's modified Eagle's medium containing 10% FBS, 100 international units/ml penicillin, and 100 pg/ml streptomycin. Cells were grown to confluence and induced with medium containing 1 μΜ dexamethasone (Sigma), 10 pg/ml human insulin (Novo Nordisk A/S), and 1 μΜ 8RL49653 (Novo Nordisk A/S) for 1 h.

Clones of fragments for Northern blotting:

Fra-1 was cloned by reverse transcription PCR from RNA isolated from 3T3-L1 cells treated with dexamethasone, insulin and BRL49653 for 1 h using the superscript II kit (Life Technologies) according to the manufacturer's instructions. Id2 and Cyr61 were cloned by reverse transcription PCR from RNA obtained from BHKTF cells treated with FVIIa for 1 h and CRL2091 cells treated with FVIIa for 6 h. The 5' and 3' primers were as follows: for Fra-1: 5'-GCGGCCGCCATGTACCGAGACTACGGGGGAACCG-3' and 5'-GCGGCCGCTCACAAAGCCAGGAGTGTAGG-3';

For Id2: 5'-CAGCATGAAAGCCTTCAGTC-3' and 5'-CTCTGGTGATGCAGGCTGAC-3';

For Cyr61: 5'-CGTCACCCTTCTCCACTTGA-3' and 5'-CTTGGTCTTGCTGCATTTCT-3'.

The PCR parameters were as follows: one cycle of denaturation at 94 °C for 10 s, annealing at 65 °C for 15 s, extension at 72 °C for 1.5 min, one cycle of denaturation at 94 °C for 10 s, annealing at 64 °C for 15 s, and extension at 72 °C for 1.5 min, one cycle of denaturation at 94 °C for 10 s, annealing at 63 °C for 15 s, and extension at 72 °C for 1.5 min, one cycle of denaturation at 94 °C for 10 s, annealing at 62 °C for 15 s, and extension at 72 °C for 1.5 min, one cycle of denaturation at 94 °C for 10 s, annealing at 62 °C for 15 s, and extension at 72 °C for 1.5 min, one cycle of denaturation at 94 °C for 15 s, annealing at 61 °C for 15 s, and extension at 72 °C for 1.5 min. °C for 1.5 min, one cycle of denaturation at 94 °C for 10 s, annealing at 60 °C for 15 s, and extension at 72 °C for 1.5 min, 40 cycles of denaturation at 94 °C for 10 s, annealing at 55 °C for 15 s, and extension at 72 °C for 1.5 min. All fragments were cloned into TOPO 2.1 (Invitrogen) and sequenced using Megabase sequencer.

Northern imprinting:

Total RNA was isolated from BHK-TF cells incubated with FVIIa, FX, ASIS, 1F44A1 or TFS5G9 using TriZol according to the manufacturer's instructions. 20 pg of RNA was size fractionated on a denaturing gel consisting of 1% agarose, 20 mM MOPS, 5 mM NaOAc, 6% formaldehyde and 1 mM EDTA, transferred by capillary blotting onto Hybond N ⁺ membrane (Amersham) and fixed by UV cross-linking. The cDNAs encoding Fra-1, Id2 or Cyr61 were

They were labeled with the Prime It kit (Stratagene) using [a- ³² P] dATP (Amersham) and hybridized using Express Hyb (Clontech) following the manufacturer's instructions, and the results were visualized autoradiographically.

Example 6 (16, test)

MAPK assay with Elkl transcription factor/luciferase reporter (PathDetect)

HeLa cells were grown to 40% confluence in T-80 dishes for one day prior to transfection. Cells were transfected with 150 ng pFA-Elk1 (Stratagene), 3 ug pFR-Luc (Stratagene), 3 pg human TF/pcDNA3 and 3 pg mouse Protease Activated Receptor 2/pcDNA3,1 + , using 36 µl FuGene (Roche) as described in the specification. The next day, cells were detached with Versene™ (Invitrogen) and plated in black 96-well plates (Packard) at a density of 20,000 cells/well. After the cells had reattached to the plate, the medium was changed to 160 µl/well of serum-free Dulbecco's modified Eagle's medium (Invitrogen) and incubated for 16 hours.

Cells were preincubated for one hour separately with: 20 µl serum-free medium (control), 20 µl 2.5 µM FFR-rFVIIa (control), 20 µl 2.5 µM anti-TF Mab B, or 20 µl 2.5 µM anti-TF Mab A. Then 20 µl 0.5 µM FVIIa was added, half of the wells were filled with medium, while the other half was filled with medium. Cells were incubated for 4 hours and then subjected to Luciferase gene assay. LucLite (Packard) reagent was added to the cells according to the manufacturer's instructions. Luciferase expression levels were read using a TopCount Microplate Scintillation instrument (Packard).

Claims (48)

PATENT CLAIMS 1. Isolated human antibody that immunologically reacts with an epitope on human TF. 2. The isolated human antibody of claim 1, which inhibits the binding of human TF to human factor VIIa. 3. The isolated human antibody according to any one of claims 1-2, which is a monoclonal antibody. 4. The isolated human antibody of any one of claims 1-3, which is a recombinant antibody. 5. The isolated human antibody of any one of claims 1-4, wherein said antibody is a Fab fragment. 6. The isolated human antibody of any one of claims 1-4, wherein said antibody is an F(ab)2 fragment. 7. The isolated human antibody of any one of claims 1-4, wherein said antibody is an F(ab)2 fragment. 8. The isolated human antibody of any one of claims 1-4, wherein said antibody is a single-chain Fv fragment. 9. The isolated human antibody of any one of claims 1-9, wherein said antibody has a Kd value for human TF of 10-15-10-8 M. 10. The isolated human antibody of any one of claims 1-9, wherein said antibody has a Kd value for human TF of 10 ¹⁵ -10 ¹⁰ M. 11. A pharmaceutical composition comprising a therapeutically effective amount of a human antibody that immunologically reacts with an epitope on human TF. 12. A pharmaceutical composition comprising a therapeutically effective amount of a human antibody that immunologically reacts with an epitope on human TF, wherein said antibody is an antibody according to any one of claims 1-10. 13. A composition comprising a human antibody that immunologically reacts with an epitope on human TF. 14. A composition comprising a human antibody that immunologically reacts with an epitope on human TF, wherein said antibody is an antibody according to any one of claims 1-10. 15. A method of treating FVIIa/TF-related diseases in a human, comprising administering to said human a therapeutically effective amount of a human antibody that immunologically reacts with an epitope on human TF. 16. A method of treating FVIIa/TF-related diseases in a human, comprising administering to said human a therapeutically effective amount of a human antibody according to any one of claims 1-10. 17. A method for producing a human antibody, characterized in that it comprises the following steps: a) production of human antibodies against human TF, b) testing the antibodies in a TF-induced blood clot formation assay and selecting the human antibody that inhibits blood clot formation in this assay when the IC50 value is less than the FFR-rFVIIa IC50 value + 1 nM, such as less than the FFR-rFVIIa IC50 value + 500 pM, preferably less than the FFR-rFVIIa IC50 value + 200 pM, preferably less than the FFR-rFVIIa IC50 value + 100 pM, preferably less than FFR-rFVIIa IC50 value + 50 pM, more preferably less than FFR-rFVIIa IC50 value + 10 pM, preferably less than FFR-rFVIIa IC50 value + 5 pM, preferably less than FFR-rFVIIa IC50 value, or testing the effect of test antibodies on FXa formation, and selecting the human antibody with an IC50 value less than FFR-rFVIIa IC50 value + 100 nM (using 0.1 nM FVIIa in the test), such as less than FFR-rFVIIa IC50 value + 10 nM, preferably less than FFR-rFVIIa IC50 value + 5 nM, preferably less than FFR-rFVIIa IC50 value + 1 nM, preferably less than FFR-rFVIIa IC50 value + 0.1 nM, preferably less than FFR-rFVIIa IC50 value; or testing the antibodies in a FVIIa/TF amidolytic system, wherein the human antibody is selected which, expressed as an IC50 value, inhibits TF induced factor VIIa amidolytic activity to an extent less than the FFR-rFVIIa IC50 +100 nM (where the FVIIa concentration in the assay is 10 nM), such as less than the FFR-rFVIIa IC50 +40 nM, preferably less than the FFRrFVIIa IC50 +20 nM, more preferably less than the FFR-rFVIIa IC50 +10 nM, preferably less than the FFR-rFVIIa IC50; or testing the test antibodies in a FVIIa competition system, and then selecting the human antibody which competes with FVIIa binding; or testing the test antibody in a TF ELISA system and selecting the human antibody that binds human TF. 18. The method of claim 17, wherein the antibody against human TF is prepared by the following steps: 69 ^: ···· «····· ·· ·* a) immunizing the mammal with human TF, b) isolation of antibodies produced by the immunized animal. 19. The method of claim 18, wherein the mammal is a mouse. 20. The 17th-19th The method according to any one of claims 1 to 4, characterized in that it comprises testing the antibodies in TF-induced blood clot formation and selecting the human antibody that inhibits blood clot formation in this test, when the IC ₅₀ value is less than the FFR-rFVIIa IC50 value + 1 nM, such as less than the FFR-rFVIIa IC50 value + 500 pM, preferably less than the FFR-rFVIIa IC50 value + 200 pM, preferably less than the FFR-rFVIIa IC50 value + 100 pM, preferably less than the FFR-rFVIIa IC50 value + 50 pM, more preferably less than the FFR-rFVIIa _IC50 value + 10 pM, preferably less than the FFR-rFVIIa IC50 value + 5 _pM , preferably less than the FFR-rFVIIa IC50 value. 21. The method according to any one of claims 17-20, characterized in that it comprises testing the effect of test antibodies on FXa formation and selecting those human antibodies with an IC50 value less than the FFRrFVIIa IC50 value + 100 nM (using 0.1 nM FVIIa in the test), such as less than the FFR-rFVIIa IC50 value + 10 nM, preferably less than the FFR-rFVIIa IC50 value + 5 nM, preferably less than the FFR-rFVIIa IC50 value + 1 nM, preferably less than the FFR-rFVIIa IC50 value + 0.1 nM, preferably less than the FFR-rFVIIa IC50 value. 22. The method according to any one of claims 17-21, characterized in that it comprises testing the FVIIa/TF amidolytic activity of the test antibodies, and that human ΊΟ selecting an antibody that inhibits TF-induced factor VIIa amidolytic activity by an IC50 value of less than the FFR-rFVIIa IC50 +100 nM (where the FVIIa concentration in the assay is 10 nM), such as less than the FFR-rFVIIa IC50 +40 nM, preferably less than the FFR-rFVIIa IC50 +20 nM, more preferably less than the FFR-rFVIIa IC50 +10 nM, preferably less than the FFR-rFVIIa IC50. 23. The method of any one of claims 17-22, characterized in that it comprises testing the test antibodies for their competition with factor VIIa and selecting the human antibody that competes with factor VIIa. 24. The method of any one of claims 17-23, characterized in that it comprises testing the test antibodies by TF ELISA and selecting a human antibody that immunologically reacts with human TF. 25. A method for producing a human antibody, characterized in that it immunologically reacts with an epitope on human TF and is capable of inhibiting the binding of human TF to coagulation factor VIIa, which method comprises the following: a) production of human antibodies against human TF, b) testing the antibodies in a TF-induced blood clot formation assay and selecting the human antibody that inhibits blood clot formation in this assay when the IC ₅₀ value is less than the FFR-rFVIIa IC50 value + 1 nM, such as less than the FFR-rFVIIa IC50 value + 500 pM, preferably less than the FFR-rFVIIa IC50 value + 200 pM, preferably less than the FFR-rFVIIa IC50 value + 100 pM, preferably less than the FFR-rFVIIa IC50 value + 50 pM, more preferably less than the FFR-rFVIIa IC50 value + 10 pM, preferably less than the FFR-.LN? L'··Ν* ··*· rFVIIa IC50 value + 5 pM, preferably less than the FFR-rFVIIa IC50 value, or testing the effect of the test antibodies on FXa formation, and selecting the human antibody whose IC50 value is less than the FFR-rFVIIa IC50 value +100 nM (using 0.1 nM FVIIa in the test), such as less than the FFR-rFVIIa IC50 value + 10 nM, preferably less than the FFR-rFVIIa IC50 value + 5 nM, preferably less than the FFR-rFVIIa IC50 value + 1 nM, preferably less than the FFRrFVIIa IC50 value + 0.1 nM, preferably less than the FFR-rFVIIa IC50 value; or testing the antibodies in a FVIIa/TF amidolytic system, wherein the human antibody is selected which, expressed as an IC50 value, inhibits TF-induced factor VIIa amidolytic activity by less than the FFR-rFVIIa IC50 +100 nM (where the FVIIa concentration in the assay is 10 nM), such as less than the FFR-rFVIIa IC50 + 40 nM, preferably less than the FFRrFVIIa IC50 + 20 nM, more preferably less than the FFR-rFVIIa IC50 + 10 nM, preferably less than the FFR-rFVIIa IC50; or testing the test antibodies in a FVIIa competition system, then selecting the human antibody that competes with FVIIa binding; or testing the test antibody in a TF ELISA system and selecting the human antibody that binds human TF. 26. A method for producing a human antibody, characterized in that it comprises the following steps: a) immunization of mice with human TF, ’J··-’’· :· b) isolation of antibody-producing cells from the immunized mouse and production of immortal cells secreting human antibodies, c) isolation of the produced antibody from the culture medium of the immortalized cells, d) testing the antibody with indirect TF ELISA, which is performed in the TF solution, and selecting human antibodies binding to human TF from the solution, e) testing antibodies in the FVIIa competition assay and selecting a human antibody capable of competing with FVIIa binding, f) testing the antibody in a FVIIa/TF amidolytic system and selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity, having an IC50 value less than the FFRrFVIIa IC50 +100 nM (where the FVIIa concentration in the assay is 10 nM), such as less than the FFR-rFVIIa IC50 +40 nM, preferably less than the FFR-rFVIIa IC50 +20 nM, more preferably less than the FFR-rFVIIa IC50 +10 nM, preferably less than the FFR-rFVIIa IC50; g) testing the antibodies in an FXa formation system and selecting a human antibody that inhibits FXa formation with an IC50 value less than the FFR-rFVIIa IC50 value + 100 nM (using 0.1 nM FVIIa in the assay), such as less than the FFR-rFVIIa IC50 value + 10 nM, preferably less than the FFR-rFVIIa IC50 value + 5 nM, preferably less than the FFR-rFVIIa IC50 value + 1 nM, preferably less than the FFRrFVIIa IC50 value + 0.1 nM, preferably less than the FFR-rFVIIa IC50 value; h) testing of antibodies in a TF-induced blood clotting system and selection of human antibodies that 73 ΙΖ· Ζ· ·-' ·*:· blood clot formation is inhibited in this assay, where the IC50 value is less than the FFR-rFVIIa IC50 value + 1 nM, such as less than the FFR-rFVIIa IC50 value + 500 pM, preferably less than the FFR-rFVIIa IC50 value + 200 pM, preferably less than the FFR-rFVIIa IC50 value + 100 pM, preferably less than the FFR-rFVIIa IC50 value + 50 pM, more preferably less than the FFR-rFVIIa IC50 value + 10 pM, preferably less than the FFR-rFVIIa IC50 value + 5 pM, preferably less than the FFR-rFVIIa IC50 value; i) selection and cultivation of immortalized cells in appropriate culture medium, thereby ensuring the production of antibodies selected in points d-h), j) isolating antibodies released from the selected immortalized cells from the culture medium. 27. The method according to claim 26, characterized in that, in addition to the above, it also comprises testing the antibody in a direct TF ELISA system and selecting a human antibody that immunologically reacts with the immobilized human TF. 28. The method according to any one of claims 26-27, characterized in that it further comprises testing the antibodies in an FXa production system performed on TF-expressing cells and selecting a human antibody that inhibits FXa production on TF-expressing cells, wherein the IC50 value is less than the FFR-rFVIIa IC50 value +100 nM (using 0.1 nM FVIIa in the assay), such as less than the FFR-rFVIIa IC50 value +10 nM, preferably less than the FFR-rFVIIa IC50 value +5 nM, preferably less than the FFRrFVIIa IC50 value +1 nM, preferably less than the FFR-rFVIIa IC50 value +1 nM, preferably less than the FFR-rFVIIa IC50 value 74 j. ·-·’··»' IC50 value + 0.1 nM, preferably less than the FFR-rFVIIa IC50 value. 29. The method according to any one of claims 26-28, characterized in that, in addition to the above, it also comprises testing the antibodies in a whole cell TF binding system and selecting the human antibody that competes with the binding of human TF and factor VIIa expressed on the surface of whole cells. 30. The method according to any one of claims 26-29, characterized in that it further comprises biosensor testing of the antibodies and selecting those human antibodies whose Kd for human TF is less than 100 nM, such as 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 one of claims 26-30, characterized in that, in addition to the above, it also comprises testing the antibodies in the MAPK signaling system and selecting those human antibodies that inhibit the activation of factor VIIa-induced MAPK signaling. 32. The method according to any one of claims 26-31, characterized in that, in addition to the above, it also comprises epitope mapping of the antibodies and selecting those human antibodies that immunologically react with the preferred epitopes on TF. 33. The method of claim 32, wherein the preferred epitope comprises the following amino acids: Trp45, Lys46 and Tyr94. the, ··· ··« · · · 75 J..».’ ‘ *···«* 34. The method of any one of claims 26-33, wherein said immortalized cell is a hybridoma. 35. A human antibody that immunologically reacts with an epitope on human TF and inhibits the binding of human coagulation factor VIIa to human TF, and can be obtained by the following method: a) immunization of mice with human TF, b) isolation of antibody-producing cells from the immunized mouse and production of immortal cells secreting human antibodies, c) isolation of the produced antibody from the culture medium of the immortalized cells, d) testing the antibody with indirect TF ELISA, which is performed in the TF solution, and selecting human antibodies binding to human TF from the solution, e) testing antibodies in the FVIIa competition assay and selecting a human antibody capable of competing with FVIIa binding, f) testing the antibody in a FVIIa/TF amidolytic system and selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity, having an IC ₅₀ value less than the FFRrFVIIa IC50 +100 nM value (where the FVIIa concentration in the assay is 10 nM), such as less than the FFR-rFVIIa IC50 +40 nM, preferably less than the FFR-rFVIIa IC50 +20 nM, more preferably less than the FFR-rFVIIa IC50 +10 nM, preferably less than the FFR-rFVIIa IC50; g) testing the antibodies in an FXa formation system and selecting the human antibody that inhibits FXa formation, where the IC50 value is less than the FFR-rFVIIa IC50 value + 100 nM (using 0.1 nM FVIIa in the assay), such as less than the FFR-rFVIIa IC50 value + 10 nM, preferably less than the FFR-rFVIIa IC50 value + 5 nM, preferably less than the FFR-rFVIIa IC50 value + 1 nM, preferably less than the FFRrFVIIa IC50 value + 0.1 nM, preferably less than the FFR-rFVIIa IC50 value; h) testing the antibodies in a TF-induced blood clot formation system, and selecting those human antibodies that inhibit blood clot formation in this assay, where the IC50 value is less than the FFR-rFVIIa IC50 value + 1 nM, such as less than the FFR-rFVIIa IC50 value + 500 pM, preferably less than the FFR-rFVIIa IC50 value + 200 pM, preferably less than the FFR-rFVIIa IC50 value + 100 pM, preferably less than the FFR-rFVIIa IC50 value + 50 pM, more preferably less than the FFR-rFVIIa IC50 value + 10 pM, preferably less than the FFR-rFVIIa IC50 value + 5 pM, preferably less than the FFR-rFVIIa IC50 value; i) selection and cultivation of immortalized cells in appropriate culture medium, thereby ensuring the production of antibodies selected in points d-h), j) isolating antibodies released from the selected immortalized cells from the culture medium. 36. A human antibody that immunologically reacts with an epitope on human TF and inhibits the binding of human coagulation factor VIIa to human TF, which can be prepared by the method of any one of claims 26-34. 37. A cell producing a human antibody that immunologically reacts with an epitope on human TF and inhibits the binding of human coagulation factor VIIa to human TF. 38. The cell of claim 37, which is an isolated lymphoid cell. 39. The cell of any one of claims 37-38, which is obtainable from a mouse. 40. The cell of claim 37, which is a hybridoma cell. 41. The cell of claim 40, wherein the hybridoma cell is obtained by fusion of the antibody-producing lymphoid cell and the immortal cell, thereby providing the antibody-producing hybridoma cell. 42. The cell of any one of claims 37-41, wherein said antibody inhibits the binding of human coagulation factor VIIa to human TF. 43. The cell of any one of claims 37-42, wherein said antibody immunologically reacts with a 3-dimensional surface comprising the following amino acids: Trp45, Lys46 and Tyr94. 44. The cell of any one of claims 37-43, wherein said antibody is a Fab fragment. 45. The cell of any one of claims 37-44, wherein said antibody is an F(ab)2 fragment. 45. The cell of any one of claims 37-44, wherein said antibody is an F(ab')2 fragment. 47. The cell of any one of claims 37-44, wherein said antibody is an scFv fragment. 48. The cell of any one of claims 37-47, wherein said antibody has a Kd value for human TF of 10 ¹⁵ 10-8 M. 49. The cell of any one of claims 37-48, wherein said antibody is ΙΟ-™ M. The Kd value for human T[F-rg is 10 ¹⁵ - H 1062 Budapest, Aiidrássy út 113. phone 461 1000 fax. 461 1099