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EP1434802A1 - Human tissue factor antibodies - Google Patents

Human tissue factor antibodies

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Publication number
EP1434802A1
EP1434802A1 EP02800043A EP02800043A EP1434802A1 EP 1434802 A1 EP1434802 A1 EP 1434802A1 EP 02800043 A EP02800043 A EP 02800043A EP 02800043 A EP02800043 A EP 02800043A EP 1434802 A1 EP1434802 A1 EP 1434802A1
Authority
EP
European Patent Office
Prior art keywords
value
rfviia
ffr
human
assay
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02800043A
Other languages
German (de)
English (en)
French (fr)
Inventor
Per-Ola Freskgaard
Jes Thorn Clausen
Brit Binow Sorensen
Marianne Kjalke
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
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Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP1434802A1 publication Critical patent/EP1434802A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • the present invention relates to isolated antibodies that immunoreacts with tissue factor (TF) to inhibit the binding of coagulation factor Vila (FVIIa) and thus an immunotherapeutic method using human antibodies against TF to inhibit thrombus formation associated with surgery, microsurgery, angioplasty or trauma or to inhibit thrombus formation and other functions of TF in abnormal haemostatic conditions associated with diseases like deep vein thrombosis, disseminated intravascular coagulation (DIC), coronary artery disease, sepsis, inflammation, atherosclerosis, or cancer. Also disclosed are a method for preparation of antibodies as well as cell lines for preparation of the human monoclonal antibodies (Mabs).
  • Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually gives rise to a fibrin clot.
  • the blood com- ponents which participate in what has been referred to as the coagulation "cascade” are pro- enzymes or zymogens, enzymatically inactive proteins which are converted to proteolytic enzymes by the action of an activator, itself an activated clotting factor.
  • Coagulation factors that have undergone such a conversion and generally referred to as “active factors,” and are designated by the addition of a lower case “a” suffix (e.g., Factor Vila).
  • Activated Factor X (“Xa”) is required to convert prothrombin to thrombin, which then converts fibrinogen to fibrin as a final stage in forming a fibrin clot.
  • the "intrinsic pathway” refers to those reactions that lead to thrombin formation through utilization of factors present only in plasma.
  • a series of protease-mediated activations ultimately generates Factor IXa which, in conjunction with Factor Villa, cleaves Factor X into Xa.
  • An identical proteolysis is effected by FVIIa and its co-factor, TF, in the "extrinsic pathway” of blood coagulation.
  • TF is a membrane bound protein and does not normally circulate in an active form in plasma. Upon vessel disruption, however, TF can complex with FVIIa to catalyze Factor X activation or Factor IX activation in the presence of Ca 2+ and phospholipid. While the relative importance of the two coagulation pathways in hemostasis is unclear, Factor VII and TF have been found to play a pivotal role in the initiation of blood coagulation.
  • Anticoagulants such as heparin, coumarin, derivatives of coumarin, indandione derivatives, or other agents may be used, for example, during kidney dialysis, or to treat deep vein thrombosis, disseminated intravascular coagulation (DIC), and a host of other medical disorders.
  • heparin treatment or extracorporeal treatment with citrate ion may be used in dialysis to prevent coagulation during the course of treatment.
  • Heparin is also used in pre- venting deep vein thrombosis in patients undergoing surgery.
  • heparin and other anticoagulants may, however, have undesirable side effects.
  • Available anticoagulants generally act throughout the body, rather than acting specifically at site of injury. Heparin, for example, may cause heavy bleeding. Furthermore, with a half-life of approximately 80 minutes, heparin is rapidly cleared from the blood, neces- sitating frequent administration. Because heparin acts as a cofactor for antithrombin III (ATIII), and ATIII is rapidly depleted in DIC treatment, it is often difficult to maintain the proper heparin dosage, necessitating continuous monitoring of ATIII and heparin levels. Heparin is also ineffective if ATIII depletion is extreme.
  • ATIII antithrombin III
  • heparin may also increase platelet aggregation and reduce platelet count, and has been implicated in the development of heparin-induced thrombocytopenia. Indandione derivatives may also have toxic side effects.
  • anticoagulants briefly described above, several naturally occurring proteins have been found to have anticoagulant activity. Also, ATIII has been proposed as a therapeutic anticoagulant.
  • Antibodies are specific immunoglobulin (lg) polypeptides produced by the vertebrate immune system in response to challenges by foreign proteins, glycoproteins, cells, or other antigenic foreign substances.
  • the sequence of events which permits the organism to overcome invasion by foreign cells or to rid the system of foreign substances is at least partially understood.
  • An important part of this process is the manufacture of antibodies which bind specifically to a particular foreign substance.
  • the binding specificity of such polypeptides to a particular antigen is highly refined, and the multitude of specificities capable of being generated by the individual vertebrate is remarkable in its complexity and variability. Millions of antigens are capable of eliciting antibody responses, each antibody almost exclusively directed to the particular antigen which elicited it.
  • Antibodies are generated in situ as a result of the differentiation of immature B lymphocytes into plasma cells, which occurs in response to stimulation by specific antigens.
  • the undifferentiated B cells the portions of DNA coding for the various regions on the immunoglobulin chains are separated in the genomic DNA. The sequences are assembled sequentially prior to expression. The resulting rearranged gene is capable of expression in the mature B lymphocyte to produce the desired antibody.
  • a uniform population of antibodies does not result.
  • the in situ immune re- sponse to any particular antigen is defined by the mosaic of responses to the various determinants which are present on the antigen.
  • Each subset of homologous antibodies is contributed by a single population of B cells, hence in situ generation of antibodies is "polyclonal”. This limited but inherent heterogeneity has been overcome in numerous particular cases by use of hybridoma technology to create "monoclonal" antibodies in cell cultures by B cell hybridomas.
  • the relatively short-lived, or mortal, splenocytes or lymphocytes from a mammal which has been injected with antigen are fused with an immortal tumor cell line, thus producing hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
  • the hybrids thus formed are segregated into single genetic strains by selection, dilution, and regrowth, and each strain thus represents a single genetic line. They therefore, produce antibodies which are assured to be homogeneous against a desired antigen. These antibodies, referencing their pure genetic parentage, are called "monoclonal".
  • Monoclonal antibodies with mono-specificity have greatly influenced immunology, and their usefulness has already been demonstrated in such sciences as biology, pharmacology, biochemistry and others. Such monoclonal antibodies have found widespread use not only as diagnostics reagents, but also therapeutically (see, for example, Ritz and Schlossman, Blood, 59:1-11 , (1982)).
  • Monoclonal antibodies produced by hybridomas while theoretically effective as dis- cussed above and clearly preferable to polyclonal antibodies because of their specificity, suffer from an important disadvantage.
  • the use of monoclonal antibodies produced in non-human animals is severely restricted where the monoclonal antibodies are to be used in humans.
  • Repeated injections of a "foreign" antibody in humans, such as a mouse antibody may lead to harmful hypersensitivity reactions.
  • a non-human derived monoclonal antibody when injected into humans, causes an anti-nonhuman antibody response.
  • Presta L. et al., Thrombosis and Haemostasis, Vol. 85 (3) pp. 379-389 (2001) relates to humanized antibody against TF.
  • the present invention fulfils this need by providing anticoagulants that do not have the side effects associated with the traditional antibodies with non-human sequences, they act specifically at sites of injury, and further provides other related advantages.
  • the present invention provides compounds, which acts to inhibit the cellular functions of TF, which is implicated in conditions like sepsis, inflammation, atherosclerosis, restenosis, or cancer.
  • the present invention relates to non-immunogenic high affinity human antibodies against human TF, which inhibits the binding of coagulation factor VII ⁇ /lla and methods for selection of therapeutically effective human antibodies against human TF.
  • the present invention relates to an isolated human antibody, which immunoreacts with an epitope present on human TF.
  • human tissue factor or “human TF” as used herein, refers to the full length polypeptide receptor comprising the amino acid sequence 1-263 of native human tissue factor.
  • antibody is intended to refer to immunoglobulin molecules and fragments thereof, that have the ability to specifically bind to an antigen (e.g., human TF).
  • Full-length antibodies comprises four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected 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
  • CL The VH and VL regions can be further subdivided into regions of hypervariabil- ity, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • an antigen e.g., human TF
  • an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antibody” include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH I domains; (ii) F(ab) 2 and F(ab') 2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546 ), which consists of a VH domain; and (vi) an isolated complementarity de- termining region (CDR).
  • CDR complementarity de- termining region
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426: and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antibody”.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with cor ⁇ - plementary domains of another chain and creating two antigen binding sites (see e.g., Hol- liger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).
  • human TF may have one or more antigenic determinants comprising (1 ) peptide antigenic determinants which consist of single peptide chains within human TF, (2) conformational antigenic determinants which consist of more than one spatially contiguous peptide chains whose respective amino acid sequences are located disjointedly along the human TF polypeptide sequence; and (3) post-translational antigenic determinants which consist, either in whole or part, of molecular structures covalently attached to human TF after translation, such as carbohydrate groups, or the like.
  • antigenic determinants comprising (1 ) peptide antigenic determinants which consist of single peptide chains within human TF, (2) conformational antigenic determinants which consist of more than one spatially contiguous peptide chains whose respective amino acid sequences are located disjointedly along the human TF polypeptide sequence; and (3) post-translational antigenic determinants which consist, either in whole or part, of molecular structures covalently attached to human TF after translation, such
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences, e.g. the so-called humanized antibodies or human/mouse chimera antibodies.
  • an "isolated human antibody”, as used herein, is intended to refer to a human anti- body that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds human TF is substantially free of antibodies that specifically bind antigens other than human TF).
  • An isolated antibody that specifically binds human TF may, however, have cross-reactivity to other antigens, such as TF molecules from other species (discussed in further detail below).
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • epitopic determinants means any antigenic determinant on an antigen to which the antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the terms “immunoreacts” or “immunoreacting”, as used herein, means any binding of an antibody to its epitope with a dissociation constant K d lower than 10 "4 M.
  • dissociation constant K d lower than 10 "4 M.
  • the terms “immunoreacts” or “immunoreacting” are used where appropriate interchangeably with the term “ specifically bind”.
  • an antibody, which inhibits the binding of human coagulation factor Vila to human TF means any antibody, which reduces the ability of human coagulation factor Vila to bind human TF compared to the ability of human coagulation factor Vila to bind human TF in the absense of the antibody.
  • affinity means the strength of the binding of an antibody to an epitope.
  • the affinity of an antibody is measured by the dissociation constant K d , defined as [Ab] x [Ag] / [Ab-Ag] where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen.
  • K d dissociation constant
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a human antibody, which immunoreacts with an epitope present on human TF.
  • a therapheutically effective amount is the effective dose to be determined by a qualified practitioner, who may titrate dosages to achieve the desired response. Factors for consideration of dose will include potency, bioavailability, desired pharmacoki- netic/pharmacodynamic profiles, condition of treatment (e.g. trauma, inflammation, septic chock), patient-related factors (e.g. weight, health, age, etc.), presence of co-administered medications, time of administration, or other factors known to a medical practitioner.
  • the dosage of a human antibody against TF administered to a patient will vary with the type and severity of the condition to be treated, but is generally in the range of 0.1-5.0 mg/kg body weight.
  • the term "subject" as used herein is intended to mean any animal, in particular mammals, such as humans, and may, where appropriate, be used interchangeably with the term “patient”.
  • the invention relates to a composition comprising a human antibody, which immunoreacts with an epitope present on human TF.
  • the invention relates to a method for treatment of a FVIIa/TF related disorder in a human, which method comprises administering to the human a therapeutically effective amount of a human antibody, which immunoreacts with an epitope present on human TF.
  • Treatment means the administration of an effective amount of a therapeutically active compound of the invention with the purpose of preventing any symptoms or disease state to develop or with the purpose of curing or easing such symptoms or disease states already developed.
  • treatment is thus meant to include prophylactic treatment.
  • FVIIa/TF related disorder as used herein means a disease or disorder, where TF and FVIIa are involved.
  • thrombotic or coagulopathic related diseases or disorders including inflammatory response and chronic thromboembolic diseases or disorders associated with fibrin formation including vascular disorders such as deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass graft (CABG), percutaneous transdermal coronary angioplastry (PTCA), stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, arteriosclerosis and restenosis following angioplastry, acute and chronic indications such as inflammation, septic chock, septicemia, hypotension, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulo- pathy (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or the prophy- lactic treatment of mammals with atherosclerotic vessels at risk for thrombosis, and other diseases or disorders.
  • vascular disorders such as deep venous thrombosis, arterial thrombosis, post surgical thrombosis
  • the FVIIa/TF related disorder is not limited to in vivo coagulopatic disorders such as those named above but includes ex vivo FVIIa/TF related processes such as coagulation that may result from the extracorporeal circulation of blood, including blood removed in-line from a patient in such processes as dialysis procedures, blood filtration, or . blood bypass during surgery.
  • FVIIa means "two chain" activated coagulation factor VII cleaved by specific cleavage at the Arg152-lle153 peptide bond.
  • FVIIa may be purified from blood or produced by recombinant means. It is evident that the practice of the methods described herein is independent of how the purified factor Vila is derived and, therefore, the pre- sent invention is contemplated to cover use of any factor Vila preparation suitable for use herein. Preferred are human FVIIa.
  • the invention relates to a method for preparation of a human antibody, which method comprises a) Preparation of human antibodies against human TF, b) testing antibodies in a TF-induced clot assay and selecting human antibody which inhibit clot formation in this assay with an IC 50 value lower than 1 nM, such as lower than 500 pM, preferably lower than 200 pM, preferably lower than 100 pM, preferably lower than 50 pM, preferably lower than 10 pM, more preferably lower than 5 pM, or testing antibodies in a FXa generation assay and selecting human antibody which inhibit FXa generation with an IC 50 value lower than 100 nM (in an assay with a FVIIa concentration of 0.1 nM) , such as lower than 10 nM, preferably lower than 5 nM, preferably lower than 1 nM, more preferably lower than 0.1 nM, or testing antibodies
  • the invention relates to a method for preparation of a human antibody, which method comprises a) Preparation of human antibodies against human TF, b) testing antibodies in a TF-induced clot assay and selecting human antibody which inhibit clot formation in this assay with an IC 50 value lower than the IC 50 value of FFR-rFVIIa + 1 nM, such as lower than the IC 50 value of FFR-rFVIIa + 500 pM, preferably lower than the IC 50 value of FFR-rFVIIa + 200 pM, preferably lower than the IC 50 value of FFR-rFVIIa + 100 pM, preferably lower than the IC 50 value of FFR-rFVIIa + 50 pM, preferably lower than the IC 50 value of FFR-rFVIIa + 10 pM, more preferably lower than the IC 50 value of FFR-rFVIIa + 5 pM, more preferably lower than the IC 50 value of FFR-rFVI
  • TF-induced clot assay is intended to mean any assay where clotting time is measured in sample comprising blood plasma and TF.
  • An example of a TF-induced clot assay is described in example 1 , assay 7.
  • FXa generation assay as used herein is intended to mean any assay where activation of FX is measured in a sample comprising TF, FVIIa, FX, calcium and phospholipids.
  • An example of a FXa generation assay is described in example 1 , assay 5.
  • FVIIa/TF amidolytic assay as used herein is intended to mean any assay where the amidolytic activity, i.e. cleavage of a small peptide substrate, of FVIIa is measured in the presence of TF.
  • An example of a FVIIa/TF amidolytic assay is described in example 1 , assay 4.
  • TF ELISA assay as used herein is intended to mean any ELISA assay comprising TF and antibodies against TF.
  • Examples of TF ELISA assays are the direct and indirect TF ELISA assays described in example 1 , assay 1 and 2.
  • direct TF ELISA assay as used herein is intended to mean any TF ELISA assay comprising immobilized TF.
  • Example of direct TF ELISA assays is described in example 1 , assay 1.
  • the term "indirect TF ELISA assay” as used herein is intended to mean any TF ELISA assay, where TF is in solution.
  • Example of direct TF ELISA assays is described in example 1 , assay 2.
  • the invention relates to a human antibody which immunoreacts with an epitope present on human TF and inhibits the binding of human coagulation factor Vila to human TF obtainable by a method comprising: a) Preparation of human antibodies against human TF, b) testing antibodies in a TF-induced clot assay and selecting human antibody which inhibit clot formation in this assay with an IC 50 value lower than 1 nM, such as lower than 500 pM, preferably lower than 200 pM, preferably lower than 100 pMschreib preferably lower than 50 pM, preferably lower than 10 pM, more preferably lower than 5 pM, or testing antibodies in a FXa generation assay and selecting human antibody which inhibit FXa generation with an IC 50 value
  • the invention relates to a human antibody which immunoreacts with an epitope present on human TF and inhibits the binding of human coagulation factor Vila to human TF obtainable by a method comprising: a) Preparation of human antibodies against human TF, b) testing antibodies in a TF-induced clot assay and selecting human antibody which inhibit clot formation in this assay with an IC 50 value lower than the IC 50 value of FFR-rFVIIa + 1 nM, such as lower than the IC 50 value of FFR-rFVIIa + 500 pM, preferably lower than the IC 50 value of FFR-rFVIIa + 200 pM, preferably lower than the IC 50 value of FFR-rFVIIa + 100 pM, such as lower than the IC 50 value of FFR-rFVIIa + 50 pM, preferably lower than the IC 50 value of FFR-rFVIIa + 10 pM, more preferably lower than the
  • FFR-rFVIIa + 10 nM preferably lower than the IC 50 value of FFR-rFVIIa + 5 nM, preferably lower than the IC 50 value of FFR-rFVIIa + 1 nM, more preferably lower than the IC 50 value of FFR-rFVIIa + 0.1 nM, more preferably lower than the IC 50 value of FFR-rFVIIa, or testing antibodies in a FVIIa/TF amidolytic assay and selecting human antibody which inhibit TF-induced FVIIa amidolytic activity, with an IC 50 value lower than the IC 50 value of FFR-rFVIIa + 100 nM (using 10 nM FVIIa in the assay), such as lower than the IC 50 value of FFR-rFVIIa + 40 nM, preferably lower than the IC 50 value of FFR-rFVIIa + 20 nM, preferably lower than the IC 50 value of FFR-rFVIIa + 10
  • the method, wherein the human antibodies against human TF are produced comprises immunization of a mammal with human TF, and isolation of antibodies produced by the immunized mammal.
  • the mammal is a mouse. It is to be understod, that the immunized mammal or mouse is capable of producing human antibodies.
  • the invention relates to a method for preparation of a human antibody, which method comprises a) immunization of mouse with human TF, b) isolation of antibody-producing cell from immunized mouse and preparation of immortal cells to secrete human antibodies, c) isolation of culture medium from immortal cells comprising produced antibodies, d) testing antibodies in an indirect TF ELISA assay comprising TF in solution and selecting human antibodies which binds human TF in solution, e) testing antibodies in a FVIIa competition assay and selecting human antibodies which competes with FVIIa binding, f) testing antibodies in a FVIIa/TF amidolytic assay and selecting human anti- bodies which inhibits TF-induced FVIIa amidolytic activity, with an IC 50 value lower than 100 nM (in an assay with a FVIIa concentration of 10 nM), such as lower than 40 nM, preferable lower than 20 nM, more preferably lower than 10 nM , g) testing antibodies in a FX
  • the invention relates to a method for preparation of a human antibody, which method comprises a) immunization of mouse with human TF, b) isolation of antibody-producing cell from immunized mouse and preparation of immortal cells to secrete human antibodies, c) isolation of culture medium from immortal cells comprising produced antibodies, d) testing antibodies in an indirect TF ELISA assay comprising TF in solution and selecting human antibodies which immunoreacts with human TF in solution, e) testing antibodies in a FVIIa competition assay and selecting human antibodies which competes with FVIIa binding, f) testing antibodies in a FVIIa/TF amidolytic assay and selecting human antibody which inhibit TF-induced FVIIa amidolytic activity, with an IC 50 value lower than the IC 50 value of FFR-rFVIIa + 100 nM (using 10 nM FVIIa
  • antibody-producing cell means any cell capable of producing an antibody. Included are hybridomas, transfected cell lines and the relatively shortlived, or mortal, splenocytes or lymphocytes from a mammal which has been injected with an antigen.
  • the invention relates to a human antibody which immunoreacts with an epitope present on human TF and inhibits the binding of human coagulation factor Vila to human TF obtainable by a method comprising: a) immunization of mouse with human TF, b) isolation of antibody-producing cells from immunized mouse and preparation of immortal cells to secrete human antibodies, c) isolation of culture medium from immortal cells comprising produced antibodies, d) testing antibodies in an indirect TF ELISA assay comprising TF in solution and selecting human antibodies which binds human TF in solution, e) testing antibodies in a FVIIa competition assay and selecting human antibodies which competes with FVIIa binding, f) testing antibodies in a FVIIa/TF amidolytic assay and selecting human antibodies which inhibits TF-induced FVIIa amidolytic activity, with an IC 50 value lower than 100 nM (in an assay with a FVIIa concentration of 10 nM), such as lower than 40 nM, prefer
  • the invention relates to a human antibody which immunoreacts with an epitope present on human TF and inhibits the binding of human coagulation factor Vila to human TF obtainable by a method comprising: a) immunization of mouse with human TF, b) isolation of antibody-producing cell from immunized mouse and preparation of immortal cells to secrete human antibodies, c) isolation of culture medium from immortal cells comprising produced antibodies, d) testing antibodies in an indirect TF ELISA assay comprising TF in solution and selecting human antibodies which immunoreacts with human TF in solution, e) testing antibodies in a FVIIa competition assay and selecting human antibodies which competes with FVIIa binding, f) testing antibodies in a FVIIa/TF amidolytic assay and selecting human antibody which inhibit TF-induced FVIIa amidolytic activity, with an IC 50 value lower than the IC- 5 0 value of FFR-rFVIIa + 100 nM (using 10 nM FVIIa
  • the immortal cell is a hybridoma cell.
  • the invention relates to a cell producing human antibodies which immunoreacts with an epitope present on human TF and inhibits the binding of human co- agulation factor Vila to human TF.
  • the cell is an isolated lymphoid cell. In a further embodiment the cell is isolated from a mouse.
  • the cell is a hybridoma cell.
  • the hybridoma cell is obtained by fusion of an antibody-producing lymphoid cell with an immortal cell to provide an antibody-producing hybridoma cell.
  • the isolated human antibody inhibits the binding of human coagulation factor Vila to human TF.
  • the isolated human antibody is a monoclonal antibody.
  • monoclonal antibody refers to a homogeneous population of immunoglobulins, i.e. the individual molecules of the antibody population are identical except for naturally occurring mutations.
  • Antibodies are normally synthesized by lymphoid cells derived from B lymphocytes of bone marrow. Lymphocytes derived from the same clone produce immunoglobulin of a single amino acid sequence. Lymphocytes can not be directly cultured over long periods of time to produce substantial amounts of their specific antibody.
  • the isolated human antibody is a recombinant antibody.
  • recombinant antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section II, below), antibodies isolated from a recombinant, combinatorial human antibody library (described further in Section III, below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human lg sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the isolated human antibody is a Fab frag- ment.
  • the isolated human antibody is a F(ab) 2 fragment.
  • the isolated human antibody is a F(ab') 2 fragment. In a further embodiment of the invention the isolated human antibody is a single chain Fv fragment.
  • the isolated human antibody has a K d for binding to human TF within the range of 10 "15 - 10 "8 M. It is to be understood, that the K d for human antibody binding to human TF referred to is as determined in an assay, wherein the human antibody is immobilized (see assay 6). In a further embodiment of the invention the isolated human antibody has a K d for binding to human TF within the range of 10 "15 - 10 "10 M.
  • the isolated human antibody has a K d for binding to human TF lower than 10 "8 M. In a further embodiment of the invention the isolated human antibody has a K d for binding to human TF lower than 10 "9 M In a further embodiment of the invention the isolated human antibody has a K d for binding to human TF lower than 10 " 10 M. In a further embodiment of the invention the isolated human antibody has a K d for binding to human TF lower than 10 "11 M. In a further embodiment of the invention the isolated human antibody has a K d for binding to human TF lower than 10 "12 M. In a further embodi- ment of the invention the isolated human antibody has a K d for binding to human TF lower than 10 "13 M. In a further embodiment of the invention the isolated human antibody has a K d for binding to human TF lower than 10 "14 M. In a further embodiment of the invention the isolated human antibody has a K d for binding to human TF lower than 10 ⁇ 15 M.
  • the method for preparation of a human an- tibody comprises testing antibodies in a TF-induced clot assay and selecting human antibody which inhibit clot formation in this assay with an IC 50 value lower than 1 nM.
  • the IC 50 value is lower than 500 pM.
  • the IC 50 value is lower than 200 pM.
  • the IC 0 value is lower than 100 pM.
  • the IC 50 value is lower than 50 pM.
  • the IC 50 value is lower than 10 pM.
  • the IC 50 value is lower than 5 pM.
  • the method for preparation of a human antibody comprises testing antibodies in a TF-induced clot assay and selecting human antibody which inhibit clot formation in this assay with an IC 50 value lower than the IC 50 value of FFR- rFVIIa + 1 nM, such as lower than the IC 50 value of FFR-rFVIIa + 500 pM, preferably lower than the IC 50 value of FFR-rFVIIa + 200 pM, preferably lower than the IC 50 value of FFR- rFVIIa + 100 pM, such as lower than the IC 50 value of FFR-rFVIIa + 50 pM, preferably lower than the IC 50 value of FFR-rFVIIa + 10 pM, more preferably lower than the IC 50 value of FFR- rFVIIa + 5 pM, more preferably lower than the IC 50 value of FFR-rFVIIa.
  • the method for preparation of a human antibody comprises testing antibodies in a FXa generation assay and selecting human antibody which inhibit FXa generation with an IC 50 value lower than 100 nM (in an assay with a FVIIa concentration of 0.1 nM)
  • the IC 50 value is lower than 10 nM.
  • the IC 50 value is lower than 5 nM.
  • the IC 50 value is lower than 1 nM .
  • the IC 50 value is lower than 0.1 nM.
  • the method for preparation of a human antibody comprises testing antibodies in a FXa generation assay and selecting human antibody which inhibit FXa generation with an IC 50 value lower than the IC 50 value of FFR-rFVIIa + 100 nM (using 0.1 nM FVIIa in the assay), such as lower than the IC 50 value of FFR-rFVIIa + 10 nM, preferably lower than the IC 50 value of FFR-rFVIIa + 5 nM, preferably lower than the IC 50 value of FFR-rFVIIa + 1 nM, more preferably lower than the IC 50 value of FFR-rFVIIa + 0.1 nM, more preferably lower than the IC 50 value of FFR-rFVIIa.
  • the method for preparation of a human antibody comprises testing antibodies in a FVIIa/TF amidolytic assay and selecting human antibody which inhibit TF-induced FVIIa amidolytic activity with an IC 50 value lower than 100 nM (in an assay with a FVIIa concentration of 10 nM).
  • the IC 50 value is lower than 40 nM.
  • the IC 50 value is lower than 20 nM.
  • the IC 50 value is lower than 10 nM.
  • the IC 50 value is lower than 5 nM.
  • the method for preparation of a human antibody comprises testing antibodies in a FVIIa/TF amidolytic assay and selecting human antibody which inhibit TF-induced FVIIa amidolytic activity, with an IC 50 value lower than the IC 50 value of FFR-rFVIIa + 100 nM (using 10 nM FVIIa in the assay), such as lower than the IC 50 value of FFR-rFVIIa + 40 nM, preferably lower than the IC 50 value of FFR-rFVIIa + 20 nM, preferably lower than the IC 50 value of FFR-rFVIIa + 10 nM, more preferably lower than the IC 50 value of FFR-rFVIIa.
  • the method for preparation of a human an- tibody comprises testing antibodies in a FVIIa competition assay and selecting human antibody which compete with FVIIa binding.
  • the method for preparation of a human antibody comprises testing antibodies in a TF ELISA assay comprising TF and selecting human antibody which bind human TF.
  • the method for preparation of a human antibody comprises testing antibodies in a direct TF ELISA assay comprising immobilized TF and selecting human antibodies which binds immobilized human TF.
  • the method for preparation of a human antibody comprises testing antibodies in an indirect TF ELISA assay comprising TF in solution and selecting human antibodies which binds human TF in solution
  • the method for preparation of a human antibody comprises testing antibodies in a FXa generation assay on TF expressing cell and selecting human antibodies which inhibits FXa generation on TF expressing cell with an IC 50 value lower than 500 nM (in an assay with a FVIIa concentration of 1 nM).
  • the IC 50 value is lower than 100 nM.
  • the IC 50 value is lower than 50 nM
  • the IC 50 value is lower than 10 nM
  • the IC 50 value is lower than 5 nM.
  • the method for preparation of a human antibody comprises testing antibodies in a FXa generation assay on TF expressing cell and se- lecting human antibodies which inhibits FXa generation on TF expressing cell with an IC 50 value lower than the IC 50 value of FFR-rFVIIa + 500 nM (using 1 nM FVIIa in the assay), such as lower than the IC 50 value of FFR-rFVIIa + 100 nM, preferably lower than the IC 50 value of FFR-rFVIIa + 50 nM preferably lower than the IC 50 value of FFR-rFVIIa + 10 nM, more preferably lower than the IC 50 value of FFR-rFVIIa + 5 nM, more preferably lower than the IC 50 value of FFR-rFVIIa.
  • TF expressing cell mean any mammalian cell, that expresses human TF.
  • the method for preparation of a human antibody comprises testing antibodies in a whole cell TF binding assay and selecting human antibodies which competes with FVIIa binding to human TF expressed on the cell surface of whole cells.
  • the method for preparation of a human antibody comprises testing antibodies in a biosensor assay and selecting human antibodies with a K value for binding to human TF lower than 100 nM.
  • the K value for binding to human TF is lower than 10 nM.
  • the K d value for binding to human TF is lower than 5 nM.
  • the K d value for binding to human TF is lower than 1 nM.
  • the K d value for binding to human TF is lower than 0.5 nM.
  • the K d value for binding to human TF is lower than 10 "10 M.
  • the K value for binding to human TF is lower than 10 "11 M.
  • the K 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.
  • the method for preparation of a human antibody comprises testing antibodies in a MAPK signalling assay and selecting human anti- bodies which inhibits FVIIa-induced activation of the MAPK signalling.
  • the human antibody inhibits FVIIa-induced activation of the MAPK signalling with 98 %.
  • the human antibody inhibits FVIIa-induced activation of the MAPK signalling with 90 %.
  • the human antibody inhibits FVIIa-induced activation of the MAPK signalling with 70 %.
  • the human antibody inhibits FVIIa-induced activation of the MAPK signalling with 50 %.
  • the human antibody inhibits FVIIa- induced activation of the MAPK signalling with 30 %.
  • MAPK signalling is intended to mean a cascade of intracellular events that mediate activation of Mitogen-Activated-Protein-Kinase (MAPK) or homologues thereof in response to various extracellular stimuli.
  • MAPK Mitogen-Activated-Protein-Kinase
  • Three distinct groups of MAP kinases have been identified in mammalian cells: 1 ) extracellular-regulated kinase (Erk1/2 or p44/42), 2) c-Jun N-terminal kinase (JNK) and 3) p38 kinase.
  • the Erk1/2 pathway involves phosphorylation of Erk 1 (p 44) and/or Erk 2 (p 42).
  • Activated MAP kinases e.g.
  • p44/42 MAPK can translocate to the nucleus where they can phosphorylate and activate transcription factors including (Elk 1) and signal transducers and activators of transcription (Stat). Erk1/2 can also phosphorylate the kinase p90RSK in the cytoplasm or in the nucleus, and p90RSK then can activate several transcription factors.
  • protein kinase is intended to indicate an enzyme that is capable of phosphorylating serine and/or threonine and/or tyrosine in peptides and/or proteins.
  • FVIIa-induced activation of the MAPK signalling is intended to indicate that FVIIa binds to TF in a mammalian cell and thereby induce MAPK signalling.
  • the method for preparation of a human antibody comprises testing antibodies in a gene expression analysis assay (assay 15) and selecting human antibodies which inhibits FVIIa induced up-regulation of genes independently selected from the list comprising Fra-1 , Id2, and Cyr61. It is to be understood, that antibodies against TF, which inhibits the activity of TF may bind different epitopes present on TF and may inhibit both the binding of FVIIa or the binding of FX or FXa to human TF. It is an object of the present invention to select antibodies, which inhibits the binding of FVIIa to human TF and thereby the FVIIa induced intracellular signalling.
  • the method for preparation of a human antibody comprises testing antibodies in a human cancer assay (assay 13) and selecting human antibodies which inhibits growth or metastasis of human cancers.
  • the isolated human antibody inhibits FVIIa induced up-regulation of genes independently selected from the list comprising Fra-1 , Id2, and Cyr61. In a further embodiment of the invention the isolated human antibody does not inhibit binding of FX or FXa to human TF.
  • the isolated human antibody inhibits the intracellular activity of TF.
  • the method for preparation of a human antibody comprises testing antibodies in an epitope mapping assay and selecting human antibodies which binds preferred epitopes on TF.
  • the preferred epitope comprises one or more of the residues Trp45, Lys46 and Tyr94.
  • the preferred epitope comprises the residue Trp45.
  • the preferred epitope comprises the residue Lys46.
  • the preferred epitope comprises the residue Tyr94.
  • the isolated human antibody binds to an epitope within the interface between TF and FVIIa.
  • the residues in TF that are responsible for the interaction between the protease domain of FVIIa and TF determined from the X-ray structure are; Ser39, Gly43, Trp45, Ser47, Phe50, Arg 74, Phe76, Tyr94, Pro92.
  • This interface between the protease domain of FVIIa and TF is characterized as being a complex interface region containing many intermolecular hydrogen bonds allowing many fine contacts between TF and FVIIa to obtain high specificity in binding process of FVIIa.
  • the present invention also relates to high affinity human monoclonal antibodies to TF.
  • the TF surface containing the contact interface for the protease domain of FVIIa holds a specific topology that are prone to react to create protein-protein interactions, wherein another type of protein-protein interaction is the complex formation between an antibody and a protein ligand.
  • monoclonal antibodies directed against this epitope on human TF gives high affinity Mabs.
  • One aspect of the present invention is high affinity human monoclonal antibodies, that are immunoreacting with the contact interface for the protease domain of FVIIa.
  • Human TF antibodies of the present invention act as antagonists for TF-mediated induction of coagulation, thus inhibiting the binding of coagulation FVIIa to TF and thereby blocking the production of thrombin and the subsequent deposition of fibrin.
  • Human TF anti- bodies are particularly useful for administration to humans to treat a variety of conditions involving intravascular coagulation.
  • human TF antibodies may be useful for inhibiting TF activity resulting in, for example, the inhibition of blood coagulation, thrombosis or platelet deposition.
  • human TF antibodies according to the present invention which acts to inhibit the cellular functions of TF, the signalling function of TF, may be useful in conditions like sepsis, inflammation, atherosclerosis, restenosis, or cancer.
  • Human TF antibodies may be useful in a variety of diseases. Included are thrombotic or coagulopathic related diseases or disorders including inflammatory response and chronic thromboembolic diseases or disorders associated with fibrin formation including vascular disorders such as deep venous thrombosis, arterial thrombosis, post surgical thrombosis, coronary artery bypass graft (CABG), percutaneous transdermal coronary angioplastry (PTCA), stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, arteriosclerosis and restenosis following angioplastry, acute and chronic indications such as inflammation, septic chock, septicemia, hypotension, adult respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), disseminated intravascular coagulopathy (DIC), pulmonary embolism, pathological platelet deposition, myocardial infarction, or the prophylactic treatment of mammals with atherosclerotic vessels at risk for thrombosis, venoocclusive disease following peripheral blood progen
  • the human TF antibodies may be used to prevent the occurrence of thromboembolic complications in identified high risk patients, such as those undergoing surgery or those with congestive heart failure.
  • the human TF antibodies may be particularly useful in the treatment of intimal hyperplasia or restenosis due to acute vascular injury.
  • Acute vascular injuries are those which occur rapidly (i.e. over hours to months, in contrast to chronic vascular injuries (e.g. atherosclerosis) which develop over a lifetime.
  • Acute vascular injuries often result from surgical procedures such as vascular reconstruction, wherein the techniques of angioplasty, endarterectomy, atherectomy, vascular graft emplacement or the like are employed.
  • Hyperplasia may also occur as a delayed response in response to, e.g., graft emplacement or organ transplantation. Since human TF antibodies is more selective than heparin, binding only TF which has been exposed at sites of injury, and because human TF antibodies does not destroy or inhibit other coagulation proteins, it will be more effective and less likely to cause bleeding complications than heparin when used prophylactically for the prevention of deep vein thrombosis.
  • the human TF antibodies of the present invention is able to bind selectively to cell-surface TF and inhibit its functional activity by inhibiting the binding of coagulation FVIIa to TF.
  • Human TF antibodies which maintain binding to TF inhibit platelet accumulation at the site of vascular injury by blocking the production of thrombin and the subsequent deposition of fibrin.
  • human TF antibodies Due to the ability of human TF antibodies to block thrombin generation and limit platelet deposition at sites of acute vascular injury, human TF antibodies which maintain binding to TF thereby inhibiting FVIIa binding can be used to inhibit vascular restenosis.
  • Compositions comprising human TF antibodies are particularly useful in methods for treating patients when formulated into pharmaceutical compositions, where they may be given to individuals suffering from a variety of disease states to treat coagulation-related conditions.
  • Such human TF antibodies, capable of binding TF and inhibiting FVIIa binding to TF may possess a longer plasma half-life and thus a correspondingly longer period of anticoagulant activity when compared to other anticoagulants.
  • compositions are those commonly treated with anticoagulants, such as, for example, deep vein thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIC), fibrin deposition in lungs and kidneys associated with sepsis, antiphospholipid syndrome (APS), atherosclerosis and myocardial infarction.
  • anticoagulants such as, for example, deep vein thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIC), fibrin deposition in lungs and kidneys associated with sepsis, antiphospholipid syndrome (APS), atherosclerosis and myocardial infarction.
  • the compositions can be used to inhibit vascular restenosis as occurs following mechanical vascular injury, such as injury caused by surgery, microsurgery, balloon angioplasty, endarterectomy, reductive atherectomy, stent placement, laser therapy or rotablation, or as occurs secondary to vascular grafts, stents, bypass grafts or organ transplants.
  • a method of inhibiting coagulation, vascular restenosis or platelet deposition comprises administering to a patient a composition comprising human TF antibodies in an amount sufficient to effectively inhibit coagulation, vascular restenosis or platelet deposition.
  • the methods also find use in the treatment of acute closure of a coronary artery in an individual (e.g. acute myocardial infarction), which comprises administering the human TF antibodies, in conjunction with tissue plasminogen activator or streptokinase, and can accelerate tPA induced thrombolysis.
  • the human TF antibodies are given prior to, in conjunction with, or shortly following administration of a thrombolytic agent, such as tissue plasminogen activator.
  • a thrombolytic agent such as tissue plasminogen activator.
  • TF may be produced by immunizing transgenic mice (Obtained from Medarex) carrying parts of the human immune system rather than the mouse system with human TF. Splenocytes from these transgenic mice are used to produce hybridomas that secrete human monoclonal antibodies as described (see, e.g., 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, L. L. et al. 1994 Nature Genet.
  • Human monoclonal antibodies directed against human TF may also be produced by phage display.
  • Human antibody libraries can be constructed from immunized persons and displayed on the surface of filamentous phage.
  • Figure 1 Schematic presentation of an examplified screening assay for selection of human monoclonal high affinity antibodies against TF.
  • Figure 5 An example of screening antibodies by assay no. 4. Inhibition of sTF/FVIIa amidolytic activity by FFR-rFVIIa (closed circles) and the human anti TF monoclonal antibody HuTF-31 F2 (open circles).
  • Figure 6. An example of screening antibodies by assay no. 5. Inhibition of factor Xa generation by FFR-rFVIIa (closed circles) and the human anti-TF monoclonal antibody HuTF-31 F2 (open circles).
  • Figure 7. An example of screening antibodies by assay no. 7. Inhibition of human TF-induced clotting by FFR-rFVIIa (closed circles) and the human anti TF monoclonal antibody HuTF- 31 F2 (open circles).
  • Figure 8 An example of screening antibodies by assay no. 10. Only anti-TF monoclonal antibodies preventing FVIIa binding inhibits TF/FVI la-mediated signaling.
  • FIG. 9 Human anti TF Mab inhibit FVIIa induced phosphorylation of p44/42 MAPK (assay no. 10). BHK cell transfected with TF were serum-starved for 2 hr to make cells quiescent. The antibodies HuMab 30F5 (500 nM) and HuMab 31 F2 (500 nM) were added to the cells 15 min prior addition of FVIIa (15 nM). Cells were lysed and proteins were separate on SDS- PAGE and transferred to nitrocellulose by electroblotting. Western blot analysis was performed using polyclonal phospho-specific antibodies to p44/42 MAPK. Secondary antibodies were anti-rabbit IgG conjugated to Horse Radish Peroxidase.
  • Figure 10 An example of screening antibodies by assay no. 16. The figure demonstrates the inhibition of TF intracellular activity in TF expressing cells by monoclonal antibodies against TF. Anti TF Mab B inhibits TF intracellular activity, while Anti-TF Mab A do not.
  • FIG. 11 An example of screening antibodies by assay no. 12. Velocity profile of thromboe- lastograms obtained with 0.5 nM of FFR-rFVIIa and the human anti-TF antibody HuTF-31 F2.
  • Human TF can be isolated from human brain as described by Rao, L.V.M., Thrombosis Research, 51 :373-384 1988.
  • Lipidated recombinant human TF can also be used as human thromboplastin reagent.
  • Rat, rabbit, baboon, and pig 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 homogenisator. After 30 min incubation at 45°C with occasional shaking, the samples are centrifuged 20 min at 2000 x g. The precipitate are discarded, and the supernatant is aliquoted and stored at -80°C until use.
  • Relipidated TF may be obtained by reconstitution of recombinant human full length TF (American Diagnostica #4500) into phospholipid vesicles (PC/PS 75/25).
  • Biotinylated human TF is produced as follows: Biotin-NHS (n-succinimido biotin,
  • FVIIa used is recombinant human FVIIa prepared as described by Thim, L. et al. Biochem 27: 7785-7793, 1988.
  • sTF Recombinant human soluble TF 1-209 expressed in E. coli and purified essentially as described by Freskgard, P.-O. et al. Protein Sci. 5, 1531-1540 (1996).
  • S2288 reconstituted in H 2 O to 17.24 mg/ml (Chromogenix)
  • FX Purified human plasma FX (HFX, Enzyme Research Laboratories Ltd.)
  • FXa Purified human plasma FX activated with Russel's Viper Venom (HFXa, Enzyme Research Laboratories Ltd.)
  • Chromozyme X Dissolved in H 2 O to 1.25 mg/ml. (Boehringer Mannheim) 125 l-FVIIa is obtained by standard radiolabelling procedures.
  • FFR-rFVIIa FVIIa blocked in the active site with D-Phe-L-Phe-L-Arg-chloromethyl ketone. Prepared as described by Sorensen B. B. et al. J.Biol.Chem. 272: 11863-11868, 1997. Immunization.
  • Human TF is emulsified in Freunds Complete Adjuvant.
  • HuMab mice or hybrids therof (Medarex) are given 40 ⁇ g by a subcutaneous injection. After 14 and 28 days, and eventually more times with intervals of 14 days the mice are boosted with a similar injection of 20 ⁇ g of TF in Incomplete Freunds Adjuvant. Ten days after the last injection a blood sample is taken and sera are tested for human TF specific antibodies by TF ELISA (Assay 1 and 2).
  • mice with positive serum test from assay 1-3 are boosted with 20 ⁇ g of human TF by an intravenous injection and sacrificed after three days. The spleen is removed aseptically and dispersed to a single cell suspension.
  • Fox-myeloma cells are grown in CD Hybridoma medium (Gibco 11279-023 ). Fusion of spleen cells and myeloma cells (P3x63 Ag8.653, ATCC CRL-1580), and the Sp2/0 (ATCC CRL-1581) myeloma cell lines for our fusions are done by the PEG- methods (Kohler, G & Milstein C. (1976), European J. Immunology, 6:511-19). Cells are seeded in microtiter plates and incubated at 37 °C. Medium is changed three times over the next two weeks. 100 ⁇ l of supernatant from hybridoma cells is removed from each well and tested for TF specific antibodies in TF ELISA (Assay 1 and 2).
  • Nunc immunoplates are coated overnight at 4°C with 1 ⁇ g/ml of human sTF in PBS. Plates are blocked with blocking buffer (TBS with 5 mM CaCI 2 and 2% BSA) and washed in TBS+ 0.05 % Tween 20, and the supematants from the hybridoma cells are added. After incubation at room temperature for 1 hour, plates are washed and anti-human IgG labelled with horseradish peroxidase (HRPO) is added. After another hour of incubation, plates are washed and developed with TMB-substate (Kem-EN-Tec) as described by the manufactures. Absorbance at 450 nm is measured on an ELISA-reader. Indirect TF ELISA assay (Assay 2):
  • Nunc-immunoplates are coated with 0.5 ⁇ g/ml of goat anti-human IgG (Southern Biotechnology Associates, Cat-#2040-1 ) in PBS and incubated overnight at 4°C. Plates are blocked with blocking buffer (TBS with 5 mM CaCI 2 and 2% BSA) and washed in TBS+ 5 mM CaCI 2 + 0.05 % Tween 20. Culture supematants from the hybridoma cells are added and the plates incubated for 1 hour at room temperature. After another wash, biotinylated human sTF are added at a concentration of 1 ⁇ g/ml, and incubated for 1 hour. After washing, 100 ⁇ l of a Streptavidin-HRPO solution is added and incubated for 1 hour. Plates are developed with TMB-substate as described for assay 1.
  • Nunc-immunoplates are incubated with human sTF (cone 5 ⁇ g/ml in PBS) over night, 4 °C. Plates are washed and blocked in TBS buffer with 5 mM CaCI 2 and 2 % BSA. Anti-human TF Mabs are added and plates are incubation for 2 hours. Plates are washed before biotinylated human FVIIa are added (1 ⁇ g/ml in TBS buffer with 5 mM CaCI 2 and 2 % BSA) and the plates incubated for 1 hour. Plates are washed before addition of HRPO-labeled Streptavidin and incubated for 45 min. Plates are washed again before development with TMB substrate (Kem-EN-Tec) as described by the manufactures.
  • TMB substrate Kerm-EN-Tec
  • Inhibition of FVIIa/sTF amidolytic activity (Assay 4): Inhibition of FVIIa-TF catalyzed amidolytic activity by anti-human TF Mabs is tested employing soluble human TF (10 nM), recombinant human FVIIa (10 nM) and increasing concentrations of Mabs (0.0122 - 50 nM).
  • Varying concentrations of anti-human TF Mabs or FFR-rFVIIa are preincubated with 10 nM sTF and 10 nM FVIIa in BSA buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 5 mM CaCI 2 and 1 mg/ml BSA) for 60 min at room temperature before addition of substrate S2288 (1.2 mM, Chromogenix). The colour development is measured continuously for 30 min at 405 nm. Amidolytic activity is presented as mOD/ in. IC 50 values for inhibition of FVIIa/TF amidolytic activity by the Mabs may be calculated. The IC 50 value for FFR-rFVIIa is 7 +/- 3 nM in this assay.
  • FXa generation (Assay 5). Lipidated TF (10 pM), FVIIa (100 pM) and anti-TF Mabs or FFR-rFVIIa (0 - 50 nM) in BSA buffer (see assay 4) are incubated 60 min at room temperature before FX (50 nM ) is added. The reaction is stopped after another 10 min by addition of 1 /a volume stopping buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 20 mM EDTA). The amount of FXa generated is de- termined by adding substrate S2765 (0.6 mM, Chromogenix, and measuring absorbance at 405 nm continuously for 10 min. IC 50 values for Mab inhibition of FVIIa/lipidated TF-mediated activation of FX may be calculated. The IC 5 o value for FFR-rFVIIa is 51 +/- 26 pM in this assay.
  • Biosensor assay 6 (Assay 6):
  • Antibodies are tested on the Biacore instrument by passing a standard solution of anti-human TF Mab over a chip with immobilized antibody to human IgG. This is followed by different concentrations of sTF in 10 mM hepes pH 7.4 containing 150 mM NaCl, 10 mM CaCI 2 and 0.0003 % polysorbate 20. K d values are calculated from the sensorgrams using the integrated Biacore evaluation software.
  • TF-dependent clotting assay (Assay 7): The assay is carried out on an ACL300 Research clotting apparatus (ILS Laboratories). Dilutions of anti-human TF Mabs in 50 mM imidazole, pH 7.4, 100 mM NaCl, 0.1 % BSA are mixed with 25 mM CaCI 2 in the ratio of 2 to 5 and added to sample cups in the clotting apparatus. Thromboplastin from human, rat, rabbit, baboon, or pig diluted with the imidazole buffer to give clotting time of approximately 30 sec in samples without antibody is placed in reagent reservoir 2, and human, rat, rabbit, baboon, or pig plasma, in reagent reservoir 3.
  • Monolayers of cells expressing human TF e.g. human lung fibroblasts 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 , are employed as TF source in FVIIa/TF catalyzed activation of FX.
  • human lung fibroblasts 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
  • Confluent cell monolayers in a 24-, 48- or 96-well plate are washed one time in buffer A (10 mM Hepes, pH 7.45, 150 mM NaCl, 4 mM KCI, and 11 mM glucose) and one time in buffer B (buffer A supplemented with 1 mg/ml BSA and 5 mM Ca 2+ ).
  • buffer A 10 mM Hepes, pH 7.45, 150 mM NaCl, 4 mM KCI, and 11 mM glucose
  • buffer B buffer A supplemented with 1 mg/ml BSA and 5 mM Ca 2+
  • FX (135 nM
  • varying concentrations of Mab (or FFR-rFVIIa) in buffer B are simultaneously added to the cells.
  • the cells are preincubated 15 min with anti-TF Mabs or FFR- rFVIIa before addition of rFVIIa and FX.
  • FXa formation is allowed for 15 min at 37°C. 50- ⁇ l aliquots are removed from each well and added to 50 ⁇ l stopping buffer (Buffer A supplemented with 10 mM EDTA and 1 mg/ml BSA). The amount of FXa generated is determined by transferring 50 ⁇ l of the above mixture to a microtiter plate well and adding 25 ⁇ l Chromo- zym X (final concentration 0.6 mM) to the wells. The absorbance at 405 nm is measured con- tinuously and the initial rates of colour development are converted to FXa concentrations using a FXa standard curve. The IC 50 value for FFR-rFVIIa is 1.5 nM in this assay.
  • Binding studies are employed using cells expressing human TF, e.g. human lung fi- broblasts 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.
  • Confluent monolayers in 24-well tissue culture plates are washed once with buffer A (see assay 8) and once with buffer B (see assay 8). The monolayers are preincubated 2 min with 100 ⁇ l cold buffer B.
  • Varying concentrations of Mabs (or FFR-rFVIIa) and radiolabelled FVIIa are simultaneously added to the cells (final volume 200 ⁇ l).
  • the plates are incubated for 2 hours at 4 °C.
  • the unbound material is removed, the cells are washed 4 times with ice-cold buffer B and lysed with 300 ⁇ l lysis buffer (200 mM NaOH, 1 % SDS and 10 mM EDTA). Radioactivity is measured in a gamma counter (Cobra, Packard Instruments).
  • the binding data are analyzed and curve fitted using GraFit4 (Erithacus Software, Ltd., (U.K.).
  • the IC 50 value for FFR-rFVIIa is 4 nM in this assay.
  • the amount of phosphorylated p44/42 MAPK and/or Akt, and/or p90RSK is deter- mined by quantitative detection of chemiluminescence (Fujifilm LAS-1000) from western blot analysis.
  • Cells expressing human TF e.g. CCD1102KerTr, NHEK P166, human glioblastoma cell line U87, or human breast cancer cell line MDA-MB231 , are cultured in medium with 0 - 0.1 % FCS for 24 or 48 hours prior to the experiment to make cells quiescent. At the day of the experiment the cells must be 70-80% confluent.
  • the experiment is performed by prein- cubating the cells with excess Mab or FFR-rFVIIa in medium without serum for 30 min at 37°C before addition of 10 - 100 nM FVIIa and incubating for 10 min.
  • cells are treated with 10 % FCS for 10 minutes.
  • Cells are washed 2 times in ice-cold PBS before cells are lysed in lysis buffer (20 mM Tris, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium-fluoride, 10 mM sodium ⁇ -glycerophosphate, 5 mM sodium pyrophosphate, 150 mM NaCl, pH 7.5 containing 0.1 mM 4-(2-aminoethyl)benzene- sulfonyl fluoride (AEBSF) and 1 mM benzamidine.
  • AEBSF 4-(2-aminoethyl)benzene- sulfonyl fluoride
  • lysates were mixed with SDS-sample buffer and loaded on a SDS-polyacrylamide gel. A standard biotinylated protein marker is loaded on each gel. Proteins separated on the SDS-polyacrylamide gel were transferred to nitrocellulose by electroblotting, and the kinases p44/42 MAPK, Akt and p90RSK were visualized by immunoblotting with phosphospecific antibodies, and chemiluminiscence is quaniti- ated by Fujifilm LAS1000.
  • sTF variants I22C, W45C, K46C, Y94C, F140C, W158C, K201C
  • sTF variants I22C, W45C, K46C, Y94C, F140C, W158C, K201C
  • Refolding is achieved after 1-hour incubation at room temperature by dropwise diluting the denatured protein into a 1 L solution containing 50 mM Tris-HCI, 0.25 M NaCl, pH 8.0 with gentle stirring for approximately 2 hours. Purification is performed using Q- Sepharose Fast Flow (Pharmacia, Uppsala, Sweden) and FVIIa affinity chromatography as described by Freskg ⁇ rd et al. (1996). The homogeneity of the protein is verified by SDS- PAGE. The concentration is measured at A 28 o nm and determined using a calculated extinction coefficient of 37440 M "1 cm "1 (Gill and von Hippel, 1989).
  • Human thromboplastin e.g. Innovin, Dade Behring, final dilution 50,000 x
  • CaCI 2 final concentration 16.7 mM
  • anti-TF Mabs incubated 15 min at room temperature.
  • Citrate-stabilized human whole blood 280 ⁇ l
  • RoTEG sample cups Pentapharm
  • 40 ⁇ l throm- boplastin/CaCI 2 /anti-TF Mab mixture Thromboelastography is followed for one hour in a RoTEG apparatus (Pentapharm).
  • Velocity profiles are obtained from the thrombograms using CoagPro SoftwareTM (MedScience, Arhus, Denmark).
  • mice Primary growth and liver metastasis of colon cancer Healthy female athymic mice (nu/nu) aged 7-8 weeks are used. To destroy the residual immunoresistance of the nude mice to the human cell implantation, the mice are routinely irradiated at 5 Gy 2 days before human tumor grafting (Vogel et al., 1997). Mice are challenged by tumor grafting of LS174T humancolon carcinoma 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).
  • FCS fetal calf serum
  • the cells are harvested with trypsin-EDTA, washed twice, and resuspended in serumfree RPMI supplemented with sodium-heparinate solution (1 U/ml).
  • a small left subcostal incision is then carried out in mice under anesthesia and 3 3 106 LS174T cells in 50 m lof phosphate-buffer ed saline (PBS) are injected into the spleen.
  • PBS phosphate-buffer ed saline
  • the spleen vessels are ligated and the spleen is surgically removed. This procedure will lead to a stable incidence of liver metastasis (more than 95%).
  • the treatment with anti-TF Mabs will be initiated immediately after implantation and will last for the remaining study period.
  • liver samples are fixed overnight in AFA (5% acetic acid, 75% ethyl alcohol, 2% formalin, 18% water), transferred to 100% ethanol, and embedded in paraffin, and 5-m m sections are prepared for histological quantification of metastatic nodules, for immunohisto- chemistry and apoptosis quantification.
  • AFA 5% acetic acid, 75% ethyl alcohol, 2% formalin, 18% water
  • mice 60 homozygotous nu/nu 6 weeks old NMRI males. Groups Mice are randomly allocated in four groups of 15 and treated with solutions A,
  • Termination At a weight loss of > 20% or other objective signs of severe toxicity the animal is terminated.
  • Parameters Tumor size in two orthogonal diameters is recorded daily during the growth phase. The body weight is recorded initially and 2-3 times per week. Postmortem determination of metastasis formation in the liver.
  • MDA-MB-231 Human breast carcinoma cells MDA-MB-231 (ATCC HTB26) are cultured in Dul- becco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). MDA-MB-231 cells (3 3 106) are injected subcutaneously in nude mice (7- to 8-week-old female mice). Primary tumor growth and metastasis is evaluated as described previously (Li et al., Human Gene Therapy 12: 515-526, 2001)
  • Aim To examine the effect on macroscopical growth and lung metastasis of MDA- MB-231 mammary tumors in nude mice of anti-TF Mabs given bolus injection i.v.; 10 mg/kg. Mice: 60 homozygotous nu/nu 6 weeks old NMRI males.
  • mice are randomly allocated in four groups of 15 and treated with solutions A,
  • Termination At a weight loss of > 20% or other objective signs of severe toxicity the animal is terminated.
  • Parameters Tumor size in two orthogonal diameters is recorded daily during the growth phase. The body weight is recorded initially and 2-3 times per week. Postmortem determination of metastasis formation in the lung.
  • the tumor cell line MG U373 is a human glioblastoma multiforme cell line, with high angiogenic activity, high vascular density and fast growth in nude mice. Tumors are inoculated in the flanks, following standard procedures (see enclosed protocols for experimental plan). The mice are observed twice daily for signs of toxicity and the tumors are measured daily in two perpendicular diameters.
  • mice are 7-week-old males obtained from M&B (Ry, Denmark). Animals are kept in a gnotobiotic environment and they receives sterile food pellets and drinking water ad libitum. Three different studies is conducted with the glioma tumor model:
  • Aim To examine the effect on macroscopical growth of U373 tumors in nude mice of anti-TF Mabs given bolus injection i.v.; 10 mg/kg. Mice: 60 homozygotous nu/nu 6 weeks old NMRI males.
  • mice are randomly allocated in four groups of 15 and treated with solutions A,
  • Termination At a weight loss of > 20% or other objective signs of severe toxicity the animal is terminated.
  • Parameters Tumor size in two orthogonal diameters is recorded daily during the growth phase. The body weight is recorded initially and 2-3 times per week.
  • Aim To examine the effect on macroscopic growth of U373 tumors in nude mice of anti-TF Mabs given bolus injection i.v.; 10 mg/kg. after pretherapeutic tumor growth has been established. Mice: 60 homozygotous nu/nu 6 weeks old NMRI males.
  • mice are randomly allocated in four groups of 15 and treated with solutions A, B, or C. Treatment starts when 6 consecutive (daily) measurements show
  • Termination Treatment lasts until the tumors have grown beyond the maximal size of approximately 1.0 cm 3 , i.e. no tumor diameter larger than 15 mm or until Gompertzian regrowth has been established by 6 consecutive measurements. At time of termination tumors from each group are excised for histological and immunochemical evaluation. At a weight loss of > 20% or other objective signs of severe toxicity the animal is terminated Parameters: Tumor size in two orthogonal diameters is recorded daily during the growth phase. The body weight is recorded initially and 2 times per week. Study 111-3:
  • Aim To examine the effect of anti-TF Mabs on growth of intracranial U373 tumors in nude mice.
  • mice 60 homozygotous nu/nu 6 weeks old NMRI males.
  • Tumor U373 implanted orthotopically in the right hemisphere following standard procedures. Groups Mice are randomly allocated in four groups of 15 and treated with solutions A,
  • Termination Mice with signs of chronic neurological impairment are euthanized.
  • Data Survival (i.e. time to neurological impairment) is quantified by Kaplan-Meyer statistics.
  • a 0.5 ml matrigel plug will be located subcutaneously under the abdominal skin.
  • b-FGF 5 ng
  • haemoglobin angiogenesis
  • cDNA microarray analyses a specific up-regulation of three genes in BHK-TF cells treated with FVIIa has been observed. These include: Fra-1 , a gene coding for Fos related antigen 1 , Id2, a gene encoding a member of the helix-loop-helix class of proteins, and Cyr61 encoding an extracellular matrix signalling protein.
  • the following assay is designed to screen for anti-TF Mabs which prevents FVIIa induced up-regulation of Fra-1 , Id2 or Cyr61.
  • BHK-TF cells created as described by Poulsen L.K. et al., J Biol. Chem. 273, 6228- 6232, 1998, are grown in Dulbecco's modified Eagle's medium containing 10% FCS, 100 lU/ml penicillin, and 100 ⁇ g/ml streptomycin to obtain 95-100% confluence, washed and grown for additional 16-18 hs in medium without FCS. The cells are again washed and ex- posed to FCS-free medium containing 100 nM FVIIa.
  • BHK-TF cells are grown in Dulbecco's modified Eagle's medium containing 10% FCS, 100 lU/ml penicillin, and 100 ⁇ g/ml streptomycin to obtain 95-100% confluence, washed and grown for additional 16-18 hs in medium without FCS. The cells are again washed and exposed to FCS-free medium containing 100 nM FVIIa for 1 h.
  • CRL2091 cells ATCC are grown in Is- cove's modified Dulbecco's medium containing 10% FBS, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin to 95-100% confluence.
  • Murine 3T3-L1 cells are maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin. Cells are grown to confluence and induced to with media containing 1 ⁇ M dexamethasone (Sigma), 10 ⁇ g/ml human insulin (Novo Nordisk A/S), and 1 ⁇ M BRL49653 (Novo Nordisk A/S) for 1 h.
  • dexamethasone Sigma
  • 10 ⁇ g/ml human insulin Novo Nordisk A/S
  • 1 ⁇ M BRL49653 Novo Nordisk A/S
  • Fra-1 is cloned by reverse transcription PCR from RNA isolated from 3T3-L1 cells treated for 1 h with dexamethasone, insulin, and BRL49653 using the superscript II kit (Life Technologies) according to the manufacturer's instructions.
  • Id2 and Cyr61 are cloned by reverse transcription PCR from RNA isolated from BHK-TF cells treated for 1 h with FVIIa and from CRL2091 cells treated for 6 hours with FVIIa, respectively.
  • the upstream and downstream primers are: 5'-GCGGCCGCCATGTACCGAGACTACGGGGAACCG-3' and 5'- GCGGCCGCTCACAAAGCCAGGAGTGTAGG-3' for Fra-1 , 5'-
  • Parameters for PCR are one cycle of denaturing at 94 °C for 10 s, annealing at 65 °C for 15 s, and extension at 72 °C for 1.5 min, one cycle of denaturing at 94 °C for 10 s, annealing at 64 °C for 15 s, and extension at 72 °C for 1.5 min, one cycle of denaturing at 94 °C for 10 s, annealing at 63 °C for 15 s, and extension at 72 °C for 1.5 min, one cycle of denaturing at 94 °C for 10 s, annealing at 62 °C for 15 s, and extension at 72 °C for 1.5 min, one cycle of denaturing at 94 °C for 10 s, annealing at 61 °C for 15 s, and extension at 72 °C for 1.5 min, one cycle of denaturing at 94 °C for 10 s, annealing at 60 °C for 15 s, and extension at
  • RNA are isolated from BHK-TF cells incubated with FVIIa, FX, ASIS, 1F44A1 or TF8- 5G9 using TriZol following the instructions of the vendor. 20 ⁇ g of RNA are size-fractionated in a denaturing gel containing 1% agarose, 20 mM MOPS, 5 mM NaOAc, 6% formaldehyde, and 1 mM EDTA, transferred to a Hybond N + membrane (Amersham) by capillary blotting and immobilized by UV crosslinking.
  • cDNA encoding Fra-1 , Id2 or Cyr61 are labelled with the Prime It kit (Stratagene) using [ ⁇ - 32 P] dATP (Amersham) and hybridized using Express Hyb (Clontech) following the manufacturer's instructions and results are visualized by autoradio- graphy.
  • HeLa cells are seeded to 40 % confluence in a T-80 flask one day prior to transfec- tion.
  • Cells are transfected with 150 ng pFA-Elk1 (Stratagene), 3 ⁇ g pFR-Luc (Stratagene), 3 ⁇ g human TF/pcDNA3 and 3 ⁇ g mouse Protease Activated Receptor 2/pcDNA3,1+ using 36 ⁇ l FuGene (Roche) as described in the manual.
  • the following day the cells are detached by VerseneTM (Invitrogen) and seeded in black 96 well view plates (Packard) at a cell density of 20.000 cells per well. After the cells had reattached to the plate, the medium is replaced with 160 ⁇ l per well serum-free Dulbeccos Modified Eagle Medium (Invitrogen) and incubated for 16 hours.
  • Cells are preincubated for 1 hour with either 20 ⁇ l serum-free medium (control), 20 ⁇ l 2,5 ⁇ M FFR-rFVIIa (control), 20 ⁇ l 2,5 ⁇ M anti TF Mab B or 20 ⁇ l 2,5 ⁇ M anti TF Mab A.
  • 20 ⁇ l 0,5 ⁇ M FVIIa is added to half of the wells and medium to the other half.
  • the cells are subjected to the Luciferase gene assay.
  • LucLite (Packard) reagent is added to the cells as described by the manufacturer. Luciferase expression levels are read on a TopCount Microplate Scintillation (Packard).

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US20060235209A9 (en) 1997-03-10 2006-10-19 Jin-An Jiao Use of anti-tissue factor antibodies for treating thromboses
US7749498B2 (en) 1997-03-10 2010-07-06 Genentech, Inc. Antibodies for inhibiting blood coagulation and methods of use thereof
US5986065A (en) 1997-03-10 1999-11-16 Sunol Molecular Corporation Antibodies for inhibiting blood coagulation and methods of use thereof
US20030109680A1 (en) 2001-11-21 2003-06-12 Sunol Molecular Corporation Antibodies for inhibiting blood coagulation and methods of use thereof
US20040180051A1 (en) * 2001-03-26 2004-09-16 Koji Suzuki Blood rheology improving agents
AU2003277832A1 (en) * 2002-10-31 2004-05-25 Novo Nordisk A/S Humanized tissue factor antibodies
EP1587549A2 (en) * 2003-01-22 2005-10-26 Novo Nordisk A/S Radiolabelled tissue factor binding agent and the use thereof
BRPI0410875A (pt) * 2003-05-30 2006-07-04 Centocor Inc método de inibição do crescimento de tumor com anticorpos de fator antitecido
US9708410B2 (en) 2003-05-30 2017-07-18 Janssen Biotech, Inc. Anti-tissue factor antibodies and compositions
US7605235B2 (en) 2003-05-30 2009-10-20 Centocor, Inc. Anti-tissue factor antibodies and compositions
WO2005004793A2 (en) * 2003-06-19 2005-01-20 Tanox Inc. Compositions and methods for treating coagulation related disorders
CA2542372A1 (en) * 2003-08-29 2005-03-10 Centocor, Inc. Method of promoting graft survival with anti-tissue factor antibodies
JP2007523099A (ja) * 2004-02-20 2007-08-16 ノボ　ノルディスク　アクティーゼルスカブ 組み合わせ療法
TWI496790B (zh) 2006-09-08 2015-08-21 艾伯維巴哈馬有限公司 介白素-１３結合蛋白質
CN101423552B (zh) * 2008-02-29 2012-05-16 复旦大学 一种人源抗人组织因子Fab及其制备方法
AU2016277670B2 (en) * 2008-12-09 2019-02-07 Genmab A/S Human antibodies against tissue factor
AU2013203150B2 (en) * 2008-12-09 2016-09-22 Genmab A/S Human antibodies against tissue factor
UA109633C2 (uk) * 2008-12-09 2015-09-25 Антитіло людини проти тканинного фактора
WO2010131235A1 (en) * 2009-05-15 2010-11-18 University Of The Free State Inhibitory antibody fragments to human tissue factor
BR112012031727B1 (pt) * 2010-06-15 2022-03-29 Genmab A/S Conjugado de droga-anticorpo, composição farmacêutica, e, uso do conjugado de droga- anticorpo
US8722044B2 (en) 2011-03-15 2014-05-13 Janssen Biotech, Inc. Human tissue factor antibody and uses thereof
CN106467574B (zh) * 2015-08-20 2019-09-20 复旦大学 靶向于组织因子的抗体、其制备方法和用途
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