JP2005512970A - Human tissue factor antibody - Google Patents

Abstract

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

Description

Field of Invention

  The present invention relates to an isolated antibody that immunoreacts with tissue factor (TF) and inhibits the binding of coagulation factor VIIa (FVIIa), as well as surgery, microsurgery, angioplasty, Or inhibits thrombus formation associated with trauma or is associated with diseases such as deep vein thrombosis, generalized intravascular coagulation syndrome (DIC), coronary artery disease, sepsis, inflammation, atherosclerosis, or cancer The present invention relates to immunotherapy that inhibits thrombus formation and other functions of TF in abnormal hemostatic conditions. Also disclosed are methods for preparing antibodies, as well as cell lines for preparing human monoclonal antibodies.

Background of the Invention

  Blood clotting is a process composed of complex interactions of various blood components or factors that ultimately produce a fibrin clot. In general, blood components involved in what is called a coagulation “cascade” are proenzymes or enzyme sources, enzymatically inactive proteins that are converted to proteolytic enzymes by the action of activators, activated coagulation The factor itself. A clotting factor, commonly referred to as an “active factor” that undergoes such a transformation, is represented by the addition of a subscript “a” (eg, Factor VIIa).

Activated factor X (“Xa”) is required to convert prothrombin to thrombin, which then converts fibrinogen to fibrin as the final step in forming a fibrin clot. There are two systems, or pathways, that promote factor X activation. “Intrinsic pathway” refers to a reaction that leads to thrombin formation through the use of factors present only in plasma. A series of protease-mediated activation ultimately produces factor IXa, which binds to factor VIIIa and degrades factor X into factor Xa. In the “extrinsic pathway” of blood clotting, the same proteolysis is performed by FVIIa and its cofactor TF. TF is a membrane-bound protein and does not normally circulate in the active form in plasma. However, when blood vessels are destroyed, TF forms a complex with FVIIa and catalyzes the activation of factor X or factor IX in the presence of Ca ²⁺ and phospholipids. Although the relative importance of the two clotting pathways in hemostasis is unknown, Factor VII and TF have been found to play a central role in the initiation of blood clotting.

  It is often necessary to selectively block the coagulation cascade in patients. Anticoagulants such as heparin, coumarins, coumarin derivatives, indandione derivatives, or other drugs, such as during renal dialysis or deep vein thrombosis, generalized intravascular coagulation syndrome (DIC), and many It can be used to treat other medical diseases. For example, heparin therapy or extracorporeal therapy with citrate ions can be used to prevent clotting during dialysis treatment. Heparin is also used in preventing deep vein thrombosis in patients undergoing surgery.

  However, treatment with heparin and other anticoagulants can have undesirable side effects. Available anticoagulants generally act throughout the body rather than specifically at the site of injury. For example, heparin can cause massive bleeding. In addition, heparin is rapidly removed from the blood with a half-life of about 80 minutes and therefore needs to be administered frequently. Because heparin functions as a cofactor for antithrombin III (ATIII) and ATIII is rapidly consumed in the treatment of DIC, it is often difficult to maintain an appropriate heparin dose, and a continuous level of ATIII and heparin levels Monitoring is necessary. Also, if ATIII deficiency is severe, heparin is ineffective. In addition, long-term use of heparin can increase platelet aggregation and reduce platelet count, leading to the development of heparin-induced thrombocytopenia. Indandione derivatives can also have 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.

  International application WO 92/15686 relates to inactivated factor VIIa for inhibiting blood coagulation.

  An antibody is a specific immunoglobulin (Ig) polypeptide produced by the vertebrate immune system in response to challenge with a foreign protein, glycoprotein, cell, or other foreign antigenic substance. At least in part, a series of events that allow an organism to overcome invasion by an exogenous cell or to remove a system of exogenous material is understood. An important part of this process is making antibodies that specifically bind to certain foreign substances. The binding specificity of such polypeptides for a particular antigen is very precise, and the high specificity that can be generated by individual vertebrates is notable for their complexity and variability. Millions of antigens can elicit antibody responses, and each antibody is induced almost exclusively to the specific antigen that elicited each antibody.

  Two main sources of vertebrate antibodies are currently available, in situ production by mammalian B lymphocytes and cell culture production by B cell hybrids. Antibodies are generated in situ as a result of the differentiation of immature B lymphocytes into plasma cells, which occurs in response to stimulation with specific antigens. In undifferentiated B cells, portions of DNA encoding various regions in the immunoglobulin chain are separated in genomic DNA. The sequence is collected sequentially prior to expression. The resulting rearranged gene can be expressed in mature B lymphocytes to produce the desired antibody. However, even when a particular mammal is exposed to only a single antigen, a uniform population of antibodies cannot be obtained. An in situ immune response against any particular antigen is defined by a mosaic of responses against various determinants present on the antigen. Since each subset of homologous antibodies is provided by a single population of B cells, the in situ generation of antibodies is “polyclonal”.

  Such limited but inherent heterogeneity has been overcome in many specific cases through the use of hybridoma technology, creating "monoclonal" antibodies in cell culture with B cell hybridomas.

  In this process, relatively short-lived or mortal lymphocytes or splenocytes from a mammal injected with the antigen are fused with an immortalized tumor cell line, thereby immortalized and B cells Hybrid cells or “hybridomas” capable of producing the genetically encoded antibodies are produced. The hybrids thus created are separated into a single genetic strain by selection, dilution and regrowth, whereby each strain represents a single genetic line. They therefore produce antibodies that are guaranteed to be homogeneous to the desired antigen. These antibodies that inherit a pure genetic lineage are termed “monoclonal”.

  Monoclonal antibodies with a single specificity have a major impact on immunology, and their usefulness has already been demonstrated in biology, pharmacology, biochemistry and other sciences. Such monoclonal antibodies have found widespread use in therapy as well as diagnostic reagents (see, for example, Ritz and Schlossman, Blood, 59: 1-11, (1982)).

  Monoclonal antibodies produced by hybridomas are therapeutically effective as described above and are clearly preferred over polyclonal antibodies because of their specificity, but have significant drawbacks. In many applications, the use of monoclonal antibodies produced in non-human animals is severely limited when the monoclonal antibodies are used in humans. Repeated injection of “foreign” antibodies, such as mouse antibodies, into humans can lead to deleterious hypersensitivity reactions. Such non-human monoclonal antibodies elicit an anti-human antibody response when injected into humans.

The therapeutic use of mouse Mabs against TF is known from US Pat. Nos. 6,001,978 and 5,223,427.
International application WO 99/51743 relates to human / mouse chimeric monoclonal antibodies directed against human TF.

European Patent Application No. 833911 relates to CDR grafted antibodies against human TF.
Presta L. et al., Thrombosis and Haemostasis, Vol. 85 (3) pp.379-389 (2001) relates to a humanized antibody against TF.

  There remains a need in the art for improved compositions with anticoagulant activity that can be administered at relatively low doses and do not produce the undesirable side effects associated with conventional anticoagulant compositions. Yes. The present invention fulfills this need by providing an anticoagulant that does not have the side effects associated with conventional antibodies with non-human sequences, the anticoagulant acting specifically on the site of injury, Provides other related benefits. Furthermore, the present invention provides compounds that act to inhibit the cellular function of TF associated with symptoms such as sepsis, inflammation, atherosclerosis, restenosis, or cancer.

Description of the invention

  The present invention relates to a non-immunoantigenic high affinity human antibody against human TF that inhibits the binding of coagulation factor VII / VIIa, and to a method for selecting a human antibody against human TF that is effective for treatment.

  In a first aspect, the present invention relates to an isolated human antibody that immunoreacts with an epitope present on human TF.

  As used herein, the term “human tissue factor” or “human TF” refers to a full-length polypeptide receptor comprising amino acid sequences 1-263 of native human tissue factor.

As used herein, the term “antibody” refers to immunoglobulin molecules and fragments thereof that have the ability to specifically bind an antigen (eg, human TF). A full-length antibody includes four polypeptide chains, two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions are further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FR). Each VH and VL is composed of 3 CDRs and 4 FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Thus, the scope of antibody definition also includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, human TF). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antibody” include the following: (i) monovalent fragments consisting of Fab fragments, VL, VH, CL and CH I domains; ) F (ab) ₂ and F (ab ″) ₂ fragments, a bivalent fragment comprising two Fab fragments joined by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; ) An Fv fragment consisting of the single arm VL and VH domains of the antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); and (vi) isolated complements Sex determination region (CDR). In addition, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but these two domains are paired with the VL and VH regions (known as single chain Fv (scFv)). Synthetic linkers capable of creating single protein chains that form monovalent molecules can be linked using recombinant methods (eg, Bird et al. (1988) Science 242: 423-426; and Huston et al. al. (1988) Proc. Natl., Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antibody”. Other forms of single chain antibodies, such as diabody, are also included. Diabodies are those in which VH and VL domains are expressed on a single polypeptide chain but are too short to allow pairing between two domains on the same chain, making these domains separate chains Bivalent antibodies that have been paired with two complementing domains to create two antigen binding sites (eg, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci USA 90: 6444-6448; Poljak, RJ, et al., (1994) Structure 2: 1121-1123). Human TF consists of (1) a peptide antigenic determinant consisting of a single peptide chain in human TF; (2) two or more spatially adjacent amino acid sequences that are scattered on the human TF polypeptide sequence. And (3) a post-translational antigenic determinant composed entirely or partly of a molecular structure (for example, a carbohydrate group, etc.) covalently bound to human TF after translation. It will be understood that these antigenic determinants may be present.

  As used herein, the terms “human antibody” and “human antibodies” and “human TF antibody” are used for human germline. It includes antibodies having variable and constant regions derived from immunoglobulin sequences. Human antibodies of the present invention contain amino acid residues that are not encoded by human germline immunoglobulin (eg, mutations induced by random or site-directed mutagenesis in vitro or somatic mutations in vivo). For example, it may be contained in CDR, especially in CDR3. However, as used herein, the term “human antibody” refers to an antibody in which a CDR sequence derived from the germline of another mammalian species (eg, mouse) is conjugated to a human framework sequence, eg, so-called humanized. It does not include antibodies or human / mouse chimeric antibodies.

  As used herein, an “isolated human antibody” refers to a human antibody that is substantially free of other antibodies with different antigen specificities (eg, specifically binds human TF). An isolated antibody is substantially free of antibodies that specifically bind antigens other than human TF). However, an isolated antibody that specifically binds human TF may be cross-reactive with other antigens, such as TF molecules from other species (described in further detail below). ). Further, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

  As used herein, the term “epitope” means any antigenic determinant on an antigen to which an antibody binds. Epitopic determinants usually consist of surface groupings of chemically active molecules (eg amino acids or sugar side chains) and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

As used herein, the term “immunoreacts” or “immunoreacting” means that an antibody binds to its epitope with a dissociation constant K _d of less than 10 ⁻⁴ M. The term “immunoreacts” or “immunoreacting” is used interchangeably with the term “specifically binds” where appropriate.

  As used herein, the term “inhibit” means to reduce compared to a reference. As an example, an antibody that inhibits the binding of human coagulation factor VIIa to human TF is compared to the ability of human coagulation factor VIIa to bind a human antibody in the absence of the antibody, compared to the ability of human coagulation factor VIIa to bind human TF. By any antibody that reduces the ability to bind is meant.

As used herein, the term “affinity” refers to the strength with which an antibody binds to an epitope. The affinity of the antibody is [Ab] × [Ag] / [Ab-Ag] (where [Ab-Ag] is the molar concentration of the antibody-antigen complex, and [Ab] is the molar concentration of unbound antibody. in and, [Ag] is measured by the dissociation constant K _d that is defined by a a) the molar concentration of the antigen unbound. Affinity constant K _a is defined by 1 / K _d. A preferred method for determining Mab specificity and affinity by competitive inhibition is Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth.Enzymol. 92: 589-601 (1983). The entirety of which is hereby incorporated by reference.

  In a second aspect, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a human antibody that immunoreacts with an epitope present on human TF.

  The term “therapeutically effective amount” is an effective dose as determined by a qualified practitioner who can titrate a dose to achieve the desired response. Factors to consider dose include efficacy, bioavailability, desired pharmacokinetic / pharmacodynamic profile, treatment symptoms (eg trauma, inflammation, septic shock), patient related factors (eg weight, health , Age, etc.), presence of co-administered medications, time of administration, or other factors known to clinical practitioners. The dosage of human antibody to TF administered to a patient will vary depending on the type and severity of the condition to be treated, but is generally in the range of 0.1 to 5.0 mg / kg body weight.

  As used herein, “subject” means any animal, particularly a mammal, such as a human, and may be used interchangeably with the term “patient” where appropriate.

  In a third aspect, the present invention relates to a composition comprising a human antibody that immunoreacts with an epitope present on human TF.

  In a fourth aspect, the present invention is a method for treating a FVIIa / TF-related disorder, comprising administering to a human a therapeutically effective amount of a human antibody that immunoreacts with an epitope present on human TF. Relates to a method comprising:

  “Treatment” involves the administration of an effective amount of a therapeutically active compound of the present invention to prevent an onset symptom or disease state or to cure or alleviate an already onset symptom or disease state. Means that. Thus, the term “treatment” includes prophylactic treatment.

  As used herein, the term “FVIIa / TF related disorder” refers to a disease or disorder involving TF and FVIIa. Diseases or disorders associated with thrombosis or coagulation disorders such as chronic thromboembolic diseases or disorders associated with inflammatory response and fibrin formation such as vascular disorders such as deep vein thrombosis, arterial thrombosis, post-surgical thrombosis, Coronary artery bypass graft (CABG), percutaneous transluminal coronary angioplasty (PTCA), stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, arteriosclerosis and restenosis after angioplasty, chronic and For acute indications such as inflammation, septic shock, sepsis, hypotension, adult respiratory distress syndrome (ARDS), generalized intravascular coagulation syndrome (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or thrombosis This includes prophylactic treatment of mammals with atherosclerotic blood vessels at risk, and other diseases or disorders. FVIIa / TF related disorders are not limited to in vivo clotting disorders such as those mentioned above, but extracorporeal blood such as blood removed in-line from the patient in processes such as dialysis, blood filtration, or blood bypass during surgery. Includes ex vivo FVIIa / TF related processes such as coagulation that can occur by circulation.

The term “Factor VIIa” or “FVIIa” means “double-stranded” activated coagulation factor VII cleaved by specific cleavage at the peptide bond of Arg152-Ile153. FVIIa may be purified from blood or produced by recombinant means. The implementation of the method described herein is independent of how purified factor VIIa is derived, so that the present invention provides for the preparation of any factor VIIa suitable for use in the present invention. It is envisioned to encompass usage. Human FVIIa is preferred.
The term “FVII” means “single chain” coagulation factor VII.

In a further aspect, the present invention relates to a method for preparing a human antibody comprising the following a) to b):
a) preparing a human antibody against human TF;
b) antibodies are tested in a TF induced clot assay, in which IC ₅₀ values lower than 1 nM, eg IC ₅₀ values lower than 500 pM, preferably IC ₅₀ values lower than 200 pM, preferably lower than 100 pM ₅₀ values, preferably 50 less an IC ₅₀ value than pM, preferably lower an IC ₅₀ value than 10 pM, more preferably lower an IC ₅₀ value than 5 pM, selecting a human antibody which inhibit clot formation, or
The antibody is tested in an FXa production assay and an IC ₅₀ value lower than 100 nM (using an FVIIa concentration of 0.1 nM in the assay), eg an IC ₅₀ value lower than 10 nM, preferably an IC ₅₀ value lower than 5 nM, preferably Selecting a human antibody that inhibits FXa production with an IC ₅₀ value lower than 1 nM, more preferably an IC ₅₀ value lower than 0.1 nM, or
The antibodies are tested in an FVIIa / TF amidolytic assay and an IC ₅₀ value lower than 100 nM (for example using an FVIIa concentration of 10 nM in the assay), eg an IC ₅₀ value lower than 40 nM, preferably an IC ₅₀ value lower than 20 nM Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity, preferably with an IC ₅₀ value lower than 10 nM, or
Testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding, or testing antibodies in TF ELISA assays that include TF and selecting human antibodies that bind human TF.

It is understood that the specific IC ₅₀ values referred to in the TF induction clot assay are when normal human plasma is used.

In a further aspect, the present invention relates to a method for preparing a human antibody comprising the following a) to b):
a) preparing a human antibody against human TF;
tested antibodies in b) TF-induced clot assay in said assay, FFR-rFVIIa IC ₅₀ values +1 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +500 pM lower an IC ₅₀ value, preferably FFR the IC ₅₀ values of -RFVIIa +200 pM lower an IC ₅₀ value, preferably an IC ₅₀ value +100 lower an IC ₅₀ value than pM of FFR-rFVIIa, preferably less an IC ₅₀ value than an IC ₅₀ value +50 pM of FFR-rFVIIa, preferably FFR-rFVIIa IC ₅₀ values +10 lower an IC ₅₀ value than pM, more preferably lower an IC ₅₀ value than an IC ₅₀ value +5 pM of FFR-rFVIIa, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, Selecting a human antibody that inhibits clot formation, or
The antibodies were tested in an FXa production assay and (using 0.1 nM FVIIa) FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 100 nM, for example FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 10 nM, preferably FFR-rFVIIa IC ₅₀ values +5 nM lower than an IC ₅₀ value, preferably less an IC ₅₀ value than an IC ₅₀ value +1 nM of FFR-rFVIIa, IC ₅₀ and more preferably less than an IC ₅₀ value +0.1 nM of FFR-rFVIIa value, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, selecting a human antibody which inhibit FXa generation, or
Antibodies were tested in a FVIIa / TF amidolytic assay (using said 10 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values less than +40 nM the IC ₅₀ values, preferably FFR-rFVIIa IC ₅₀ values +20 nM lower than an IC ₅₀ value, preferably less an IC ₅₀ value than an IC ₅₀ value +10 nM of FFR-rFVIIa, more preferably less than an IC ₅₀ value of FFR-rFVIIa Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity with an IC ₅₀ value, or
Testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding, or testing antibodies in TF ELISA assays that include TF and selecting human antibodies that immunoreact with human TF.

It is understood that the specific IC ₅₀ values referred to in the TF induction clot assay are when normal human plasma is used.

  As used herein, the term “TF induced clot assay” is intended to mean any assay that measures clotting time in a sample containing plasma and TF. An example of a TF induction clot assay is described in Example 1, Assay 7.

  The term “FXa production assay” as used herein is intended to mean any assay that measures FX activation in a sample containing TF, FVIIa, FX, calcium and phospholipids. An example of an FXa production assay is described in Example 1, Assay 5.

  As used herein, the term “FVIIa / TF amidolytic assay” is intended to mean any assay that measures FVIIa amidolytic activity (ie, cleavage of small peptide substrates) in the presence of TF. An example of an FVIIa / TF amidolysis assay is described in Example 1, Assay 4.

  The term “TF ELISA assay” as used herein is intended to mean any ELISA assay involving TF and antibodies to TF. Examples of ELISA assays are the TF ELISA direct and indirect assays described in Example 1, Assays 1 and 2.

  As used herein, the term “TF ELISA direct assay” is intended to mean any TF ELISA assay that includes immobilized TF. An example of a TF ELISA direct assay is described in Example 1, Assay 1.

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

In a further aspect, the present invention is a human antibody that immunoreacts with an epitope present on human TF and inhibits human clotting factor VIIa binding to human TF, comprising the following a) to b): Regarding human antibodies obtained by the method:
a) preparing a human antibody against human TF;
b) antibodies are tested in a TF induced clot assay, in which IC ₅₀ values lower than 1 nM, eg IC ₅₀ values lower than 500 pM, preferably IC ₅₀ values lower than 200 pM, preferably lower than 100 pM ₅₀ values, preferably 50 less an IC ₅₀ value than pM, preferably lower an IC ₅₀ value than 10 pM, more preferably lower an IC ₅₀ value than 5 pM, selecting a human antibody which inhibit clot formation, or
The antibody is tested in an FXa production assay and an IC ₅₀ value lower than 100 nM (using an FVIIa concentration of 0.1 nM in the assay), eg an IC ₅₀ value lower than 10 nM, preferably an IC ₅₀ value lower than 5 nM, preferably Selecting a human antibody that inhibits FXa production with an IC ₅₀ value lower than 1 nM, more preferably an IC ₅₀ value lower than 0.1 nM, or
The antibodies are tested in an FVIIa / TF amidolytic assay and an IC ₅₀ value lower than 100 nM (for example using an FVIIa concentration of 10 nM in the assay), eg an IC ₅₀ value lower than 40 nM, preferably an IC ₅₀ value lower than 20 nM Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity, preferably with an IC ₅₀ value lower than 10 nM, or
Testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding, or testing antibodies in TF ELISA assays that include TF and selecting human antibodies that bind human TF.

It is understood that the specific IC ₅₀ values referred to in the TF induction clot assay are when normal human plasma is used.

In a further aspect, the present invention is a human antibody that immunoreacts with an epitope present on human TF and inhibits human clotting factor VIIa binding to human TF, comprising the following a) to b): Regarding human antibodies obtained by the method:
a) preparing a human antibody against human TF;
tested antibodies in b) TF-induced clot assay in said assay, FFR-rFVIIa IC ₅₀ values +1 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +500 pM lower an IC ₅₀ value, preferably FFR the IC ₅₀ values of -rFVIIa +200 pM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +100 pM lower an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +50 lower an IC ₅₀ value than pM, preferably FFR the IC ₅₀ values +10 lower an IC ₅₀ value than pM of -RFVIIa, more preferably less an IC ₅₀ value than an IC ₅₀ value +5 pM of FFR-rFVIIa, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, clot Selecting a human antibody that inhibits formation, or
The antibodies were tested in the FXa production assay (using 0.1 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 100 nM, eg FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 10 nM , preferably an IC ₅₀ value +5 lower an IC ₅₀ value than nM of FFR-rFVIIa, more preferably less an IC ₅₀ value than an IC ₅₀ value +1 nM of FFR-rFVIIa, more preferably an IC ₅₀ value of FFR-rFVIIa +0.1 nM low an IC ₅₀ value, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, selecting a human antibody which inhibit FXa generation, or
Tested antibodies in FVIIa / TF amidolytic assay (using 10 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, an IC ₅₀ value of FFR-rFVIIa lower than +40 nM the IC ₅₀ values, preferably FFR-rFVIIa IC ₅₀ values +20 nM lower than an IC ₅₀ value, preferably less an IC ₅₀ value than an IC ₅₀ value +10 nM of FFR-rFVIIa, more preferably less than an IC ₅₀ value of FFR-rFVIIa Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity with an IC ₅₀ value, or
Testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding, or testing antibodies in TF ELISA assays that include TF and selecting human antibodies that immunoreact with human TF.

It is understood that the specific IC ₅₀ values referred to in the TF induction clot assay are when normal human plasma is used.

  In one embodiment of the invention, a method of producing a human antibody against human TF comprises immunizing a mammal with human TF and isolating the antibody produced by the immunized mammal. In a preferred embodiment, the mammal is a mouse. It will be appreciated that the immunized mammal or mouse can produce human antibodies.

In a further aspect, the present invention relates to a method for preparing a human antibody comprising the following a) to j):
a) immunizing mice with human TF;
b) isolating antibody-producing cells from the immunized mouse and preparing immortalized cells that secrete human antibodies;
c) isolating a medium derived from immortalized cells containing the produced antibody;
d) testing the antibodies in a TF ELISA indirect assay comprising TF in dissolved state and selecting human antibodies that bind human TF in dissolved state;
e) testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding;
f) antibodies are tested in the FVIIa / TF amidolysis assay, using an IC ₅₀ value lower than 100 nM (for example using an FVIIa concentration of 10 nM in the assay), eg an IC ₅₀ value lower than 40 nM, preferably lower than 20 nM Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity with a ₅₀ value, more preferably with an IC ₅₀ value lower than 10 nM,
g) testing antibodies in FXa generation assay (using FVIIa concentration of 0.1 nM in the assay) lower an IC ₅₀ value than 100 nM, for example less than 10 nM an IC ₅₀ value, preferably less than 5 nM an IC ₅₀ value, preferably less an IC ₅₀ value than 1 nM, more preferably lower an IC ₅₀ value than 0.1 nM, selecting a human antibody which inhibit FXa generation,
h) antibodies are tested in a TF-induced clot assay, in which an IC ₅₀ value lower than 1 nM, such as an IC ₅₀ value lower than 500 pM, preferably an IC ₅₀ value lower than 200 pM, preferably an IC lower than 100 pM ₅₀ values, preferably 50 less an IC ₅₀ value than pM, preferably lower an IC ₅₀ value than 10 pM, more preferably lower an IC ₅₀ value than 5 pM, selecting a human antibody which inhibit clot formation,
i) after steps dh, selecting immortalized cells producing the selected antibody and culturing in an appropriate medium;
j) isolating the selected antibody from the medium of the selected immortalized cells.

It is understood that the specific IC ₅₀ values referred to in the TF induction clot assay are when normal human plasma is used.

In a further aspect, the present invention relates to a method for preparing a human antibody comprising the following a) to j):
a) immunizing mice with human TF;
b) isolating antibody-producing cells from the immunized mouse and preparing immortalized cells that secrete human antibodies;
c) isolating a medium derived from immortalized cells containing the produced antibody;
d) testing antibodies in a TF ELISA indirect assay comprising TF in dissolved state and selecting human antibodies that immunoreact with human TF in dissolved state;
e) testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding;
tested antibodies in f) FVIIa / TF amidolytic assay (the using 10 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +40 nM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +20 nM lower than an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +10 nM lower than an IC ₅₀ value, more preferably an IC ₅₀ value of FFR-rFVIIa Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity with a lower IC ₅₀ value;
tested antibodies in g) FXa generation assay, less than (the using 0.1 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa an IC ₅₀ value +10 nM of IC ₅₀ values, preferably an IC ₅₀ value +5 lower an IC ₅₀ value than nM of FFR-rFVIIa, preferably less an IC ₅₀ value than an IC ₅₀ value +1 nM of FFR-rFVIIa, more preferably an IC ₅₀ value of FFR-rFVIIa +0.1 less than nM an IC ₅₀ value, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, selecting a human antibody which inhibit FXa generation,
tested antibodies in h) TF-induced clot assay in said assay, FFR-rFVIIa IC ₅₀ values +1 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +500 pM lower an IC ₅₀ value, preferably FFR the IC ₅₀ values of -rFVIIa +200 pM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +100 pM lower an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +50 lower an IC ₅₀ value than pM, preferably FFR the IC ₅₀ values +10 lower an IC ₅₀ value than pM of -RFVIIa, more preferably less an IC ₅₀ value than an IC ₅₀ value +5 pM of FFR-rFVIIa, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, clot Selecting a human antibody that inhibits formation,
i) after steps dh, selecting immortalized cells producing the selected antibody and culturing in an appropriate medium;
j) isolating the selected antibody from the medium of the selected immortalized cells.

It is understood that the specific IC ₅₀ values referred to in the TF induction clot assay are when normal human plasma is used.

  The term “antibody producing cell” as used herein means any cell capable of producing an antibody. Included are hybridomas, transfected cell lines, and relatively short-lived or mortal splenocytes or lymphocytes derived from mammals injected with antigen.

In a further aspect, the present invention is a human antibody that immunoreacts with an epitope present on human TF and inhibits human clotting factor VIIa binding to human TF, comprising the following a) to j): Regarding human antibodies obtained by the method:
a) immunizing mice with human TF;
b) isolating antibody-producing cells from the immunized mouse and preparing immortalized cells that secrete human antibodies;
c) isolating a medium derived from immortalized cells containing the produced antibody;
d) testing the antibodies in a TF ELISA indirect assay comprising TF in dissolved state and selecting human antibodies that bind human TF in dissolved state;
e) testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding;
f) antibodies are tested in the FVIIa / TF amidolysis assay, using an IC ₅₀ value lower than 100 nM (for example using an FVIIa concentration of 10 nM in the assay), eg an IC ₅₀ value lower than 40 nM, preferably lower than 20 nM Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity with a ₅₀ value, more preferably with an IC ₅₀ value lower than 10 nM,
g) testing antibodies in FXa generation assay (using FVIIa concentration of 0.1 nM in the assay) lower an IC ₅₀ value than 100 nM, for example less than 10 nM an IC ₅₀ value, preferably less than 5 nM an IC ₅₀ value, preferably less an IC ₅₀ value than 1 nM, more preferably lower an IC ₅₀ value than 0.1 nM, selecting a human antibody which inhibit FXa generation,
h) antibodies are tested in a TF-induced clot assay, in which an IC ₅₀ value lower than 1 nM, such as an IC ₅₀ value lower than 500 pM, preferably an IC ₅₀ value lower than 200 pM, preferably an IC lower than 100 pM ₅₀ values, preferably 50 less an IC ₅₀ value than pM, preferably lower an IC ₅₀ value than 10 pM, more preferably lower an IC ₅₀ value than 5 pM, selecting a human antibody which inhibit clot formation,
i) after steps dh, selecting immortalized cells producing the selected antibody and culturing in an appropriate medium;
j) isolating the selected antibody from the medium of the selected immortalized cells.

It is understood that the specific IC ₅₀ values referred to in the TF induction clot assay are when normal human plasma is used.

In a further aspect, the present invention is a human antibody that immunoreacts with an epitope present on human TF and inhibits human clotting factor VIIa binding to human TF, comprising the following a) to j): Regarding human antibodies obtained by the method:
a) immunizing mice with human TF;
b) isolating antibody-producing cells from the immunized mouse and preparing immortalized cells that secrete human antibodies;
c) isolating a medium derived from immortalized cells containing the produced antibody;
d) testing antibodies in a TF ELISA indirect assay comprising TF in dissolved state and selecting human antibodies that immunoreact with human TF in dissolved state;
e) testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding;
tested antibodies in f) FVIIa / TF amidolytic assay (the using 10 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +40 nM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +20 nM lower than an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +10 nM lower than an IC ₅₀ value, more preferably an IC ₅₀ value of FFR-rFVIIa Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity with a lower IC ₅₀ value;
tested antibodies in g) FXa generation assay, less than (the using 0.1 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa an IC ₅₀ value +10 nM of IC ₅₀ values, preferably an IC ₅₀ value +5 lower an IC ₅₀ value than nM of FFR-rFVIIa, preferably less an IC ₅₀ value than an IC ₅₀ value +1 nM of FFR-rFVIIa, more preferably an IC ₅₀ value of FFR-rFVIIa +0.1 less than nM an IC ₅₀ value, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, selecting a human antibody which inhibit FXa generation,
tested antibodies in h) TF-induced clot assay in said assay, FFR-rFVIIa IC ₅₀ values +1 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +500 pM lower an IC ₅₀ value, preferably FFR the IC ₅₀ values of -rFVIIa +200 pM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +100 pM lower an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +50 lower an IC ₅₀ value than pM, preferably FFR the IC ₅₀ values +10 lower an IC ₅₀ value than pM of -RFVIIa, more preferably less an IC ₅₀ value than an IC ₅₀ value +5 pM of FFR-rFVIIa, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, clot Selecting a human antibody that inhibits formation,
i) after steps dh, selecting immortalized cells producing the selected antibody and culturing in an appropriate medium;
j) isolating the selected antibody from the medium of the selected immortalized cells.

It is understood that the specific IC ₅₀ values referred to in the TF induction clot assay are when normal human plasma is used.

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

  In a further aspect, the present invention relates to a cell producing a human antibody that immunoreacts with an epitope present on human TF and inhibits human clotting factor VIIa binding to human TF.

In one embodiment, the cell is an isolated lymphoid cell.
In a further embodiment, the cells are isolated from the mouse.

  In a further embodiment, the cell is a hybridoma cell. In one embodiment, hybridoma cells are obtained by fusing antibody-producing lymphoid cells and immortalized cells to provide antibody-producing hybridoma cells.

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

  The term “monoclonal antibody” as used herein refers to a homogeneous population of immunoglobulins, ie, the individual molecules of an antibody population are identical except for naturally occurring mutations. Antibodies are usually synthesized by lymphoid cells derived from bone marrow B lymphocytes. Lymphocytes from the same clone produce an immunoglobulin of a single amino acid sequence. Lymphocytes cannot be cultured directly over a long period of time to produce significant amounts of specific antibodies. However, according to Kohler et al., 1975, Nature, 256: 495, the somatic cell fusion process (specifically, the fusion process between lymphocytes and myeloma cells) yields hybridoma cells that are grown in culture. However, it has been demonstrated to produce specific antibodies referred to as “monoclonal antibodies”. Myeloma cells are lymphocyte tumor cells and are known to be “non-producing” strains or produce antibodies on their own depending on the cell line.

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

  The term “recombinant antibody” as used herein includes all human antibodies prepared, expressed, produced or isolated by recombinant means, eg recombinantly transfected into a host cell. Antibodies expressed using expression vectors (further described in Section II below), antibodies isolated from recombinant combinatorial human antibody libraries (further described in Section III below), transgenic for human immunoglobulin genes With splicing of antibodies isolated from animals (eg mice) (eg Taylor, LD, et al. (1992) Nucl. Acids Res. 20: 6287-6295) or other human immunoglobulin gene sequences to other DNA sequences Antibodies prepared, expressed, generated or isolated by any other means are included. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies undergo in vitro mutagenesis, whereby the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from human germline VH and VL sequences. Is a sequence that is not naturally occurring in the germline repertoire of human antibodies 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 has a K _d for binding to human TF in the range of 10 ⁻¹⁵ to 10 ⁻⁸ M. It is understood that the K _d for binding of the mentioned human antibody to human TF is measured in an assay that immobilizes the human antibody (see Assay 6).

In a further embodiment of the invention, the isolated human antibody has a K _d for binding to human TF in the range of 10 ⁻¹⁵ to 10 ⁻¹⁰ M.

In a further embodiment of the invention, the isolated human antibody has a K _d for binding to human TF of less than 10 ⁻⁸ M. In a further embodiment of the invention, the K _d for binding to human TF is lower than 10 ⁻⁹ M. In a further embodiment of the invention, the K _d for binding to human TF is lower than 10 ⁻¹⁰ M. In a further embodiment of the invention, the K _d for binding to human TF is lower than 10 ⁻¹¹ M. In a further embodiment of the invention, the K _d for binding to human TF is lower than 10 ⁻¹² M. In a further embodiment of the invention, the K _d for binding to human TF is lower than 10 ⁻¹³ M. In a further embodiment of the invention, the K _d for binding to human TF is lower than 10 ⁻¹⁴ M. In a further embodiment of the invention, the K _d for binding to human TF is lower than 10 ⁻¹⁵ M.

In a further embodiment of the invention, the method of preparing a human antibody comprises testing the antibody in a TF induced clot assay, wherein selecting a human antibody that inhibits clot formation with an IC ₅₀ value of less than 1 nM. Including. In a further embodiment, the IC ₅₀ value is lower than 500 pM. In a further embodiment, the IC ₅₀ value is lower than 200 pM. In a further embodiment, the IC ₅₀ value is less than 100 pM. In a further embodiment, the IC ₅₀ value is less than 50 pM. In a further embodiment, the IC ₅₀ value is less than 10 pM. In a further embodiment, the IC ₅₀ value is lower than 5 pM.

In a further aspect of the present invention, a method for preparing human antibodies are tested antibodies in the TF-induced clot assay, in this assay, FFR-rFVIIa IC ₅₀ values +1 nM lower than an IC ₅₀ value, for example of FFR-rFVIIa the IC ₅₀ values +500 pM lower an IC ₅₀ value, preferably an IC ₅₀ value +200 pM lower an IC ₅₀ value of FFR-rFVIIa, preferably an IC ₅₀ value +100 lower an IC ₅₀ value than pM of FFR-rFVIIa, for example of FFR-rFVIIa the IC ₅₀ values +50 lower an IC ₅₀ value than pM, preferably FFR-rFVIIa IC ₅₀ values +10 lower an IC ₅₀ value than pM, more preferably FFR-rFVIIa IC ₅₀ values +5 lower an IC ₅₀ value than pM, more preferably FFR in low an IC ₅₀ value than an IC ₅₀ value of -RFVIIa, comprising selecting the human antibody which inhibit clot formation.

It is understood that the specific IC ₅₀ values referred to in the TF induction clot assay are when normal human plasma is used.

In a further embodiment of the invention, the method of preparing a human antibody tests the antibody in an FXa production assay and inhibits FXa production with an IC ₅₀ value lower than 100 nM (using a 0.1 nM FVIIa concentration in the assay). Selecting a human antibody. In a further embodiment, the IC ₅₀ value is less than 10 nM. In a further embodiment, the IC ₅₀ value is less than 5 nM. In a further embodiment, the IC ₅₀ value is less than 1 nM. In a further embodiment, the IC ₅₀ value is less than 0.1 nM.

In a further embodiment of the invention, the method of preparing a human antibody tests the antibody in an FXa production assay and uses an IC ₅₀ value of FFR-rFVIIa + IC ₅₀ value lower than 100 nM (using 0.1 nM FVIIa in the assay). , for example, FFR-rFVIIa IC ₅₀ values +10 nM lower than an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +5 nM lower than an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +1 nM lower than an IC ₅₀ value , more preferably FFR-rFVIIa IC ₅₀ values +0.1 lower an IC ₅₀ value than nM, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, selecting a human antibody which inhibit FXa generation Including.

In a further embodiment of the invention, the method of preparing a human antibody tests the antibody in an FVIIa / TF amidolysis assay and induces TF with an IC ₅₀ value lower than 100 nM (using a 10 nM FVIIa concentration in the assay). Selecting a human antibody that inhibits FVIIa amidolytic activity. In a further embodiment, the IC ₅₀ value is less than 40 nM. In a further embodiment, the IC ₅₀ value is less than 20 nM. In a further embodiment, the IC ₅₀ value is less than 10 nM. In a further embodiment, the IC ₅₀ value is less than 5 nM.

In a further embodiment of the invention, the method of preparing a human antibody tests the antibody in an FVIIa / TF amidolysis assay and uses an IC ₅₀ value of FFR-rFVIIa +100 nM (using 10 nM FVIIa in the assay). the IC ₅₀ values, for example, FFR-rFVIIa IC ₅₀ values +40 lower an IC ₅₀ value than nM, preferably FFR-rFVIIa IC ₅₀ values +20 nM lower than an IC ₅₀ value, preferably less than an IC ₅₀ value +10 nM of FFR-rFVIIa the IC ₅₀ values, and more preferably at lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, comprising selecting the human antibody which inhibit TF-induced FVIIa amidolytic activity.

  In a further embodiment of the invention, the method of preparing a human antibody comprises testing the antibody in an FVIIa competition assay and selecting a human antibody that competes with FVIIa binding.

  In a further embodiment of the invention, the method of preparing a human antibody comprises testing the antibody in a TF ELISA assay comprising TF and selecting a human antibody that binds human TF.

  In a further aspect of the invention, the method of preparing a human antibody comprises testing the antibody in a TF ELISA direct assay comprising immobilized TF and selecting a human antibody that binds immobilized human TF.

  In a further aspect of the invention, the method of preparing a human antibody comprises testing the antibody in a TF ELISA indirect assay comprising TF in a dissolved state and selecting a human antibody that binds human TF in a dissolved state.

In a further embodiment of the invention, a method of preparing a human antibody tests an antibody in an FXa production assay on TF expressing cells, with an IC ₅₀ value lower than 500 nM (using a 1 nM FVIIa concentration in the assay). Selecting a human antibody that inhibits FXa production on TF expressing cells. In a further embodiment, the IC ₅₀ value is less than 100 nM. In a further embodiment, the IC ₅₀ value is less than 50 nM. In a further embodiment, the IC ₅₀ value is less than 10 nM. In a further embodiment, the IC ₅₀ value is less than 5 nM.

In a further embodiment of the invention, the method of preparing a human antibody comprises testing an antibody in an FXa production assay on TF expressing cells and using an IC ₅₀ value of FFR-rFVIIa + 500 nM (using 1 nM FVIIa in the assay). lower an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +50 nM lower than an IC ₅₀ value, preferably ₅₀ values +10 nM IC of FFR-rFVIIa lower an IC ₅₀ value, more preferably less an IC ₅₀ value than an IC ₅₀ value +5 nM of FFR-rFVIIa, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, inhibits FXa generation on TF expressing cells Selecting a human antibody to be treated.

  "TF expressing cell" means any mammalian cell that expresses human TF.

  In a further embodiment of the invention, the method of preparing a human antibody tests the antibody in a complete cell TF binding assay and selects a human antibody that competes with FVIIa that binds to human TF expressed on the cell surface of the complete cell. Including that.

In a further embodiment of the invention, the method of preparing a human antibody comprises testing the antibody in a biosensor assay and selecting a human antibody having a K _d value for binding to human TF of less than 100 nM. In a further embodiment, the K _d value for binding to human TF is less than 10 nM. In a further embodiment, the K _d value for binding to human TF is lower than 5 nM. In a further embodiment, the K _d value for binding to human TF is less than 1 nM. In a further embodiment, the K _d value for binding to human TF is less than 0.5 nM. In a further embodiment, the K _d value for binding to human TF is lower than 10 ⁻¹⁰ M. In a further embodiment, the K _d value for binding to human TF is lower than 10 ⁻¹¹ M. In a further embodiment, the K _d value for binding to human TF is lower than 10 ⁻¹² M. In a further embodiment, the K _d value for binding to human TF is lower than 10 ⁻¹³ M.
In a further embodiment, the K _d value for binding to human TF is lower than 10 ⁻¹⁴ M. In a further embodiment, the K _d value for binding to human TF is lower than 10 ⁻¹⁵ M.

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

  The term “MAPK signaling” is intended to mean a cascade of intracellular events that mediate activation of mitogen-activated protein kinase (MAPK) or its homologues in response to various extracellular stimuli. Three different groups of MAP kinases have been identified in mammalian cells: 1) extracellular regulated kinases (Erk1 / 2 or p44 / 42), 2) c-Jun N-terminal kinase (JNK), and 3) p38 Kinase. The Erk1 / 2 pathway involves phosphorylation of Erk1 (p44) and / or Erk2 (p42). Activated MAP kinases, such as p44 / 42 MAPK, are translocated to the nucleus where they phosphorylate transcription factors such as (Elk1) and signal transducer and activator of transcription (Stat). Can be activated. Erk1 / 2 can also phosphorylate the kinase p90RSK in the cytoplasm or nucleus, and p90RSK can then activate several transcription factors.

  The term “protein kinase” refers to an enzyme capable of phosphorylating serine and / or threonine and / or tyrosine with peptides and / or proteins.

  The term “FVIIa-induced activation of MAPK signaling” indicates that FVIIa binds to TF in mammalian cells, thereby inducing MAPK signaling.

  In a further embodiment of the invention, the method of preparing a human antibody comprises testing for an antibody in a gene expression analysis assay (Assay 15) and a gene selected independently from a list comprising Fra-1, Id2, and Cyr61. Selecting a human antibody that inhibits FVIIa-induced upregulation.

  It is understood that an antibody against TF that inhibits the activity of TF binds various epitopes present on TF and can inhibit both FVIIa binding to human TF and FX or FXa binding. The object of the present invention is to select antibodies that inhibit the binding of FVIIa to human TF, thereby inhibiting FVIIa-induced intracellular signaling.

  In a further embodiment of the invention, the method of preparing a human antibody comprises testing the antibody in a human cancer assay (Assay 13) and selecting a human antibody that inhibits human cancer growth or metastasis.

  In a further embodiment of the invention, the isolated human antibody inhibits FVIIa-induced upregulation of a gene selected independently from the list comprising Fra-1, Id2, and Cyr61.

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

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

  In a further aspect of the invention, the method of preparing a human antibody comprises testing the antibody in an epitope mapping assay and selecting a human antibody that binds a preferred epitope on TF. In one embodiment, preferred epitopes include one or more of Trp45, Lys46 and Tyr94 residues. In one embodiment, preferred epitopes include Trp45 residues. In one embodiment, preferred epitopes include Lys46 residues. In one embodiment, preferred epitopes include Tyr94 residues.

  In a further embodiment of the invention, the isolated human antibody binds to an epitope within the interface of TF and FVIIa.

  The residues in TF responsible for the interaction between the protease domain of FVIIa and TF determined from the X-ray structure (Banner et al. 1996 Nature, 380: 41-46) are Ser39, Gly43, Trp45, Ser47, Phe50, Arg74, Phe76, Tyr94, Pro92. The interface between the protease domain of FVIIa and TF allows for many fine contacts between TF and FVIIa, and a complex containing many intermolecular hydrogen bonds that acquire high specificity in the FVIIa binding process. Characterized as boundary area.

  The present invention also relates to a high affinity human monoclonal antibody against TF. The TF surface containing the contact interface of the protease domain of FVIIa retains a specific topology that is susceptible to react to create protein-protein interactions, where another type of protein-protein interaction is an antibody And protein ligand complex formation. Thus, monoclonal antibodies against this epitope on human TF provide high affinity Mabs.

  One aspect of the invention is a high affinity human monoclonal antibody that immunoreacts with the contact interface of the protease domain of FVIIa.

  The human TF antibody of the present invention acts as an antagonist of TF-mediated coagulation induction and inhibits the binding of coagulation FVIIa to TF, thereby blocking thrombin production and subsequent fibrin deposition. Human TF antibodies are particularly effective for administration to humans to treat various conditions associated with intravascular coagulation. As such, human TF antibodies may be effective in inhibiting TF activity, for example leading to inhibition of blood clotting, thrombosis or platelet deposition. Furthermore, the human TF antibody of the present invention acts to inhibit the signaling function of TF, which is a cellular function of TF, and may be effective for symptoms such as sepsis, inflammation, atherosclerosis, restenosis or cancer. .

  Human TF antibodies may be effective for various diseases. Diseases or disorders associated with thrombosis or coagulation disorders such as chronic thromboembolic diseases or disorders associated with inflammatory response and fibrin formation such as vascular disorders such as deep vein thrombosis, arterial thrombosis, post-surgical thrombosis, Coronary artery bypass graft (CABG), percutaneous transluminal coronary angioplasty (PTCA), stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, arteriosclerosis and restenosis after angioplasty, chronic and Acute indications such as inflammation, septic shock, sepsis, hypotension, adult respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), generalized intravascular coagulation syndrome (DIC), pulmonary embolism, disease Preventive treatment of mammals with atherosclerotic blood vessels at risk of experimental platelet deposition, myocardial infarction, or thrombosis, venous occlusion disease after peripheral blood progenitor cell (PBPC) transplantation, hemolytic uremic disease Syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), and includes other diseases or disorders. Human TF antibodies can be used to prevent the development of thromboembolic complications in patients identified as high risk, such as patients undergoing surgery or patients with congestive heart failure. Human TF antibodies can be particularly effective in treating intimal hyperplasia or restenosis due to acute vascular injury. Acute vascular injury is one that develops rapidly (ie, over a period of hours to months), as opposed to chronic vascular injury (atherosclerosis) that occurs throughout life. Acute vascular injury is often caused by surgical procedures such as vascular reconstruction where techniques such as angioplasty, arterial endarterectomy, atherectomy, and vascular graft placement are employed. Hyperplasia can also occur as a delayed reaction in response to, for example, graft placement or organ transplantation. Since human TF antibody is more selective than heparin and binds only TF exposed at the site of injury, and human TF antibody does not destroy or inhibit other coagulation proteins, When used prophylactically to prevent it from occurring, it is more effective than heparin and tends to cause less bleeding complications.

  As shown in the Examples below, the human TF antibody of the present invention can selectively bind to cell surface TF and inhibit its functional activity by inhibiting the binding of coagulation FVIIa to TF. Human TF antibodies that maintain binding to TF block the accumulation of platelets at the site of vascular injury by blocking thrombin production and subsequently blocking fibrin deposition.

  Human TF antibodies that inhibit FVIIa binding by maintaining binding to TF because human TF antibodies have the ability to block thrombin generation and limit platelet deposition at the site of acute vascular injury Can be used to inhibit vascular restenosis.

  Compositions containing human TF antibodies are particularly effective in methods of treating patients when formulated into pharmaceutical compositions, where the compositions are administered to individuals suffering from various disease states and coagulated. Related symptoms can be treated. Such human TF antibodies can bind TF and inhibit FVIIa binding to TF, have a longer plasma half-life compared to other anticoagulants, and correspondingly longer durations. Can exhibit anticoagulant activity. The medical indications for this composition are those commonly treated with anticoagulants such as deep vein thrombosis, pulmonary embolism, stroke, generalized intravascular coagulation syndrome (DIC), pulmonary associated with sepsis And fibrin deposition in the kidneys, antiphospholipid syndrome (APS), atherosclerosis and myocardial infarction. The composition may be used for mechanical vascular injury, eg, surgery, microsurgery, balloon angioplasty, arterial endarterectomy, reducing atherectomy, stent placement, laser treatment or rotablation. Can be used to inhibit vascular restenosis that occurs after injury caused by or secondary to vascular grafts, stents, bypass grafts, or organ transplants. Thus, the composition can be used to inhibit platelet deposition and related disorders. Thus, a method of inhibiting clotting, vascular restenosis or platelet deposition includes, for example, a composition comprising human TF antibody in an amount sufficient to effectively inhibit clotting, vascular restenosis or platelet deposition. Administration of the product to the patient. The method also finds use in treating acute coronary artery closure (eg, acute myocardial infarction) in an individual, which comprises administering human TF antibody together with tissue plasminogen activator or streptokinase. Can promote tPA-induced thrombolysis. Human TF antibody is administered before, with or immediately after administration of a thrombolytic agent, such as tissue plasminogen activator.

  According to the present invention, human monoclonal antibodies against human TF can be generated by immunizing a transgenic mouse (obtained from Medarex) that has part of the human immune mechanism rather than the mouse mechanism with human TF. . Spleen cells derived from these transgenic mice are used to produce hybridomas that secrete the above-described human monoclonal antibodies (eg, Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT Publication WO 91/10741; Lonberg et al. International application WO 92/03918; Kay et al. International application 92/03917; Lonberg, N. et al. 1994 Nature 368: 856-859; Green, LL et al. 1994 Nature Genet. 7: 13- 21; Morrison, SL et al. 1994 Proc. Natl. Acad. Sci. USA 81: 6851-6855; Bruggeman et al. 1993 Year Immunol 7: 33-40; Tuaillon et al. 1993 PNAS 90: 3720-3724; Bruggeman et al. 1991 Eur J Immunol 21: 1323-1326).

  A human monoclonal antibody against human TF can be prepared by phage display. Human antibody libraries can be constructed from immunized humans and displayed on the surface of filamentous phage. High affinity human single chain Fv (ScFv) and Fab antibody fragments can often be obtained using panning techniques in which the antigen of interest is immobilized on a solid surface (ie, a microtiter plate or bead). It has been isolated from a library (Barbas CF, III and Burton, DR Trends. Biotechnol. 1996, 14: 230-234; Aujame L. et al, Hum. Antibodies 1997, 8: 155-68). In addition, phage display of large and naive libraries has made it possible to directly isolate human antibodies without immunization (DeHaard H. J. et al J. Biol. Chem. 1999, 18218-18230).

  The invention is explained in more detail in the examples with reference to the accompanying drawings.

  The invention is further illustrated by the following examples, which should not be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the examples described below can be material for understanding the invention in its various forms, either separately or in any combination.

Example 1
Preparation of human Mab immunospecific for human TF
The reagent human TF can be isolated from human brain as described in Rao, LVM, Thrombosis Research, 51: 373-384 1988.

  Lipidated 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 with a manual glass homogenizer. After incubating at 45 ° C. for 30 minutes with occasional shaking, the sample is centrifuged at 2000 × g for 20 minutes. Discard the precipitate and aliquot the supernatant and store at -80 ° C until use.

  Relipidated TF can be obtained by reconstitution of full length recombinant human TF (American Diagnostica # 4500) into phospholipid vesicles (PC / PS 75/25).

Biotinylated human TF is made as follows: Biotin-NHS (n-succinimid biotin, Sigma H-1759) is dissolved in DMF (dimethylformamide) at a concentration of 1.7 mg / mL. 1 mg / mL human TF in 0.1 M NaHCO ₃ buffer is added to 60 μL of biotin-NHS solution and allowed to react for 4 hours at room temperature. The reaction solution containing biotinylated TF is dialyzed overnight against PBS buffer.

The FVlla used is recombinant human FVIIa prepared as described in Thim, L. et al. Biochem 27: 7785-7793, 1988.
sTF: soluble recombinant human TF expressed in E. coli and essentially purified as described by Freskgard, P. -O. et al. Protein Sci. 5, 1531-1540 (1996).
S2288: Reconstituted to 17.24 mg / mL in H ₂ 0 (Chromogenix).
FX: Purified human plasma FX (HFX, Enzyme Research Laboratories Ltd.).
FXa: Purified human plasma FX (HFXa, Enzyme Research Laboratories Ltd.) activated with Russel's Viper Venom.
Chromozym (Chromozyme) X: dissolved in 1.25 mg / mL in H ₂ 0 (Boehringer Mannheim)
¹²⁵ I-FVIIa is obtained by standard radioisotope labeling methods.
FFR-rFVIIa: FVIIa blocked in the active site with D-Phe-L-Phe-L-Arg-chloromethyl ketone. Prepared as described in Sorensen BB et al. J. Biol. Chem. 272: 11863-11868, 1997.

Immunized human TF is emulsified in Freund's complete adjuvant. HuMab mice or their hybrids (Medarex) are administered 40 μg by subcutaneous injection. After 14 and 28 days, the mice are boosted with similar injections of 20 μg TF in Freund's incomplete adjuvant multiple times at 14-day intervals thereafter. Ten days after the last injection, a blood sample is taken and the serum is tested for human TF specific antibodies by TF ELISA (Assays 1 and 2).

Fusion assay 1-3 positive serum test mice are boosted with 20 μg human TF by intravenous infusion and sacrificed 3 days later. The spleen is removed aseptically and dispersed in a single cell suspension.

  Fox myeloma cells are grown in CD hybridoma medium (Gibco 11279-023).

  For fusion, spleen cells and myeloma cells (P3x63 Ag8.653, ATCC CRL-1580) and Sp2 / 0 (ATCC CRL-1581) myeloma cell line are fused by the PEG method ( Kohler, G & Milstein C. (1976), European J. Immunology, 6: 511-19). Cells are seeded in microtiter plates and incubated at 37 ° C. The medium is changed 3 times over the next 2 weeks. 100 μL of supernatant from hybridoma cells is removed from each well and tested for TF-specific antibodies in a TF ELISA (Assay 1 and 2).

Example 2
The various assays used in the screening of serum and culture supernatants for selected antibodies specific for screening are as follows:

Direct TF ELISA assay (Assay 1):
Nunc immunoplates are coated overnight at 4 ° C. with 1 μg / mL human sTF in PBS. The plate is blocked with blocking buffer (TBS containing 5 mM CaCl ₂ and 2% BSA), washed with TBS + 0.05% Tween 20, and the supernatant from the hybridoma cells is added. After 1 hour incubation at room temperature, the plate is washed and anti-human IgG labeled with horseradish peroxidase (HRPO) is added. After an additional 1 hour incubation, the plates are washed and developed with TMB substrate (Kem-EN-Tec) as described by the manufacturer. Absorbance at 450 nm is measured with an ELISA reader.

Indirect TF ELISA assay (Assay 2):
Nunc immunoplates are coated with 0.5 μg / mL goat anti-human IgG (Southern Biotechnology Associates, Cat- # 2040-1) in PBS and incubated overnight at 4 ° C. The plate is blocked with blocking buffer (TBS containing 5 mM CaCl ₂ and 2% BSA) and washed with TBS + 5 mM CaCl ₂ + 0.05% Tween 20. Culture supernatant from hybridoma cells is added and the plate is incubated for 1 hour at room temperature. After further washing, biotinylated human sTF is added at a concentration of 1 μg / mL and incubated for 1 hour. After washing, 100 μL of streptavidin-HRPO solution is added and incubated for 1 hour. Plates are developed with TMB substrate as described in Assay 1.

FVIIa competition assay (Assay 3):
Nunc immunoplates are incubated overnight at 4 ° C. with human sTF (concentration 5 μg / mL in PBS). Plates were washed and blocked with TBS buffer containing 5 mM CaCl ₂ and 2% BSA. Anti-human TF Mab is added and the plate is incubated for 2 hours. Plates were washed and then added biotinylated human FVIIa (5 mM CaC1 TBS buffer at 1 [mu] g / mL containing ₂ and 2% BSA), incubated for 1 hour plate. The plate is washed and then HRPO labeled streptavidin is added and incubated for 45 minutes. The plate is washed again and then developed with TMB substrate (Kem-EN-Tec) as described by the manufacturer.

Inhibition of FVIIa / sTF amidolytic activity (Assay 4):
FVIIa-TF catalyzed amidolytic activity is inhibited by anti-human TF Mab, soluble human TF (10 nM), recombinant human FVIIa (10 nM) and increasing concentrations of Mab (0.0122-50 nM) Test with. RT anti-human TF Mab or FFR-rFVIIa in varying concentrations, BSA buffer with (50 mM Hepes, pH 7.4,100 mM NaCl, 5 mM CaC1 2 and 1 mg / mL BSA) in at 10 nM sTF and 10 nM FVIIa For 60 minutes, followed by addition of substrate S2288 (1.2 mM, Chromogenix). Color development is measured continuously at 405 nm for 30 minutes. Amidolytic activity is expressed in mOD / min. IC ₅₀ values for inhibition of FVIIa / TF amidolytic activity by Mab can be calculated. The IC ₅₀ value for FFR-rFVIIa is 7 +/- 3 nM in this assay.

Inhibition of FXa production (Assay 5):
Lipidated TF (10 pM), FVIIa (100 pM) and anti-TF Mab or FFR-rFVIIa (0-50 nM) are incubated for 60 minutes at room temperature in BSA buffer (see Assay 4) followed by FX (50 nM ) Is added. After another 10 min, the reaction is stopped by adding 1/2 volume of stop buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 20 mM EDTA). The amount of FXa produced is determined by adding substrate S2765 (0.6 mM, Chromogenix) and measuring absorbance at 405 nm for 10 minutes continuously. IC ₅₀ values for Mab inhibition of FVIIa / lipidated TF-mediated FX activation can be calculated. The IC ₅₀ value for FFR-rFVIIa is 51 +/− 26 pM in this assay.

Biosensor assay (Assay 6):
The antibody is tested on a Biacore instrument by passing a standard solution of anti-human TF Mab over a chip immobilized with an antibody against human IgG. This is followed by passage of various concentrations of sTF in 10 mM hepes pH 7.4 containing 150 mM NaCl, 10 mM CaCl 2 and 0.0003% polysorbate 20. Kd values are calculated from sensorgrams using integrated Biacore evaluation software.

TF-dependent coagulation assay (Assay 7):
The assay is performed on an ACL300 research coagulator (ILS Laboratories). A dilution of anti-human TF Mab in 50 mM imidazole, pH 7.4, 100 mM NaCl, 0.1% BSA is mixed with 25 mM CaCl ₂ at a ratio of 2-5 and added to the sample cup of the coagulator. Thromboplastin from human, rat, rabbit, baboon, or pig, which is diluted with imidazole buffer and shows a clotting time of about 30 seconds in a sample containing no antibody, is placed in reagent reservoir 2, and human, rat, rabbit, baboon, Or place porcine plasma in reagent reservoir 3. During the analysis, 70 μL of antibody and CaCl ₂ mixture is transferred to 25 μL thromboplastin reagent and preincubated for 900 seconds, after which 60 μL plasma is added and the clotting time is measured. Set the maximum coagulation time to 400 seconds. Dilution of thromboplastin is used as a standard curve to convert clotting time to TF activity relative to controls without the addition of anti-TF Mab or FFR-rFVIIa. The IC ₅₀ value for FFR-rFVIIa is 4.4 +/− 0.4 pM in this assay.

Inhibition by Mab of FX activation catalyzed by FVIIa / cell surface TF (Assay 8):
Monolayer cells expressing human 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 is used as a source of TF in FX activation catalyzed by FVIIa / TF. Confluent cell monolayers in 24-, 48- or 96-well plates are washed once with buffer A (10 mM Hepes, pH 7.45, 150 mM NaCl, 4 mM KCl, and 11 mM glucose) Wash once with buffer B (buffer A supplemented with 1 mg / mL BSA and 5 mM Ca ²⁺ ). FVIIa (1 nM), FX (135 nM) and varying concentrations of Mab (or FFR-rFVIIa) in buffer B are added simultaneously to the cells. Alternatively, cells are preincubated with anti-TF Mab or FFRrFVIIa for 15 minutes, followed by the addition of rFVIIa and FX. Generate FXa for 15 minutes at 37 ° C. Remove 50 μL from each well and add to 50 μL stop buffer (buffer A supplemented with 10 mM EDTA and 1 mg / mL BSA). The amount of FXa produced is transferred to 50 μL of the above mixture microtiter plate and determined by adding 25 μL Chromozym X (final concentration 0.6 mM) to the wells. The absorbance at 405 nm is measured continuously and the initial rate of color development is converted to FXa concentration using the FXa standard curve. The IC ₅₀ value for FFR-rFVIIa is 1.5 nM in this assay.

Inhibition of ¹²⁵ I-FVIIa binding to cell surface TF by Mab (Assay 9):
Binding studies include cells expressing human 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. A confluent monolayer in a 24-well tissue culture plate is washed once with buffer A (see assay 8) and once with buffer B (see assay 8). Monolayer is preincubated with 100 μL of cold buffer B for 2 minutes. Varying concentrations of Mab (or FFR-rFVIIa) and radiolabeled FVIIa (0.5 nM ¹²⁵ I-FVIIa) are added simultaneously to the cells (final volume 200 μL). Incubate the plate at 4 ° C. for 2 hours. At the end of the incubation, unbound material is removed and the cells are washed 4 times with ice cold buffer B and lysed with 300 μL lysis buffer (200 mM NaOH, 1% SDS and 10 mM EDTA). Radioactivity is measured with a gamma counter (Cobra, Packard Instruments). Binding data is analyzed and curves are fitted using GraFit4 (Erithacus Software, Ltd., (UK). The IC ₅₀ value for FFR-rFVIIa is 4 nM in this assay.

Inhibition of FVIIa / TF-induced p44 / 42 MAPK activation by Mab (Assay 10):
The amount of phosphorylated p44 / 42 MAPK and / or Akt, and / or p90RSK is determined by quantitative detection of chemiluminescence derived from Western blot analysis (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, in medium containing 0-0.1% FCS prior to experiments to quiesce the cells. Incubate for 24 or 48 hours. On the day of the experiment, the cells should be 70-80% confluent. Experiments are performed by pre-incubating cells with excess Mab or FFR-rFVlla for 30 minutes at 37 ° C. in serum-free medium, followed by addition of 10-100 nM FVlla and incubation for 10 minutes. As a positive control for cell signaling, cells are treated with 10% FCS for 10 minutes. Cells are washed twice with ice-cold PBS, then cells are lysed with buffer (20 mM Tris, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 10 mM sodium glycerophosphate, 5 mM Sodium pyrophosphate, 150 mM NaCl, pH 7.5, the buffer contains 0.1 mM 4- (2-aminoethyl) benzenesulfonyl fluoride (AEBSF) and 1 mM benzamidine, 1 mM sodium orthovanadate immediately before use, Dissolve in 5 μg / mL leupeptin and 10 μg / mL aprotinin). The lysate is mixed with SDS-sample buffer and loaded onto an SDS-polyacrylamide gel. A standard marker of biotinylated protein is loaded onto each gel. Proteins separated on SDS-polyacrylamide gels were transferred to nitrocellulose by electroblotting, and kinases p44 / 42 MAPK, Akt and p90RSK were visualized by immunoblotting using phospho-specific antibodies, and chemiluminescence was analyzed by Fujifilm LAS1000. Quantify.

Epitope mapping assay (Assay 11):
Preparation of soluble TF (sTF) variants
sTF mutants (I22C, W45C, K46C, Y94C, F140C, W158C, K201C) were reverse PCR (QuikChange, Stratagene) using a wild-type plasmid (Freskgard et al. Protein Sci. 5, 1531-1540, 1996) as a template. , La Jolla, CA, USA). Wild type and mutants are expressed and purified in E. coli with some modifications as described elsewhere (Freskgard et al., Protein Sci. 5, 1531-1540, 1996). Cell lysis is performed by X-press (Biox, Sweden) technique in 10 mM Tris-HCl buffer, pH 7.5, and then resuspended in the same buffer supplemented with 1 mg DNAse. The solution is centrifuged at 11000 × g for 20 minutes at 4 ° C. and the inclusion bodies are denatured in 75 mL of 6 M GuHCl, 0.5 M NaCl, 20 mM Tris-HCl, pH 8.0. Refolding is achieved after 1 hour incubation at room temperature by diluting the denatured protein in a 1 L solution containing 50 mM Tris-HCl, 0.25 M NaCl, pH 8.0 with gentle agitation for approximately 2 hours. Is done. Purification is performed using Q-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden) and FVIIa affinity chromatography as described in Freskgard et al. (1996). Protein homogeneity is confirmed by SDS-PAGE. The concentration is measured at A _{280 nm} and determined using the calculated extinction coefficient of 37440 M ⁻¹ cm ⁻¹ (Gill and von Hippel, 1989).

MaxiSorp plates (Nunc-Immuno) are coated with wild type sTF and mutant (10 μg / mL) in TBS and blocked with blocking buffer (TBS containing 0.1% Tween 20 and 0.5% BSA). Wash plate with wash buffer (TBS and 0.1% Tween 20). Anti-human TF Mab is added at a concentration of 1 ng / mL in blocking buffer and incubated for 1 hour. The plate is then washed (6x) with wash buffer. Antibody binding is then detected with TMB _plus -substrate (Kem Tech Cat. 4390A) using HRP-labeled anti-human IgG (Helica Biosystems, Inc) at a 1: 2000 dilution in blocking buffer. . The final ELISA signal (OD _450-620 ) is used as a measure of the binding of each antibody to all sTF variants.

Thrombus elasticity record (Assay 12):
Human thromboplastin (eg Innovin, Dade Behring, final dilution 50,000 ×) is mixed with CaCl ₂ (final concentration 16.7 mM) and anti-TF Mab and incubated for 15 minutes at room temperature. Citrate stabilized human whole blood (280 μL) is added to the RoTEG sample cup (Pentapharm), pre-heated at 37 ° C. for 5 minutes, and then 40 μL of thromboplastin / CaCl ₂ / anti-TF Mab mixture is added. Thromboelastography is followed for 1 hour with a RoTEG device (Pentapharm). Velocity profiles are obtained from thrombograms using CoagPro Software (MedScience, Arhus, Denmark).

Example 3
Human cancer assay. To investigate the effect of treatment with human anti-TF Mab on human cancer growth and metastasis in a mouse model (Assay 13):
treatment:
Human anti-TF Mab is administered intravenously by bolus injection; 10 mg / kg = 0.1 mg / 10 g; the injection volume is 0.1 mL per 10 g mouse for all three treatment solutions:
A. Excipient control
B. 1 mg / mL human FFR-rFVIIa
C. 1 mg / mL anti-TF Mab

Model description:
I. Colon cancer primary growth and liver metastasis
Use healthy female athymic mice (nulnu) 7-8 weeks old. To destroy the remaining immune resistance of nude mice to human cell transplantation, mice are regularly irradiated with 5 Gy two days before transplanting human tumors (Vogel et al., 1997). Tumor transplantation of LS174T human colon cancer cells (ATCC CCL 188) cultured in RPMI 1640 with 15% fetal calf serum (FCS) as described (Li et al., Human Gene Therapy 10: 3045-3053, 1999) The mice are challenged with Briefly, cells are harvested with trypsin-EDTA, washed twice and resuspended in serum-free RPMI supplemented with sodium-heparinate solution (1 U / mL). Subsequently, a small excision under the left rib of the mouse is performed under anesthesia, and 3 3 106 LS174T cells in 50 mL of phosphate buffer saline (PBS) are injected into the spleen. After 3-5 minutes, the spleen vessels are connected and the spleen is surgically removed. With this technique, liver metastases develop stably (over 95%). Treatment with anti-TF Mab begins immediately after transplantation and continues for the remainder of the study. Mice are sacrificed 15 and 30 days after tumor cell inoculation, the liver is removed and weighed, and the number of tumor nodules visible on the liver surface is counted. Liver samples were fixed overnight in AFA (5% acetic acid, 75% ethyl alcohol, 2% formalin, 18% water), transferred to 100% ethanol, embedded in paraffin, and histological quantification of metastatic nodules Therefore, prepare 5 mm sections for immunohistochemistry and apoptosis quantification.

Study I-1:
Objective: To investigate the effect of anti-TF Mab injected intravenously at 10 mg / kg on macroscopic growth and liver metastasis of LS174T colon tumor in nude mice.
Mice: 60 homozygous nu / nu 6 week old NMRI males.
Groups: Mice are randomly assigned to 4 groups of 15 animals and treated with solutions A, B, or C.
Termination: Animals are terminated with more than 20% weight loss or other objective sign of severe toxicity.
Parameters: Tumor size is recorded daily at two orthogonal diameters during the growth phase. Body weight is recorded first and 2-3 times a week. The formation of metastases in the liver is determined postmortem.

II. Primary Breast Cancer Growth and Lung Metastases Human breast cancer cells MDA-MB-231 (ATCC HTB26) are cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS). MDA-MB-231 cells (3 3 106) are injected subcutaneously into nude mice (7-8 week old female mice). Primary tumor growth and metastasis are assessed as previously described (Li et al., Human Gene Therapy 12: 515-526, 2001).

Study II-1:
Objective: To investigate the effect of intravenous 10 mg / kg bolus-injected anti-TF Mab on gross growth and lung metastasis of MDA-MB-231 breast tumor in nude mice.
Mice: 60 homozygous nu / nu 6 week old NMRI males.
Groups: Mice are randomly assigned to 4 groups of 15 animals and treated with solutions A, B, or C.
Termination: Animals are terminated with more than 20% weight loss or other objective sign of severe toxicity.
Parameters: Tumor size is recorded daily at two orthogonal diameters during the growth phase. Body weight is recorded first and 2-3 times a week. The formation of metastases in the lung is determined postmortem.

III. Glioma tumor Xenograft primary proliferating tumor cell line MG U373 is a human glioblastoma polymorphic cell line with high angiogenic activity, high vascular density, and rapidity in nude mice Show proliferative. Tumors are inoculated into the flank according to standard procedures (see protocol enclosed with experimental plan). Mice are observed twice daily for signs of toxicity and tumors are measured daily with two vertical diameters.

  Tumors are transplanted on the flank of nu / nu homozygous nude mice with NMRI background. Mice are 7 week old males obtained from M & B (Ry, Denmark). Animals are kept in a gnotobiotic environment and ingested sterilized food pellets and drinking water as appropriate.

Three studies will be performed using a glioma model:
Study III-1:
Objective: To investigate the effect of intravenous 10 mg / kg bolus injected anti-TF Mab on macroscopic growth of U373 tumor in nude mice.
Mice: 60 homozygous nu / nu 6 week old NMRI males.
Groups: Mice are randomly assigned to 4 groups of 15 animals and treated with solutions A, B, or C.
Termination: Animals are terminated with more than 20% weight loss or other objective sign of severe toxicity.
Parameters: Tumor size is recorded daily at two orthogonal diameters during the growth phase. Body weight is recorded first and 2-3 times a week.

Study III-2:
Objective: To investigate the effect of intravenous 10 mg / kg bolus-injected anti-TF Mab on macroscopic growth of U373 tumor in nude mice after confirmation of pre-treatment tumor growth.
Mice: 60 homozygous nu / nu 6 week old NMRI males.
Groups: Mice are randomly assigned to 4 groups of 15 animals and treated with solutions A, B, or C. Treatment begins when six continuity (daily) measurements indicate Gompertzian growth. This corresponds to 100-200 mm ³ .
Termination: Treatment continues until the tumor grows beyond a maximum size of about 1.0 cm ³ (ie not a tumor diameter greater than 15 mm) or until Gompertz regrowth is confirmed by 6 continuity measurements. At termination, tumors are excised from each group for histological and immunochemical evaluation. Animals are terminated with more than 20% weight loss or other objective signs of severe toxicity.
Parameters: Tumor size is recorded daily at two orthogonal diameters during the growth phase. Body weight is recorded first and twice a week.

Study III-3:
Objective: To investigate the effect of anti-TF Mab on intracranial U373 tumor growth in nude mice.
Mice: 60 homozygous nu / nu 6 week old NMRI males.
Tumor: U373 implanted orthotopically into the right hemisphere according to standard procedures.
Groups: Mice are randomly assigned to 4 groups of 15 animals and treated with solutions A, B, or C.
Termination: Euthanize mice showing signs of chronic neurological deficits.
Data: Survival (ie, time to neurological defect) is quantified by Kaplan-Meyer statistics.

Example 4 (Assay 14)
In mice in which the TF gene has been knocked out and the human TF gene has been inserted (mTF-KO / hTF-KI mice), a 0.5 mL matrigel plug is placed subcutaneously in the abdominal skin. Matrigel incorporates b-FGF (5 ng), and after 1 week, the formation of new patented blood vessels in the gel is quantified by measuring hemoglobin content (angiogenesis). The ability of human anti-TF Mab to inhibit (% inhibition of hemoglobin content) can be assessed after single or repeated parenteral administration of the protein.

Example 5 (Assay 15)
Gene expression analysis assay to distinguish between antibodies that interfere with FVIIa binding to TF and antibodies that interfere with FX binding to TF
Specific up-regulation of 3 genes in BHK-TF cells treated with FVIIa was observed by cDNA microarray analysis. These genes include Fra-1, a gene encoding Fos-related antigen 1, Id2 encoding a member of a helix-loop helix class protein, and Cyr61 encoding an extracellular matrix signaling protein. The following assays were designed to test anti-TF Mabs that interfere with FVIIa-induced Fra-1, Id2 or Cyr61 upregulation.

Cell culture reagents are purchased from GIBCO-BRL Life Technologies unless otherwise noted.
BHK-TF cells were made as described in Poulsen LK et al., J Biol. Chem. 273, 6228-6232, 1998 and contain 10% FCS, 100 IU / mL penicillin, and 100 μg / mL streptomycin Grow in Dulbecco's modified Eagle medium to obtain 95-100% confluence, wash and grow for 16-18 hours in medium without FCS. Cells are washed again and exposed to FCS free medium containing 100 nM FVIIa.

  To clone the fragment for Northern blot analysis, the cells are treated as follows. BHK-TF cells are grown in Dulbecco's modified Eagle's medium containing 10% FCS, 100 IU / mL penicillin, and 100 μg / mL streptomycin to obtain 95-100% confluence, wash, and 16-16 Grow in medium without FCS for 18 hours. Cells are washed again and exposed to FCS-free medium containing 100 nM FVIIa for 1 hour. CRL2091 cells (ATCC) are grown in Iscove's modified Dulbecco's medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin to 95-100% confluence. Cells are then serum starved for 16-18 hours and treated with FBS-free medium containing 100 nM FVIIa for 6 hours. Murine 3T3-L1 cells (ATCC) are maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells are grown to confluence and induced for 1 hour in medium containing 1 μM dexamethasone (Sigma), 10 μg / mL human insulin (Novo Nordisk A / S), and 1 μM BRL49653 (Novo Nordisk A / S).

Fragment cloning for Northern blot analysis
Fra-1 was reverse transcription PCR from RNA isolated from 3T3-L1 cells treated with dexamethasone, insulin, and BRL49653 for 1 hour using the Superscript 11 kit (Life Technologies) according to the manufacturer's instructions. To clone. Id2 and Cyr61 are cloned by reverse transcription PCR from RNA isolated from BHK-TF cells treated with FVIIa for 1 hour and CRL2091 cells treated with FVIIa for 6 hours, respectively. The upstream and downstream primers are as follows: 5'-GCGGCCGCCATGTACCGAGACTACGGGGAACCG-3 'and 5'-GCGGCCGCTCACAAAGCCAGGAGTGTAGG-3' for Fra-1, 5'-CAGCATGAAAGCCTTCAGTC-3 'and 5'-CTCTGGTGATGCAGGCTGAC-3 'For Cyr61, 5'-CGTCACCCTTCTCCACTTGA-3' and 5'-CTTGGTCTTGCTGCATTTCT-3 '. PCR parameters were 94 ° C for 10 seconds denaturation, 65 ° C for 15 seconds annealing, and 72 ° C for 1.5 minutes extension, 94 ° C for 10 seconds denaturation, 64 ° C for 15 seconds annealing, and 1 cycle of extension at 72 ° C for 1.5 min, denaturation at 94 ° C for 10 seconds, annealing at 63 ° C for 15 seconds, and 1 cycle of extension at 72 ° C for 1.5 minutes, denaturation at 94 ° C for 10 seconds, at 62 ° C One cycle of 15 seconds of annealing and extension at 72 ° C for 1.5 minutes, one cycle of denaturation at 94 ° C for 10 seconds, annealing at 61 ° C for 15 seconds and 1.5 minutes at 72 ° C, 10 seconds at 94 ° C Denaturation, annealing at 60 ° C for 15 seconds, and extension for 1.5 minutes at 72 ° C for 1 cycle, denaturation at 94 ° C for 10 seconds, annealing at 55 ° C for 15 seconds, and extension for 1.5 minutes at 72 ° C for 40 cycles It is. All fragments are cloned into TOPO 2.1 (Invitrogen) and sequenced using a Megabase sequencer.

Northern blot analysis Total RNA is isolated from BHK-TF cells incubated with FVIIa, FX, ASIS, 1F44A1 or TF85G9 using TriZol according to the manufacturer's instructions. 20 μg RNA was size fractionated on a denaturing gel containing 1% agarose, 20 mM MOPS, 5 mM NaOAc, 6% formaldehyde, and 1 mM EDTA, transferred to a high-bond N ⁺ membrane (Amersham) by capillary blotting, Fix by UV cross-linking. Label the cDNA encoding Fra-1, Id2 or Cyr61 with Prime It kit (Stratagene) using [α- ³² P] dATP (Amersham) and use Express Hyb (Clontech) according to the manufacturer's instructions. And the results are visualized by autoradiography.

Example 6 (Assay 16)
MAPK assay via Elk1 transcription factor / luciferase reporter (PathDetect)
HeLa cells are seeded 40% confluent in T-80 flasks one day prior to transfection. Cells were prepared using 36 μL FuGene (Roche) as described in the manual, 150 ng pFA-Elk1 (Stratagene), 3 μg pFR-Luc (Stratagene), 3 μg human TF / pcDNA3 and 3 μg mouse protease activated receptor (Protease Activated Receptor) Receptor) Transfect with 2 / pcDNA3,1 +. The next day, cells are detached with Versene ^™ (Invitrogen) and plated in black 96 well view plates (Packard) at a cell density of 20,000 cells per well. After the cells are reattached to the plate, the medium is replaced with 160 μL per well of serum free Dulbecco's Modified Eagle Medium (Invitrogen) and incubated for 16 hours.

  Cells are preincubated for 1 hour with either 20 μL serum free medium (control), 20 μL 2,5 μM FFR-rFVIIa (control), 20 μL 2,5 M anti-TF Mab B or 20 μL 2,5 μM anti-TF Mab A. Add 20 μL 0.5 μM FVIIa to half of the wells and add the medium to the other half. After 4 hours of incubation, the cells are subjected to a luciferase gene assay. LucLite (Packard) reagent is added to the cells as described by the manufacturer. The expression level of luciferase is read by TopCount Microplate Scintillation (Packard).

Schematic diagram of an example screening assay for selecting human monoclonal high affinity antibodies to TF. Detailed schematic of screening assays 1-3 described in Example 1. Detailed schematic of screening assays 4-7 described in Example 1. FIG. Detailed schematic diagram of screening assays 8-10 described in Example 1. FIG. An example of screening an antibody by assay number 4. Inhibition of sTF / FVIIa amidolytic activity by FFR-rFVIIa (black circle) and human anti-TF monoclonal antibody HuTF-31F2 (white circle). An example of screening an antibody by assay number 5. Inhibition of factor Xa production by FFR-rFVIIa (black circle) and human anti-TF monoclonal antibody HuTF-31F2 (white circle). An example of screening an antibody by assay number 7. Inhibition of human TF-induced coagulation by FFR-rFVIIa (black circle) and human anti-TF monoclonal antibody HuTF-31F2 (white circle). An example of screening an antibody by assay number 10. Only anti-TF monoclonal antibodies that interfere with FVIIa binding inhibit TF / FVIIa-mediated signaling. Human anti-TF Mab inhibits FVIIa-induced phosphorylation of p44 / 42 MAPK (assay number 10). BHK cells transfected with TF were serum starved for 2 hours to quiesce the cells. The antibodies HuMab 30F5 (500 nM) and HuMab 31F2 (500 nM) were added to the cells 15 minutes before the addition of FVIIa (15 nM). Cells were lysed and proteins were separated by SDS-PAGE and transferred to nitrocellulose by electroblotting. Western blot analysis was performed using a polyclonal phospho-specific antibody against p44 / 42 MAPK. The secondary antibody was anti-rabbit IgG conjugated with horseradish peroxidase. Chemiluminescence detection was performed using a cooled CCD camera. The band on the digital image was quantified and the band obtained using FVIIa was set to 100%. A 50% reduction in phosphorylated band was observed when cells were preincubated with HuMab 30F5 (500 nM) and a 25% reduction was observed when cells were preincubated with HuMab 31F2 (500 nM). In conclusion, this experiment shows that human antibodies against TF (30F5 and 31F2) partially inhibit FVIIa-induced phosphorylation of p44 / 42 MAPK. Similar results were obtained with 50 nM FVIIa. An example of screening an antibody by assay number 16. This figure demonstrates that monoclonal antibodies against TF inhibit TF intracellular activity in TF expressing cells. Anti-TF Mab B inhibits TF intracellular activity, but does not inhibit anti-TF Mab A. An example of screening an antibody by assay number 12. Velocity profile of thromboelastograms obtained with 0.5 nM FFR-rFVIIa and human anti-TF antibody HuTF-31F2.

Claims (49)

  An isolated human antibody that immunoreacts with an epitope present on human TF.   2. The isolated human antibody of claim 1, which inhibits human clotting factor VIIa binding to human TF.   The isolated human antibody according to any one of claims 1 to 2, which is a monoclonal antibody.   The isolated human antibody according to any one of claims 1 to 3, which is a recombinant antibody.   The isolated human antibody according to any one of claims 1 to 4, wherein the antibody is a Fab fragment. The isolated human antibody according to any one of claims 1 to 4, wherein the antibody is an F (ab) ₂ fragment. The isolated human antibody according to any one of claims 1 to 4, wherein the antibody is an F (ab '') ₂ fragment.   The isolated human antibody of any one of claims 1 to 4, wherein the antibody is a single chain Fv fragment. 9. The isolated human antibody of any one of claims 1-8, wherein the antibody has a _Kd for binding to human TF in the range of ^10-15 to ^10-8M . 10. The isolated human antibody of any one of claims 1-9, wherein the antibody has a _Kd for binding to human TF in the range of ^10-15 to ^10-9 M.   A pharmaceutical composition comprising a therapeutically effective amount of a human antibody that immunoreacts with an epitope present on human TF.   11. A pharmaceutical composition comprising a therapeutically effective amount of a human antibody that immunoreacts with an epitope present on human TF, wherein the antibody is one of claims 1-10. Pharmaceutical composition.   A composition comprising a human antibody that immunoreacts with an epitope present on human TF.   11. A composition comprising a human antibody that immunoreacts with an epitope present on human TF, wherein the antibody is one of claims 1-10.   A method of treating a FVIIa / TF-related disorder in a human comprising administering to said human a therapeutically effective amount of a human antibody that immunoreacts with an epitope present on human TF.   11. A method of treating a FVIIa / TF-related disorder in a human comprising administering to said human a therapeutically effective amount of an antibody according to any one of claims 1-10. A method for preparing a human antibody comprising the following a) to b):
a) preparing a human antibody against human TF;
tested antibodies in b) TF-induced clot assay in said assay, FFR-rFVIIa IC ₅₀ values +1 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +500 pM lower an IC ₅₀ value, preferably FFR the IC ₅₀ values of -rFVIIa +200 pM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +100 pM lower an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +50 lower an IC ₅₀ value than pM, preferably FFR the IC ₅₀ values +10 lower an IC ₅₀ value than pM of -RFVIIa, more preferably less an IC ₅₀ value than an IC ₅₀ value +5 pM of FFR-rFVIIa, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, clot Selecting a human antibody that inhibits formation, or
The antibodies were tested in an FXa production assay and (using 0.1 nM FVIIa) FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 100 nM, for example FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 10 nM, preferably FFR-rFVIIa IC ₅₀ values +5 nM lower than an IC ₅₀ value, preferably less an IC ₅₀ value than an IC ₅₀ value +1 nM of FFR-rFVIIa, IC ₅₀ and more preferably less than an IC ₅₀ value +0.1 nM of FFR-rFVIIa value, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, selecting a human antibody which inhibit FXa generation, or
Antibodies were tested in a FVIIa / TF amidolytic assay (using said 10 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values less than +40 nM the IC ₅₀ values, preferably FFR-rFVIIa IC ₅₀ values +20 nM lower than an IC ₅₀ value, preferably less an IC ₅₀ value than an IC ₅₀ value +10 nM of FFR-rFVIIa, more preferably less than an IC ₅₀ value of FFR-rFVIIa Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity with an IC ₅₀ value, or
Testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding, or testing antibodies in TF ELISA assays that include TF and selecting human antibodies that immunoreact with human TF. 18. The method of claim 17, wherein the human antibody against human TF is
a) immunization of mammals with human TF,
b) A method produced by a method comprising isolation of antibodies produced by the immunized mammal.   The method of claim 18, wherein the mammal is a mouse. A method according to any one of claims 17 to 19, comprising
Tested antibodies in the TF-induced clot assay in said assay, FFR-rFVIIa IC ₅₀ values +1 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +500 pM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +200 pM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +100 pM lower an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +50 lower an IC ₅₀ value than pM, preferably FFR-rFVIIa IC ₅₀ values +10 lower an IC ₅₀ value than pM, more preferably lower an IC ₅₀ value than an IC ₅₀ value +5 pM of FFR-rFVIIa, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, clot formation Selecting a human antibody to inhibit. 21. The method according to any one of claims 17 to 20, comprising:
The antibodies were tested in the FXa production assay and (with 0.1 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 100 nM, eg FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 10 nM , preferably an IC ₅₀ value +5 lower an IC ₅₀ value than nM of FFR-rFVIIa, more preferably less an IC ₅₀ value than an IC ₅₀ value +1 nM of FFR-rFVIIa, more preferably an IC ₅₀ value of FFR-rFVIIa +0.1 nM low an IC ₅₀ value, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, the method includes selecting a human antibody which inhibit FXa generation. The method according to any one of claims 17 to 21, comprising:
Antibodies were tested in a FVIIa / TF amidolytic assay (using said 10 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values less than +40 nM the IC ₅₀ values, preferably FFR-rFVIIa IC ₅₀ values +20 nM lower than an IC ₅₀ value, preferably less an IC ₅₀ value than an IC ₅₀ value +10 nM of FFR-rFVIIa, more preferably less than an IC ₅₀ value of FFR-rFVIIa Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity with an IC ₅₀ value. A method according to any one of claims 17-22,
Testing the antibody in an FVIIa competition assay and selecting a human antibody that competes with FVIIa binding. 24. A method according to any one of claims 17 to 23, comprising:
A method comprising testing an antibody in a TF ELISA assay comprising TF and selecting a human antibody that immunoreacts with human TF. A human antibody that immunoreacts with an epitope present on human TF and inhibits the binding of human coagulation factor VIIa to human TF, obtained by a method comprising the following a) to b):
a) preparing a human antibody against human TF;
tested antibodies in b) TF-induced clot assay in said assay, FFR-rFVIIa IC ₅₀ values +1 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +500 pM lower an IC ₅₀ value, preferably FFR the IC ₅₀ values of -rFVIIa +200 pM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +100 pM lower an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +50 lower an IC ₅₀ value than pM, preferably FFR the IC ₅₀ values +10 lower an IC ₅₀ value than pM of -RFVIIa, more preferably less an IC ₅₀ value than an IC ₅₀ value +5 pM of FFR-rFVIIa, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, clot Selecting a human antibody that inhibits formation, or
The antibodies were tested in the FXa production assay and (with 0.1 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 100 nM, eg FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 10 nM , preferably an IC ₅₀ value +5 lower an IC ₅₀ value than nM of FFR-rFVIIa, more preferably less an IC ₅₀ value than an IC ₅₀ value +1 nM of FFR-rFVIIa, more preferably an IC ₅₀ value of FFR-rFVIIa +0.1 nM low an IC ₅₀ value, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, selecting a human antibody which inhibit FXa generation, or
The antibodies were tested in the FVIIa / TF amidolytic assay and (using 10 nM FVIIa) FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 100 nM, eg FFR-rFVIIa IC ₅₀ value + IC ₅₀ value lower than 40 nM , preferably IC ₅₀ values +20 nM lower IC ₅₀ value of FFR-rFVIIa, preferably lower IC ₅₀ values than the IC ₅₀ value +10 nM of FFR-rFVIIa, more preferably ₅₀ values less IC than IC ₅₀ values of FFR-rFVIIa Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity, or
Testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding, or testing antibodies in TF ELISA assays that include TF and selecting human antibodies that immunoreact with human TF. A method for preparing a human antibody comprising the following a) to j):
a) immunizing mice with human TF;
b) isolating antibody-producing cells from the immunized mouse and preparing immortalized cells that secrete human antibodies;
c) isolating a medium derived from immortalized cells containing the produced antibody;
d) testing antibodies in a TF ELISA indirect assay comprising TF in dissolved state and selecting human antibodies that immunoreact with human TF in dissolved state;
e) testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding;
tested antibodies in f) FVIIa / TF amidolytic assay (using said 10 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ of values +40 nM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +20 nM lower than an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +10 nM lower than an IC ₅₀ value, more preferably an IC ₅₀ value of FFR-rFVIIa Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity with a lower IC ₅₀ value;
tested antibodies in g) FXa generation assay, less than (the using 0.1 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa an IC ₅₀ value +10 nM of IC ₅₀ values, preferably an IC ₅₀ value +5 lower an IC ₅₀ value than nM of FFR-rFVIIa, preferably less an IC ₅₀ value than an IC ₅₀ value +1 nM of FFR-rFVIIa, more preferably an IC ₅₀ value of FFR-rFVIIa +0.1 less than nM an IC ₅₀ value, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, selecting a human antibody which inhibit FXa generation,
tested antibodies in h) TF-induced clot assay in said assay, FFR-rFVIIa IC ₅₀ values +1 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +500 pM lower an IC ₅₀ value, preferably FFR the IC ₅₀ values of -rFVIIa +200 pM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +100 pM lower an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +50 lower an IC ₅₀ value than pM, preferably FFR the IC ₅₀ values +10 lower an IC ₅₀ value than pM of -RFVIIa, more preferably less an IC ₅₀ value than an IC ₅₀ value +5 pM of FFR-rFVIIa, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, clot Selecting a human antibody that inhibits formation,
i) after steps dh, selecting immortalized cells producing the selected antibody and culturing in an appropriate medium;
j) isolating the selected antibody from the medium of the selected immortalized cells. 27. The method of claim 26, comprising:
A method further comprising testing the antibody in a TF ELISA direct assay comprising immobilized TF and selecting a human antibody that immunoreacts with immobilized human TF. A method according to any one of claims 26 to 27, comprising:
Tested antibodies in FXa generation assay on TF expressing cells, (wherein using 1 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +500 lower an IC ₅₀ value than nM, for example FFR-rFVIIa an IC ₅₀ value +100 nM of lower an IC ₅₀ value, preferably an IC ₅₀ value +50 nM lower than an IC ₅₀ value of FFR-rFVIIa, preferably FFR-rFVIIa IC ₅₀ values +10 nM lower than an IC ₅₀ value, more preferably an IC ₅₀ value of FFR-rFVIIa +5 lower an IC ₅₀ value than nM, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, further comprising the selecting human antibody which inhibit FXa generation on TF expressing cells. A method according to any one of claims 26 to 28, comprising:
A method further comprising testing the antibody in a complete cell TF binding assay and selecting a human antibody that competes with FVIIa for binding to human TF expressed on the cell surface of the complete cell. 30. A method according to any one of claims 26 to 29, comprising:
The antibody is tested in a biosensor assay and the K _d for binding to human TF is lower than 100 nM, such as lower than 10 nM, preferably lower than 5 nM, preferably lower than 1 nM, more preferably lower than 0.5 nM A method further comprising selecting a human antibody. A method according to any one of claims 26 to 30, comprising
A method further comprising testing the antibody in a MAPK signaling assay and selecting a human antibody that inhibits FVIIa-induced activation of MAPK signaling. 32. A method according to any one of claims 26-31, comprising:
The method further comprising testing the antibody in an epitope mapping assay and selecting a human antibody that immunoreacts with a preferred epitope on TF.   34. The method of claim 32, wherein the preferred epitope comprises Trp45, Lys46 and Tyr94 residues.   The method according to any one of claims 26 to 33, wherein the immortalized cell is a hybridoma cell. A human antibody that immunoreacts with an epitope present on human TF and inhibits the binding of human coagulation factor VIIa to human TF, obtained by a method comprising the following a) to j):
a) immunizing mice with human TF;
b) isolating antibody-producing cells from the immunized mouse and preparing immortalized cells that secrete human antibodies;
c) isolating a medium derived from immortalized cells containing the produced antibody;
d) testing antibodies in a TF ELISA indirect assay comprising TF in dissolved state and selecting human antibodies that immunoreact with human TF in dissolved state;
e) testing antibodies in FVIIa competition assays and selecting human antibodies that compete with FVIIa binding;
tested antibodies in f) FVIIa / TF amidolytic assay (using said 10 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ of values +40 nM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +20 nM lower than an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +10 nM lower than an IC ₅₀ value, more preferably an IC ₅₀ value of FFR-rFVIIa Selecting a human antibody that inhibits TF-induced FVIIa amidolytic activity with a lower IC ₅₀ value;
tested antibodies in g) FXa generation assay, less than (the using 0.1 nM FVIIa in the assay) FFR-rFVIIa IC ₅₀ values +100 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa an IC ₅₀ value +10 nM of IC ₅₀ values, preferably an IC ₅₀ value +5 lower an IC ₅₀ value than nM of FFR-rFVIIa, preferably less an IC ₅₀ value than an IC ₅₀ value +1 nM of FFR-rFVIIa, more preferably an IC ₅₀ value of FFR-rFVIIa +0.1 less than nM an IC ₅₀ value, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, selecting a human antibody which inhibit FXa generation,
tested antibodies in h) TF-induced clot assay in said assay, FFR-rFVIIa IC ₅₀ values +1 nM lower than an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +500 pM lower an IC ₅₀ value, preferably FFR the IC ₅₀ values of -rFVIIa +200 pM lower an IC ₅₀ value, preferably FFR-rFVIIa IC ₅₀ values +100 pM lower an IC ₅₀ value, for example, FFR-rFVIIa IC ₅₀ values +50 lower an IC ₅₀ value than pM, preferably FFR the IC ₅₀ values +10 lower an IC ₅₀ value than pM of -RFVIIa, more preferably less an IC ₅₀ value than an IC ₅₀ value +5 pM of FFR-rFVIIa, more preferably lower an IC ₅₀ value than an IC ₅₀ value of FFR-rFVIIa, clot Selecting a human antibody that inhibits formation,
i) after steps dh, selecting immortalized cells producing the selected antibody and culturing in an appropriate medium;
j) isolating the selected antibody from the medium of the selected immortalized cells.   35. A human antibody that immunoreacts with an epitope present on human TF and inhibits the binding of human coagulation factor VIIa to human TF, which is obtained by the method according to any one of claims 26 to 34. antibody.   A cell that produces a human antibody that immunoreacts with an epitope present on human TF and inhibits the binding of human coagulation factor VIIa to human TF.   38. The cell of claim 37, wherein the cell is an isolated lymphoid cell.   The cell according to any one of claims 37 to 38, wherein the cell is isolated from a mouse.   38. The cell of claim 37, wherein the cell is a hybridoma cell.   41. The cell according to claim 40, wherein the hybridoma cell is obtained by fusing an antibody-producing lymphoid cell and an immortalized cell to provide an antibody-producing hybridoma cell.   42. The cell of any one of claims 37 to 41, wherein the antibody inhibits human clotting factor VIIa binding to human TF.   43. The cell of any one of claims 37 to 42, wherein the antibody immunoreacts with a three-dimensional surface comprising all Trp45, Lys46 and Tyr94 residues.   44. The cell of any one of claims 37 to 43, wherein the antibody is a Fab fragment. 45. The cell of any one of claims 37 to 44, wherein the antibody is an F (ab) ₂ fragment. 45. The cell of any one of claims 37 to 44, wherein the antibody is an F (ab '') ₂ fragment.   45. The cell of any one of claims 37 to 44, wherein the antibody is an scFv fragment. Wherein the antibody, 10 ^-15 to 10 ^-8 M, with a K _d for binding to human TF, cells according to any one of claims 37-47. Wherein the antibody, 10 ^-15 to 10 of ^-10 M, has a K _d for binding to human TF, cells according to any one of claims 37 to 48.

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