CN1575302A

CN1575302A - Human tissue factor antibodies

Info

Publication number: CN1575302A
Application number: CNA028208463A
Authority: CN
Inventors: P－O·弗瑞斯克加尔德; J·T·克劳森; B·B·瑟林森; M·克加尔克
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 2001-10-02
Filing date: 2002-09-30
Publication date: 2005-02-02
Also published as: EP1434802A1; NO20041802L; HUP0401658A2; JP2005512970A; BR0213046A; IL160998A0; ZA200402303B; PL368989A1; WO2003029295A1; KR20040045478A; CZ2004454A3; RU2004113373A; MXPA04003051A; CA2460917A1

Abstract

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

Description

Human tissue factor antibody

Invention field

The present invention relates to isolating can be with tissue factor (TF) thus immune response anticoagulant factor VIIa (FVIIa) bonded people antibody takes place, relate to the thrombosis in use suppresses the thrombosis relevant with operation, micrurgy, angioplasty or wound or inhibition and the disease-related as dvt formation, disseminated inravascular coagulation (DIC), coronary artery disease, Sepsis, inflammation, atherosclerosis or cancer at people's antibody of TF the abnormal hemostasis situation and the immunotherapy of other TF function thus.The clone that the invention also discloses the preparation method of antibody and be used to prepare human monoclonal antibodies (Mabs).

Background of invention

The process that blood coagulation is interacted and formed by the complexity between the different blood ingredients or the factor, this process finally produces the fibrin piece.In general, the blood ingredient that the participation what is called is solidified " cascade " is preferment or proenzyme, and they are the protein of no enzymatic activity, changes into proteolytic ferment by this effect as the activator of activated clotting factor.The thrombin that carries out this class conversion generally is called " active factor ", names (for example factor VIIa) by the subscript " a " of adding small letter.

Activation factor X (" Xa ") is that thrombogen is changed into zymoplasm is needed, and this enzyme changes into fibrin with fibrinogen then, and this is the terminal stage that forms the fibrin piece.Exist two kinds to promote factor X activatory system or approach." inherent approach " refers to by utilization and exists only in those reactions that the factor in the blood plasma forms zymoplasm.The activation of a series of proteolytic enzyme mediations finally produces factors IX a, and this factor is cracked into Xa with Factor IX a with factor X.In " the external approach " of blood coagulation, identical proteolysis is undertaken by FVIIa and cofactor TF thereof.TF is an embrane-associated protein, does not generally circulate in blood plasma with activity form.Yet when angiorrhexis, TF can be compound with FVIIa, so that Ca is being arranged ²⁺Catalysis factor X activation or factors IX activation under the situation about existing with phosphatide.Although these two kinds of relative importances of solidifying approach it be unclear that in the hemostasis, have been found that factor VII and TF play a crucial role in the initial stage of blood coagulation.

The intravital coagulation cascade of selective exclusion patient is normally requisite.For example, can in the kidney dialysis procedure, use such as heparin, tonka bean camphor, coumarin derivatives, such antithrombotics or other promoting agent of indandione derivative, or they are used for the treatment of other medical conditions of dvt formation, disseminated inravascular coagulation (DIC) and host.For example, the therapy of heparinotherapy or external use citrate ion can be used for dialysis, so that the blood coagulation that occurs in the prophylactic treatment process.The intravital dvt of patient that heparin also is used to prevent to undergo surgery forms.

Yet, use the therapy of heparin and other antithrombotics to have unwanted side effect.Can generally work for the antithrombotics that utilizes, but not act on damage location specifically at whole body.For example, heparin can cause severe bleeding.In addition, because the transformation period is about 80 minutes, heparin can be removed from blood fast, therefore essential frequent drug administration.Because heparin works as the cofactor of Antithrombin III (ATIII), and ATIII exhausts in the DIC therapy fast, thus be difficult to keep suitable heparin dosage usually, thus essential continuous monitoring ATIII and heparin level.If ATIII extremely exhausts, heparin will be invalid so.In addition, the life-time service heparin also may platelet increasing be assembled and is also reduced platelet count, has found that the development of the thrombocytopenia of bringing out with heparin is relevant.Also may there be toxic side effect in indandione derivative.Except that the antithrombotics of sketching above, have been found that the protein of several natural appearance has anticoagulating active.In addition, recommended ATIII as the treatment antithrombotics.

International Application No. WO 92/15686 relates to the inactivation factor VIIa that is used to suppress blood coagulation.

Antibody is specific immunoglobulin (Ig) the polypeptide class that is produced as the reaction that foreign protein, glycoprotein, cell or other outside antigenic substance are attacked by vertebrate immune system.Event sequence to the small part that makes organism overcome outside cell infringement or removing foreign matter system obtains understanding.The important component part of this process be produce can with specific foreign matter specificity bonded antibody.The binding specificity of this class polypeptide and specific antigen is highly accurate, and can all be significant aspect complexity and the variability by the individual numerous specificitys that produce of vertebrates.Millions of antigen can cause antibody response, and each antibody is almost only at the specific antigen that produces it.

Utilize the vertebrates antibody of two kinds of main sources at present, they are produced in position by the Mammals bone-marrow-derived lymphocyte and are produced in cell culture by B-cell heterozygote.It is that immature B cell is divided into plasmacytic result that the original position of antibody produces, and this process is the reaction that specific antigen is stimulated.In undifferentiated B cell, the DNA part of the not same district on the coding immunoglobulin chain obtains separating in genomic dna.These sequences obtain assembling successively before expression.The rearrangement gene of gained can be expressed in ripe bone-marrow-derived lymphocyte, produces required antibody.Yet,, do not produce the antibody population of homogeneous even when specific Mammals only contacts single antigen yet.To the original position immune response of any specific antigen by to being present in the reaction diversity decision of the different determinants on this antigen.Each homologous antibody hypotype is produced by single B cell mass, and therefore the antibody that produces in position is " polyclone ".

Under many particular cases, overcome this limited but inherent ununiformity by utilizing the hybridoma technology that in cell culture, produces " mono-clonal " antibody by B cell hybridoma.

In this process, make from the splenocyte of having injected antigenic mammiferous short-life relatively or non-infinite multiplication or lymphocyte and infinite multiplication tumor cell line and merge, produce not only to infinite multiplication thus but also can produce hybrid cell or " hybridoma " of the B cell antibody of genetic coding.By screening, dilution and regrowth, make the heterozygote that forms thus be separated into single genetic strain, each strain is represented single hereditary system.Therefore, their generations are the antibody of homogeneity really for required antigen.According to its pure hereditary family these antibody are called " mono-clonal ".

Monoclonal antibody with monospecific has produced remarkably influenced to immunology, and their application is confirmed in such as this class science of biology, pharmacology, biological chemistry and other subject.Have been found that this class monoclonal antibody not only can be widely used as diagnostic reagent, and can be used for the treatment of (for example, referring to Ritz and Schlossman, " blood " (Blood), 59:1-11, (1982)).

Although the monoclonal antibody that is produced by hybridoma as mentioned above in theory effectively and because of its specificity obviously is better than polyclonal antibody, there is significant defective in they.In many application, monoclonal antibody is being used for man-hour, the application of the monoclonal antibody that the non-human animal produces is severely limited.Give the people inject " xenogenesis " antibody repeatedly, such as mouse antibodies, can cause deleterious anaphylaxis.When injecting the monoclonal antibody in this inhuman source of class to the people, they can produce anti--non-human antibody's reaction.

The treatment of the mouse Mabs of known anti-TF is applied in U.S. Pat 6,001, and is on the books in 978 and US5,223,427.

International Application No. WO 99/51743 relates to the people/mouse chimeric mAb at people TF.

European patent application EP 833911 relates to the CDR grafting antibody of anti-people TF.

Presta L. etc. have described the humanized antibody of anti-TF at " thrombosis and hemostasis " (Thrombosis and Haemostasis) among the 85th volume (3) pp.379-389 (2001).

To have anticoagulating active, low dosage administration and can not produce the improvement composition that do not need side effect relevant and still have demand relatively with traditional anticoagulant compositions.The present invention has satisfied this demand by the antithrombotics with side effect relevant with the traditional antibody that carries non-human sequence is provided, and they work at damage location specifically, and other associated advantages further is provided.In addition, the invention provides the compound that can suppress with the TF cell function of disease-related as Sepsis, inflammation, atherosclerosis, restenosis or cancer.

Description of the invention

The present invention relates to the neutralizing high affinity human antibody of the non-immunogenic of anti-people TF, but their anticoagulant factor VII/VIIa combinations the invention still further relates to the screening method that people's antibody of effective anti-people TF is gone up in treatment.

Immunoreactive isolating people's antibody takes place in the epi-position that the present invention relates in aspect first and is present on the people TF.

Term used herein " human tissue factor " or " people TF " refer to the full-length polypeptide acceptor of aminoacid sequence shown in the 1-263 position of containing the natural human tissue factor.

Term used herein " antibody " in order to refer to can with antigen (for example people TF) specificity bonded immunoglobulin molecules and fragment thereof.Full length antibody contains 4 polypeptide chains, i.e. two weight (H) chains and two light (L) chains that interconnect by disulfide linkage.Each heavy chain is made up of variable region of heavy chain (this paper is abbreviated as HCVR or VH) and CH.CH is made up of three domain C H1, CH2 and CH3.Each light chain is made up of variable region of light chain (this paper is abbreviated as LCVR or VL) and constant region of light chain.Constant region of light chain is made up of a domain C L.VH and VL district further can be subdivided into the more conservative district that is called framework region (FR) and interleave therein the hypervariable region that is called complementarity-determining region (CDR).Each VH and VL are made up of three CDR and four FR of arranging from the N-terminal to the C-terminal according to following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.Therefore, the one or more fragments that in the range of definition of antibody, also comprise the antibody of maintenance and antigen (for example people TF) specificity bonded ability.Confirmed that the fragment of full length antibody can implement the antigen combined function of antibody.The example of the binding fragment that comprises in the term " antibody " comprises: (i) Fab fragment, i.e. the unit price fragment of being made up of VL, VH, CL and CH1 structural domain; (ii) F (ab) ₂And F (ab ') ₂Fragment promptly contains the segmental divalence fragment of two Fab that connects by the disulfide linkage on the hinge area; The (iii) Fd fragment of forming by VH and CH1 structural domain; The (iv) Fv fragment of forming by VL in the antibody single armed and VH structural domain; (the v) dAb fragment of forming by the VH structural domain (Ward etc. (1989) " nature " are 341:544-546 (Nature)); (vi) isolating complementarity-determining region (CDR).In addition, although segmental two structural domain VL of Fv and VH are by independent genes encoding, but can use recombination method to connect them by synthetic linker, the single protein chain that described synthetic linker can make them make VL and VH district pairing formation monovalent molecule (is called strand Fv (scFv); For example, referring to (Science) 242:423-426 of (1988) " science " such as Bird; With (Proc.Natl.Acad.Sci.USA) 85:5879-5883 of (1988) " NAS's journals " such as Huston).This class single-chain antibody is also included within term " antibody " scope.The single-chain antibody that also comprises other form is such as bi-specific antibody (diabodies).Bi-specific antibody is the bi-specific antibody of divalence, wherein on the single polypeptide chain, express VH and VL structural domain, but the joint that uses is too short two structural domains on the same chain are matched, impel the complementary structure territory pairing on these structural domains and another chain thus and (for example produce two antigen binding sites, referring to Hol-liger, P. etc. (1993) " NAS's journal " are 90:6444-6448 (Proc.Natl.Acad.Sci.USA); POIJAK, R.J. etc. (1994) " structure " are 2:1121-1123 (Structure)).Be understandable that people TF can have one or more antigenic determinants, comprising: the peptide antigenic determinant that (1) is made up of the single chain polypeptide in the people TF; (2) by a conformational antigenic determinant that above peptide chain is formed of space adjacency, described space is not continuous distribution in abutting connection with the corresponding aminoacid sequence of peptide chain at people TF peptide sequence; (3) antigenic determinant after the translation of forming by the covalently bound molecular structure of translation back and people TF, such as carbohydrate group etc. in whole or in part.

Term used herein " people's antibody " and " people TF antibody " are in order to comprise that having the ethnic group of deriving from is the variable region of immunoglobulin sequences and the antibody of constant region.People's antibody of the present invention can comprise that can't help ethnic group is immunoglobulin sequences amino acids coding residue (for example by at random external or site-specific mutagenesis or the sudden change that imports by somatic mutation in the body), for example in CDRs, particularly CDR3.But, term used herein " people's antibody " does not comprise the antibody of CDR sequence grafting on people's frame sequence that will derive from such as another such Mammals kind system of mouse, for example so-called humanized antibody or people/little mouse chimeric antibody.

" isolating people's antibody " used herein is in order to refer to be substantially free of people's antibody (for example specificity is substantially free of the antigenic antibody of specificity in conjunction with inhuman TF in conjunction with the isolated antibody of people TF) of other antibody with different antigen-specifiies.Yet specificity is in conjunction with the isolated antibody of people TF and other antigen, can have cross reactivity (further specifically discussing below) such as the TF molecule from other kind.In addition, isolated antibody can be substantially free of other cellular material and/or chemical substance.

Term used herein " epi-position " refers on the antigen antibody any antigenic determinant of bonded with it.The epi-position determinant is usually by having chemically active surface molecular group group, form such as amino acid or sugared side chain, and has specific Three Dimensions Structure and specific charge characteristic usually.

Term used herein " immune response " refers to antibody and its epi-position bonded dissociation constant K _dBe lower than 10 ^-4M.If suitable, term " immune response " can exchange use with term " specificity combines ".

Term used herein " inhibition " refers to any reduction of comparing with reference substance.As an example, suppressing human blood coagulation factor VII a and people TF bonded antibody refers to and has with this antibody not that human blood coagulation factor VII a compares any antibody that can reduce human blood coagulation factor VII a and people TF binding ability under the situation in conjunction with the ability of people TF.

Term used herein " affinity " refers to the bonding strength of antibody and epi-position.The affinity of antibody is by the dissociation constant K that is defined as [Ab] * [Ag]/[Ab-Ag] _dDetermine that wherein [Ab-Ag] is the volumetric molar concentration of antibody-antigenic complex, [Ab] is the volumetric molar concentration of unconjugated antibody, and [Ag] is unconjugated antigenic volumetric molar concentration.Affinity costant K _aBy 1/K _dDetermine.The preferred method of measuring Mabs specificity and affinity by competitive inhibition can find in following document: Harlow etc. " antibody lab guide " (Antibodies:A Laboratory Manual), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Colligan etc. edit " up-to-date immunology scheme " (Current Protocols in Immuology), Greene Publishing Assoc. and Wiley Iinterscience, N.Y., (1992,1993); And Muller " Enzymology method " is (Meth.Enzymol.)] 92:589-601 (1983), the full content of these documents is incorporated herein by reference.

The present invention relates to pharmaceutical composition in aspect second, comprising the epi-position with being present on the people TF of treatment significant quantity immunoreactive people's antibody takes place.

Term " treatment significant quantity " is to measure the determined effective dose of qualified doctor of the dosage that obtains required reaction with volumetry.The factor of considering at dosage comprises that effect, bioavailability, required pharmacokinetics/pharmacodynamics distribute, the other factors known to common administered agents, administration time or the doctor of the disease (for example wound, inflammation, septic shock) of treatment, the factor (for example body weight, health condition, age etc.) relevant with the patient, existence.The dosage of anti-TF people's antibody that the patient is given changes with the type and the different of severity of treatment disease, but generally in the scope of 0.1-5.0mg/kg body weight.

Term used herein " experimenter ", can exchange with term " patient " and use if suitable in order to finger any animal, particularly Mammals, such as the people.

The present invention relates in aspect the 3rd and comprises and be present in the composition that immunoreactive people's antibody takes place for epi-position on the people TF.

The present invention relates to the human disease's relevant with FVIIa/TF methods of treatment in one aspect of the method, this method comprise to this people treat significant quantity, can be present in the step that immunoreactive people's antibody takes place for epi-position on the people TF.

" treatment " refers to the therapeutical active compound of the present invention that gives significant quantity to prevent any symptom or disease situation and take place or to cure or alleviate this class symptom or the disease situation that has taken place.Therefore term " treatment " means and comprises prophylactic treatment.

Term used herein " FVIIa/TF relative disease " refers to disease or the illness that relates to TF and FVIIa.Comprise: disease or the illness relevant with thrombosis or coagulopathy, comprise inflammatory reaction and form relevant chronic thrombotic disease or illness with fibrin, comprise vascular disease, such as the restenosis of dvt formation, artery thrombosis, postoperative thrombosis, coronary artery bypass graft surgery (CABG), percutaneous coronary angioplasty (PTCA), apoplexy, tumor growth, metastases, blood vessel generation, thrombolysis, atherosclerosis and postangioplasty; Acute and chronic indication is such as inflammation, septic shock, septicemia, ypotension, adult respiratory distress syndrome (ARDS), disseminated inravascular coagulation (DIC), pulmonary infarction, platelet deposition, myocardial infarction; Or prophylactic treatment has the Mammals of the atherosclerotic blood vessel that is in the thrombosis danger; And other disease or illness.The FVIIa/TF relative disease is not limited to those body intravascular coagulation disorder diseases of mentioning such as above-mentioned, and comprise the FVIIa/TF correlated process that exsomatizes, such as the blood coagulation that may cause because of extracorporeal circulation of blood, described extracorporeal circulation of blood be included in such as in the such process of the blood bypass in dialysis procedure, blood filtration or the surgical procedure by the blood of online taking-up in the patient.

Term " factor VIIa " or " FVIIa " refer to by specificity cracking institute cracked " two chains " the activated clotting factor VII at Arg152-lle153 peptide bond place.FVIIa can the autoblood purifying or is produced by recombination form.Obviously how the enforcement of methods described herein and the factor VIIa that do not rely on purifying derive, and therefore should understand the application that the present invention includes any factor VIIa goods that are applicable to this paper.People FVIIa preferably.

Term " FVII " refers to " strand " proconvertin.

The present invention relates to the preparation method of people's antibody in one aspect of the method, and this method comprises the following steps:

A) people's antibody of the anti-people TF of preparation;

B) test antibody in the setting test of TF inductive, and screening suppresses people's antibody that grumeleuse forms, the IC of the people's antibody that is wherein screened in this test ₅₀Value is lower than 1nM, such as being lower than 500pM, preferably is lower than 200pM, preferably is lower than 100pM, preferably is lower than 50pM, preferably is lower than 10pM, more preferably less than 5pM; Or

Test antibody and screening suppress people's antibody of FXa generation, the IC of the people's antibody that is wherein screened in FXa generation test ₅₀Value is lower than 100nM (is in the experiment of 0.1nM in FVIIa concentration), such as being lower than 10nM, preferably is lower than 5nM, preferably is lower than 1nM, more preferably less than 0.1nM; Or

Test antibody and screening suppress the active people's antibody of TF-inductive FVIIa acid amides cracking (amidolytic), the IC of the people's antibody that is wherein screened in FVIIa/TF acid amides cracking (amidolytic) test ₅₀Value is lower than 100nM (is in the experiment of 10nM in FVIIa concentration), such as being lower than 40nM, preferably is lower than 20nM, more preferably less than 10nM; Or

Test antibody and screening combine people's antibody of competition with FVIIa in the FVIIa competition experiments; Or

Test antibody and screening are in conjunction with people's antibody of people TF in the TFELISA test that comprises TF.

Should understand specific IC related in the setting test of TF inductive ₅₀The IC that value is to use the human normal plasma to measure ₅₀Value.

A) people's antibody of the anti-people TF of preparation;

B) test antibody and screening can suppress people's antibody that grumeleuse forms, the IC of the people's antibody that is wherein screened in this test in the setting test of TF inductive ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is such as the IC that is lower than FFR-rFVIIa ₅₀Value+500pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+200pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+100pM preferably is lower than the IC of FFR-RFVIIa ₅₀Value+50pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+5pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value; Or

Test antibody and screening suppress people's antibody of FXa generation, the IC of the people's antibody that is wherein screened in FXa generation test ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 0.1nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+10nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+5nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+0.1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value; Or

Test antibody and screening can suppress people's antibody of TF-inductive FVIIa acid amides lytic activity, the IC of the people's antibody that is wherein screened in FVIIa/TF acid amides breaking test ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 10nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+40nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+20nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value; Or

Immunoreactive people's antibody takes place with people TF in test antibody and screening in the TF ELISA test that comprises TF.

Term used herein " setting test of TF inductive " is in order to refer to measure any test of setting time in the sample that comprises blood plasma and TF.The example of TF inductive setting test is described in the test 7 of embodiment 1.

Term used herein " FXa produces test " is in order to refer to that measuring the FX activatory in the sample that comprises TF, FVIIa, FX, calcium and phosphatide tests arbitrarily.FXa produces the example of test and describes in the test 5 of embodiment 1.

Term used herein " FVIIa/TF acid amides breaking test " is in order to refer at the acid amides lytic activity that has TF to exist to measure FVIIa under the situation, to be that little peptide substrates cracked is tested arbitrarily.The example of FVIIa/TF acid amides breaking test is described in the test 4 of embodiment 1.

Term used herein " TF ELISA test " comprises any ELISA test of the antibody of TF and anti-TF in order to finger.The example of TF ELISA test is the direct and indirect TF ELISA test described in embodiment 1

test

1 and 2.

Term used herein " directly TF ELISA test " is in order to refer to comprise any TF ELISA test of immobilization TF.Directly the example of TF ELISA test is described in the test 1 of embodiment 1.

Term used herein " TF ELISA test indirectly " is in any TF ELISA test of solution form in order to refer to TF.The example of TF ELISA test is described in the test 2 of embodiment 1 indirectly.

The present invention relates in one aspect of the method and is present in the epi-position generation immune response on the people TF and suppresses human blood coagulation factor VII a and people TF bonded people antibody, and this antibody can obtain by the method that comprises the following steps:

A) people's antibody of the anti-people TF of preparation;

B) test antibody and screening suppress people's antibody that grumeleuse forms, the IC of the people's antibody that is wherein screened in this test in the setting test of TF inductive ₅₀Value is lower than 1nM, such as being lower than 500pM, preferably is lower than 200pM, preferably is lower than 100pM, preferably is lower than 50pM, preferably is lower than 10pM, more preferably less than 5pM; Or

Test antibody and screening suppress people's antibody of FXa generation, the IC of the people's antibody that is wherein screened in FXa generation test ₅₀Value is lower than 100nM (is in the test of 0.1nM in FVIIa concentration), such as being lower than 10nM, preferably is lower than 5nM, preferably is lower than 1nM, more preferably less than 0.1nM; Or

Test antibody and screening can suppress people's antibody of TF-inductive FVIIa acid amides lytic activity, the IC of the people's antibody that is wherein screened in FVIIa/TF acid amides breaking test ₅₀Value is lower than 100nM (is in the test of 10nM in FVIIa concentration), such as being lower than 40nM, preferably is lower than 20nM, more preferably less than 10nM; Or

Test antibody and screening are in conjunction with people's antibody of people TF in the TF ELISA test that comprises TF.

A) people's antibody of the anti-people TF of preparation;

B) test antibody and screening suppress people's antibody that grumeleuse forms, the IC of the people's antibody that is wherein screened in this test in the setting test of TF inductive ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is such as the IC that is lower than FFR-rFVIIa ₅₀Value+500pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+200pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+100pM preferably is lower than the IC of FFR-RFVIIa ₅₀Value+50pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+5pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value; Or

Test antibody and screening can suppress people's antibody that FXa produces, the IC of the people's antibody that is wherein screened in FXa generation test ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 0.1nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+10nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+5nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+0.1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value; Or

In one embodiment of the invention, the method that produces people's antibody of anti-people TF comprises to mammalian immune inoculation people TF and separates the antibody that the Mammals by immunization produces.In preferred embodiments, described Mammals is a mouse.Mammals or the mouse that should understand immunization can produce people's antibody.

A) to mouse immune inoculation people TF;

B) separate the cell that produces antibody and prepare the infinite multiplication cell of secreting people's antibody by immunized mice;

C) isolation medium from the infinite multiplication cell of the antibody that contains generation;

D) but in the indirect TF ELISA of the TF that comprises the solution form test people's antibody of the people TF of test antibody and screening binding soln form;

E) test antibody and screening combine people's antibody of competing with FVIIa in the FVIIa competition experiments;

F) test antibody also screens people's antibody that can suppress TF-inductive FVIIa acid amides lytic activity, the IC of the people's antibody that is wherein screened in FVIIa/TF acid amides breaking test ₅₀Value is lower than 100nM (is in the test of 10nM in FVIIa concentration), such as being lower than 40nM, preferably is lower than 20nM, more preferably less than 10nM;

G) test antibody and screening can suppress people's antibody that FXa produces, the IC of the people's antibody that is wherein screened in FXa generation test ₅₀Value is lower than 100nM (is in the test of 0.1nM in FVIIa concentration), such as being lower than 10nM, preferably is lower than 5nM, preferably is lower than 1nM, more preferably less than 0.1nM;

H) test antibody and screening can suppress people's antibody that grumeleuse forms, the IC of the people's antibody that is wherein screened in this test in the setting test of TF inductive ₅₀Value is lower than 1nM, such as being lower than 500pM, preferably is lower than 200pM, preferably is lower than 100pM, preferably is lower than 50pM, preferably is lower than 10pM, more preferably less than 5pM;

I) in appropriate media, screen and cultivate the infinite multiplication cell that produces the antibody that screens behind steps d-h;

J) antibody of separation screening from the substratum of the infinite multiplication cell that screened.

A) to mouse immune inoculation people TF;

B) separate the cell that produces antibody and prepare the infinite multiplication cell of secreting people's antibody from immunized mice;

C) isolation medium from the infinite multiplication cell that contains the antibody that produces to some extent;

D) immunoreactive people's antibody can take place with the people TF of solution form in test antibody and screening in the indirect TF ELISA test that comprises the TF that is in the solution form;

F) people's antibody of test antibody and screening inhibition TF-inductive FVIIa acid amides lytic activity in FVIIa/TF acid amides breaking test, the IC of the people's antibody that is wherein screened ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 10nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+40nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+20nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value;

G) test antibody and screening can suppress people's antibody that FXa produces, the IC of the people's antibody that is wherein screened in FXa generation test ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 0.1nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+10nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+5nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+0.1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value;

H) test antibody and screening can suppress people's antibody that grumeleuse forms, the IC of the people's antibody that is wherein screened in this test in the setting test of TF inductive ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is such as the IC that is lower than FFR-rFVIIa ₅₀Value+500pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+200pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+100pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+50pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+5pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value;

I) in appropriate media, screening and cultivating the infinite multiplication cell that produces the antibody that is screened behind steps d-h;

Term used herein " produces the cell of antibody " and refers to the arbitrary cell that can produce antibody.Comprise hybridoma, cells transfected system with from splenocyte or the lymphocyte of having injected antigenic relative short-life or non-infinite multiplication.

The present invention relate in one aspect of the method can be present in the epi-position generation immune response on the people TF and suppress human blood coagulation factor VII a and people TF bonded people antibody, this antibody can obtain by the method that comprises the following steps:

A) to mouse immune inoculation people TF;

D) but in the indirect TF ELISA test that comprises the TF that is in the solution form people's antibody of the people TF of test antibody and screening binding soln form;

I) at the infinite multiplication cell that in appropriate media, screens and cultivate the antibody that generation screens behind steps d-h;

The antibody of separation screening the substratum of the infinite multiplication cell that j) screens from having.

A) to mouse immune inoculation people TF;

F) test antibody also screens people's antibody that can suppress TF-inductive FVIIa acid amides lytic activity, the IC of the people's antibody that is wherein screened in FVIIa/TF acid amides breaking test ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 10nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+40nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+20nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value;

J) antibody of separation screening from the substratum of infinite multiplication cell with screening.

In one embodiment of the invention, described infinite multiplication cell is a hybridoma.

The present invention relate in one aspect of the method generation can be present in the epi-position generation immune response on the people TF and suppress human blood coagulation factor VII a and the cell of people TF bonded people antibody.

In one embodiment, described cell is isolating lymphoidocyte.

In one embodiment, described cellular segregation is from mouse.

In another embodiment, described cell is a hybridoma.In one embodiment, can obtain described hybridoma by making the lymphoidocyte and the infinite multiplication cytogamy that produce antibody, thereby the hybridoma that produces antibody is provided.

In another embodiment of the invention, isolating people's antibody can suppress human blood coagulation factor VII a and combine with people TF.

In another embodiment of the invention, isolating people's antibody is monoclonal antibody.

Term used herein " monoclonal antibody " refers to the homogeneous colony of immunoglobulin (Ig), i.e. each molecule of all identical antibody population except that the sudden change of natural appearance.General origin comes from the lymphoidocyte synthetic antibody of the bone-marrow-derived lymphocyte of marrow.The lymphocyte that derives from identical clone produces the immunoglobulin (Ig) of single seed amino acid sequence.Lymphocyte can not directly be cultivated for a long time and produce a large amount of its specific antibodies.Yet, Kohler etc. at 1975 " natures " (Nature), confirm among the 256:495, somatocyte merges, the cell fusion process between lymphocyte and the myeloma cell particularly, can produce hybridoma, such hybridoma can be grown in culture and be produced the specific antibody that is called " monoclonal antibody ".Although known have " non-generation " strain, the myeloma cell is the lymph tumor cell that often produces antibody with the difference self of cell strain.

In another embodiment of the invention, isolating people's antibody is recombinant antibodies.

Term used herein " recombinant antibodies " is in order to comprising by recombination form preparation, expression, generation or isolating everyone antibody, such as: the antibody (further describing in the part ii below) that uses the recombinant expression vector that is transfected into host cell to express; Separate antibody (further describing in the III part below) from reorganization, combination people antibody library; Separation is from for the antibody (for example, referring to (1992) " nucleic acids research " (Nucl.Acids Res.) 20:6287-6295s such as Taylor L.D.s) of human immunoglobulin gene for genetically modified animal (for example mouse); Or by comprising with human immunoglobulin gene's sequence and other dna sequence dna any alternate manner preparation, expression, generation or the isolated antibody of montage mutually.This class recombinant human antibody has and derives from variable region and the constant region that ethnic group is an immunoglobulin sequences.Yet, in certain embodiments, can carry out vitro mutagenesis (maybe when using people Ig sequence to this class recombinant human antibody to genetically modified animal, carry out body endosome cell mutation), though so the aminoacid sequence in the VH of recombinant antibodies and VL district be that to derive from ethnic group be that VH and VL sequence and the associated antibody of people in vivo kind system form not naturally occurring sequence in the storehouse.

In another embodiment of the invention, isolating people's antibody is the Fab fragment.

In another embodiment of the invention, isolating people's antibody is F (ab) ₂Fragment.

In another embodiment of the invention, isolating people's antibody is F (ab ') ₂Fragment.

In another embodiment of the invention, isolating people's antibody is strand Fv fragment.

In another embodiment of the invention, the K that isolating people's antibody combines with people TF _d10 ^-15-10 ^-8The scope of M.Should understand the K that people's antibody of relating to combines with people TF _dAs measuring in the test, wherein with people's antibody immobilization (referring to test 6).

In another embodiment of the invention, the K that isolating people's antibody combines with people TF _d10 ^-15-10 ^-10The scope of M.

In another embodiment of the invention, the K that isolating people's antibody combines with people TF _dBe lower than 10 ^-8M.In another embodiment of the invention, the K that isolating people's antibody combines with people TF _dBe lower than 10 ^-9M.In another embodiment of the invention, the K that isolating people's antibody combines with people TF _dBe lower than 10 ^-20M.In another embodiment of the invention, the K that isolating people's antibody combines with people TF _dBe lower than 10 ^-11M.In another embodiment of the invention, the K that isolating people's antibody combines with people TF _dBe lower than 10 ^-12M.In another embodiment of the invention, the K that isolating people's antibody combines with people TF _dBe lower than 10 ^-13M.In another embodiment of the invention, the K that isolating people's antibody combines with people TF _dBe lower than 10 ^-14M.In another embodiment of the invention, the K that isolating people's antibody combines with people TF _dBe lower than 10 ^-15M.

In another embodiment of the invention, the preparation method of people's antibody be included in test antibody in the setting test of TF inductive and screening in this test, suppress grumeleuse formation, IC ₅₀Value is lower than people's antibody of 1nM.In another embodiment of the invention, described IC ₅₀Value is lower than 500pM.In another embodiment of the invention, described IC ₅₀Value is lower than 200pM.In another embodiment of the invention, described IC ₅₀Value is lower than 100pM.In another embodiment of the invention, described IC ₅₀Value is lower than 50pM.In another embodiment of the invention, described IC ₅₀Value is lower than 10pM.In another embodiment of the invention, described IC ₅₀Value is lower than 5pM.

In another embodiment of the invention, the preparation method of people's antibody comprises the following steps: test antibody and screening people's antibody that the inhibition grumeleuse forms in this test in the setting test of TF inductive, the IC of the people's antibody that is wherein screened ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is such as the IC that is lower than FFR-rFVIIa ₅₀Value+500pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+200pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+100pM is such as the IC that is lower than FFR-rFVIIa ₅₀Value+50pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+5pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value.

In another embodiment of the invention, the preparation method of people's antibody is included in that FXa produces in the test test antibody and screening can suppress FXa generation, IC in this test ₅₀Value is lower than people's antibody of 100nM (in the test of using FVIIa concentration as 0.1nM).In another embodiment of the invention, described IC ₅₀Value is lower than 10nM.In another embodiment of the invention, described IC ₅₀Value is lower than 5nM.In another embodiment of the invention, described IC ₅₀Value is lower than 1nM.In another embodiment of the invention, described IC ₅₀Value is lower than 0.1nM.

In another embodiment of the invention, the preparation method of people's antibody is included in that FXa produces in the test test antibody and screening can suppress people's antibody that FXa produces, the IC of the people's antibody that is wherein screened ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 0.1nMFVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+10nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+5nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+0.1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value.

In another embodiment of the invention, the preparation method of people's antibody is included in the FVIIa/TF acid amides breaking test test antibody and screening can suppress TF-inductive FVIIa acid amides lytic activity, IC ₅₀Value is lower than people's antibody of 100nM (in the test of using 10nM FVIIa).In another embodiment, described IC ₅₀Value is lower than 40nM.In another embodiment, described IC ₅₀Value is lower than 20nM.In another embodiment, described IC ₅₀Value is lower than 10nM.In another embodiment, described IC ₅₀Value is lower than 5nM.

In another embodiment of the invention, the preparation method of people's antibody is included in people's antibody that test antibody in the FVIIa/TF acid amides breaking test and screening can suppress TF-inductive FVIIa acid amides lytic activity, the IC of the people's antibody that is wherein screened ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 10nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+40nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+20nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value.

In another embodiment of the invention, the preparation method of people's antibody is included in test antibody in the FVIIa competition experiments and screening combines people's antibody of competing with FVIIa.

In another embodiment of the invention, the preparation method of people's antibody is included in the TF ELISA test that comprises TF test antibody and screening can be in conjunction with people's antibody of people TF.

In another embodiment of the invention, the preparation method of people's antibody is included in test antibody in the direct TF ELISA test that comprises immobilization TF and screening can be in conjunction with the step of people's antibody of immobilization people TF.

In another embodiment of the invention, but the preparation method of people's antibody is included in the step of people's antibody of the people TF of test antibody in the indirect TF ELISA test that comprises the TF that is in the solution form and screening binding soln form.

In another embodiment of the invention, the preparation method of people's antibody comprises: produce test antibody on the cell of expressing TF in the test at FXa, and screening can suppress to express generation, the IC of FXa on the cell of TF ₅₀Value is lower than people's antibody of 500nM (in the test of the concentration of using FVIIa as 1nM).In another embodiment, described IC ₅₀Value is lower than 100nM.In another embodiment, described IC ₅₀Value is lower than 50nM.In another embodiment, described IC ₅₀Value is lower than 10nM.In another embodiment, described IC ₅₀Value is lower than 5nM.

In another embodiment of the invention, the preparation method of people's antibody comprises: produce people's antibody of the generation of FXa on the cell that test antibody on the cell of expressing TF in the test and screening can suppress to express TF, the IC of the people's antibody that is wherein screened at FXa ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+500nM (using in test 1nM FVIIa) is such as the IC that is lower than FFR-RFVIIa ₅₀Value+100nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+50nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+5nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value.

Term " cell of expression TF " refers to any mammalian cell of expressing human TF.

In another embodiment of the invention, the preparation method of people's antibody is included in intact cell TF combines the people TF competition of expressing on the cell surface of intact cell with the FVIIa competition in conjunction with test antibody in the test and screening people's antibody.

In another embodiment of the invention, the preparation method of people's antibody is included in the K that test antibody in the biosensor test and screening combine with people TF _dValue is lower than the step of people's antibody of 100nM.In another embodiment, the K that combines with people TF _dValue is lower than 10nM.In another embodiment, the K that combines with people TF _dValue is lower than 5nM.In another embodiment, the K that combines with people TF _dValue is lower than 1nM.In another embodiment, the K that combines with people TF _dValue is lower than 0.5nM.In another embodiment, the K that combines with people TF _dValue is lower than 10 ^-10M.In another embodiment, the K that combines with people TF _dValue is lower than 10 ^-11M.In another embodiment, the K that combines with people TF _dValue is lower than 10 ^-12M.In another embodiment, the K that combines with people TF _dValue is lower than 10 ^-13M.In another embodiment, the K that combines with people TF _dValue is lower than 10 ^-14M.In another embodiment, the K that combines with people TF _dValue is lower than 10 ^-15M.

In another embodiment of the invention, the preparation method of people's antibody is included in the step that test antibody in the conduction test of MAPK signal and screening can suppress FVIIa-inductive MAPK signal conduction activatory people antibody.In one embodiment, inhibition is 98% to described people's antibody to FVIIa-inductive MAPK signal conduction activatory.In one embodiment, inhibition is 90% to described people's antibody to FVIIa-inductive MAPK signal conduction activatory.In one embodiment, inhibition is 70% to described people's antibody to FVIIa-inductive MAPK signal conduction activatory.In one embodiment, inhibition is 50% to described people's antibody to FVIIa-inductive MAPK signal conduction activatory.In one embodiment, inhibition is 30% to described people's antibody to FVIIa-inductive MAPK signal conduction activatory.

Term " conduction of MAPK signal " in order to refer to the mediation mitogen-activated protein kinase (Mitogen-Activated-Protein-Kinase, MAPK) or incident series connection in its homologue activatory born of the same parents of taking place in response to various born of the same parents' external stimulus.Three groups of different map kinases in mammalian cell, have been identified: 1) regulate kinases (Erk1/2 or p44/42) outside the born of the same parents; 2) the terminal kinases (JNK) of c-Jun N-; With 3) the p38 kinases.The Erk1/2 approach comprises the phosphorylation of Erk1 (p44) and/or Erk2 (p42).Activatory map kinase, for example p44/42 MAPK can be indexed into nuclear, therein they can phosphorylation and activating transcription factor, comprise (Elk1) and transcribe signal transducer and activator (Stat).Erk1/2 can also make the kinases p90RSK phosphorylation in tenuigenin or the nucleus, and p90RSK can activate several transcription factors then.

Term " protein kinase " is in order to refer to make the Serine in peptide and/or the protein and/or the enzyme of Threonine and/or tyrosine phosphorylation.

Term " FVIIa-inductive MAPK signal conduction activation " combines and induces thus the MAPK signal to conduct in order to refer to FVIIa with TF in the mammalian cell.

In another embodiment of the invention, the preparation method of people's antibody is included in gene expression analysis and tests the step that test antibody also screens people's antibody that can suppress the rise of FVIIa inductive gene in (test 15), and described gene is independently selected from the group that comprises Fra-1, Id2 and Cyr61.

Should understand suppress the active anti-TF antibody of TF can be in conjunction with the different epi-positions that are present on the TF, might not only can suppress FVIIa and combined, but also can suppress FX or FXa combines with people TF with people TF.An object of the present invention is to screen the antibody that combines, suppresses thus the conduction of FVIIa inductive intracellular signal that can suppress FVIIa and people TF.

In another embodiment of the invention, the preparation method of people's antibody is included in the step that test antibody in the human cancer test (test 13) and screening can suppress people's antibody of the growth of human cancer or transfer.

In another embodiment of the invention, isolating people's antibody can suppress FVIIa inductive gene and raise, and described gene is independently selected from the group that comprises Fra-1, Id2 and Cyr61.

In another embodiment of the invention, isolating people's antibody does not suppress combining of FX or FXa and people TF.

In another embodiment of the invention, isolating people's antibody suppresses the interior activity of born of the same parents of TF.

In another embodiment of the invention, the preparation method of people's antibody is included in test antibody in the epitope mapping test and screening can be in conjunction with the step of people's antibody of the preferred epi-position on the TF.In one embodiment, preferred epi-position comprises one or more in Trp45, Lys46 and the Typ94 residue.In one embodiment, preferred epi-position comprises residue Trp45.In one embodiment, preferred epi-position comprises residue Lys46.In one embodiment, preferred epi-position comprises residue Tyr94.

In another embodiment of the invention, isolating people's antibody can be in conjunction with the epi-position on the interface between TF and the FVIIa.

Between the proteolytic enzyme structural domain of the responsible FVIIa that determines according to the X-ray structure and the TF in the interactional TF residue (Banner etc. 1996 " nature " (Nature) 380:41-46) are: Ser39, Gly43, Trp45, Ser47, Phe50, Arg74, Phe76, Tyr94, Pro92.The proteolytic enzyme structural domain of this FVIIa and the interface between the TF be characterized as the complicated interface district of containing many intermolecular hydrogen bondings, this intermolecular hydrogen bonding makes to produce between TF and the FVIIa and well contacts and obtain high degree of specificity in the FVIIa cohesive process.

The invention still further relates to neutralizing high affinity human monoclonal antibodies at TF.The TF surface of containing the contact interface of FVIIa proteolytic enzyme structural domain has specific topological framework, be easy to react and produce protein-protein interaction, wherein the protein-protein interaction of another kind of type is that complex body between antibody and the protein ligands forms.Therefore, the monoclonal antibody at this epi-position on the people TF provides high-affinity Mabs.

One aspect of the present invention is that immunoreactive neutralizing high affinity human monoclonal antibodies takes place the proteolytic enzyme structural domain contact interface with FVIIa.

The inductive antagonist that solidifies that people TF antibody of the present invention can be used as TF-mediation works, thereby combining of anticoagulant factor FVIIa and TF blocked thrombin generation and fibrin subsequently thus and deposited.People TF antibody is particularly useful for the administration to the people, comprises the various diseases of intravascular coagulation with treatment.Like this, people TF antibody can be used to suppress the TF activity, for example causes the inhibition to blood coagulation, thrombosis or platelet deposition.In addition, the present invention works the people TF antibody that suppresses TF cell function, TF signal conduction function and can be used for disease as Sepsis, inflammation, atherosclerosis, restenosis or cancer.

People TF antibody can be used for various diseases.Comprise: disease or the illness relevant with thrombosis or coagulopathy, comprise inflammatory reaction and form relevant chronic thrombotic disease or illness with fibrin, comprise vascular disease, such as dvt formation, artery thrombosis, postoperative thrombosis, coronary artery bypass graft surgery (CABG), percutaneous coronary angioplasty (PTCA), apoplexy, tumor growth, metastases, blood vessel generation, thrombolysis, atherosclerosis and postangioplasty restenosis; Acute and chronic indication is such as inflammation, septic shock, septicemia, ypotension, adult respiratory distress syndrome (ARDS), systemic inflammatory responses syndrome (SIRS), disseminated inravascular coagulation (DIC), pulmonary infarction, pathologic platelet deposition, myocardial infarction; Or prophylactic treatment has the Mammals of venous occlusive disease, hemolytic uremic syndrome (HUS), thrombocytopenic purpura,thrombotic (TTP) and other disease or illness after the atherosclerotic blood vessel, the peripheral blood progenitor cell (PBPC) that are in the thrombosis danger are transplanted.This people TF antibody can be used for preventing the thromboembolic complication of the high-risk patient identified to take place, such as those patients that undergo surgery or suffer from those patients of congestive heart failure.The restenosis that this people TF antibody can be used for the treatment of intimal hyperplasia especially or cause because of acute vascular damage.Acute vascular damage is that those blood vessel injury diseases that (be several hours interior to some months) takes place rapidly are opposite with the chronic blood vessel injury that can take place throughout one's life (for example atherosclerosis).The acute vascular damage is usually because of these use the operation of technology such as angioplasty, endarterectomy, atherosclerotic plaque surgical blanking, blood vessel graft placement to cause such as reconstructing blood vessel.Hyperplasia for example also can be used as places or organ transplantation reaction's delayed response takes place graft.Because people's antibody has more selectivity than heparin, only be combined in the TF that injury site exposes, and because people's antibody can not destroy or suppress other solidifying egg white, so when being used for the prevention of deep venous thrombosis with precautionary approach, people TF can be more effective than heparin, and the more unlikely complication that causes bleeding.

As what confirmed in the following examples, people TF antibody of the present invention can selective binding cell surface TF and is suppressed its functionally active by anticoagulant factor FVIIa with combining of TF.Keep with TF bonded people TF antibody and can deposit the thrombocyte accumulation that suppresses the blood vessel injury site by blocking-up thrombin generation and fibrin subsequently.

Because people TF antibody can block the thrombin generation at acute vascular injury site place and limit platelet deposition, so keep and the combining, suppress thus FVIIa bonded people TF antibody and can be used to suppress vascular restenosis of TF.

The composition that comprises people TF antibody is particularly useful for treating when being mixed with pharmaceutical composition in patient's the method, wherein can be with described composition to the individual administration of suffering from the various disease situation with the treatment disease relevant with blood coagulation.Compare with other antithrombotics, this class can and suppress FVIIa and TF bonded people TF antibody may have long plasma half-life in conjunction with TF, thereby has the anticoagulating active phase of obvious longer time.The present composition at the medical science indication be usually with those indications of antithrombotics treatment, such as fibrin deposition, antiphospholipid syndrome (APS), atherosclerosis and myocardial infarction in: for example dvt formation, pulmonary infarction, apoplexy, disseminated inravascular coagulation (DIC), the lung relevant and the kidney with Sepsis.These compositions can be used to suppress the mechanicalness blood vessel injury, such as take place after the damage that causes because of operation, micrurgy, sacculus arterioplasty, endarterectomy, resetting property atherosclerotic plaque surgical blanking (reductive atherectomy), the placement of Si Tengte support, laser therapy or rotablation or as the vascular restenosis that blood vessel graft, Si Tengte support, by-pass graft thing or organ graft Secondary cases are taken place.Therefore these compositions can be used to suppress platelet deposition and relative disease.Therefore, the method that suppresses for example blood coagulation, vascular restenosis or platelet deposition comprises the composition of people TF antibody that the patient is comprised the consumption of effective anticoagulant, vascular restenosis or platelet deposition.These methods also can be applicable to treat individual coronary artery acute closed (for example acute myocardial infarction), and these methods comprise administration of human TF antibody and tissue plasminogen activator or streptokinase, and can quicken the thrombolysis of tPA inductive.Give thrombolytic agent, such as tissue plasminogen activator before, administration of human TF antibody immediately simultaneously or afterwards.

The human monoclonal antibodies that the present invention is directed to people TF can carry groups of people's immunity system but not transgenic mice (available from Medarex) the immunization people TF of mouse system produces by giving.To be used to produce the hybridoma of the described human monoclonal antibodies of secretion from the splenocyte of these transgenic mices (for example, referring to International Application No. WO such as Wood 91/00906; PCT such as Kucherlapati openly apply for WO91/10741; International Application No. WO such as Lonberg 92/03918; International applications such as Kay 92/03917; Lonberg, N. etc. 1994 " nature " are 368:856-859 (Nature); Green, L.L. etc. 1994 " natural genetics " (Nature Genet.) 7:13-21; Morrison, S.L. etc. 1994 " NAS's journal " are 81:6851-6855 (Proc.Natl.Acad.Sci.USA); Bruggeman etc. 1993 " annual immunology " are 7:33-40 (YearImmunol); 1993 PNAS 90:3720-3724 such as Tuaillon; Bruggeman etc. 1991 " European IMMUNOLOGY KEY WORDS INDEX (Eur J Immunol) 21:1323-1326).

Human monoclonal antibodies at people TF can also produce by phage display.Can make up people's antibody library by the people of immunization, and on the filobactivirus surface, show.Used panning technique from this class library, to separate neutralizing high affinity human strand Fv (ScFv) and Fab antibody fragment in many cases, wherein the antigen of being paid close attention to is fixed on solid surface, is (Barbas C.F. on microtitration flat board or the bead surface, III and Burton, D.R. " biotechnology trend " (Trends.Biotechnol.) 1996,14:230-234; " people's antibody " such as Aujame L. (Hum.Antibodies) 1997,8:155-68).The phage display in big original library also makes without immunization directly isolating human antibodies become that possible (DeHaard H.J. etc. " journal of biological chemistry " (J.Biol.Chem.) 1999,18218-18230).

The accompanying drawing summary

With reference to accompanying drawing the present invention is described particularly further in an embodiment, wherein

Accompanying drawing 1. is used to screen the typical shaker test sketch of the human monoclonal high-affinity antibody of anti-TF.

The detail drawing of accompanying drawing 2. shaker test 1-3 as described in example 1 above.

The detail drawing of accompanying drawing 3. shaker test 4-7 as described in example 1 above.

The detail drawing of accompanying drawing 4. shaker test 8-10 as described in example 1 above.

The example of accompanying drawing 5. use-testings 4 screening antibody.FFR-rFVIIa (solid circles) and the anti-TF monoclonal antibody of people HuTF-31 F2 (empty circles) are to the restraining effect of sTF/FVIIa acid amides lytic activity.

The example of accompanying drawing 6. use-testings 5 screening antibody.FFR-rFVIIa (solid circles) and people be anti--restraining effect that TF monoclonal antibody HuTF-31 F2 (empty circles) produces factor Xa.

The example of accompanying drawing 7. use-testings 7 screening antibody.FFR-rFVIIa (solid circles) and the anti-TF monoclonal antibody of people HuTF-31 F2 (empty circles) are to the restraining effect of people TF-inductive blood coagulation.

The example of accompanying drawing 8. use-testings 10 screening antibody.Only prevent the FVIIa bonded anti--the TF monoclonal antibody can suppress the signal conduction of TF/FVIIa-mediation.

The anti-TF Mab of accompanying drawing 9. people suppresses FVIIa inductive p44/42 MAPK phosphorylation (test 10).To carry out serum starvation 2 hours with the bhk cell of TF transfection, so that the cell dormancy.Adding FVIIa (15nM) 15 minutes before, antibody HuMab 30F5 (500nM) and HuMab31 F2 (500nM) are joined in the described cell.Make lysis and make protein separate on the SDS-PAGE and forward on the cellulose nitrate by electroblotting.Use is carried out western blot analysis at the polyclone phosphoric acid specific antibody of p44/42 MAPK.Two anti-anti-tame rabbit iggs for puting together with horseradish peroxidase.Use refrigerative CCD-photographic camera that chemoluminescence is detected.The band that band on the digitized image is carried out quantitatively also will using FVIIa to obtain is set at 100%.When with cell during with HuMab 30F5 (500nM) pre-incubation, the band of observing phosphorylation has reduced 50%, and when with cell during with HuMab 31 F2 (500nM) pre-incubation, the band of observing phosphorylation has reduced 25%.This experiment shows that finally people's antibody (30F5 and 31 F2) of anti-TF can partly suppress FVIIa inductive p44/42 MAPK phosphorylation.Use 50nM FVIIa to obtain similar results.

The example of accompanying drawing 10. use-testings 16 screening antibody.The monoclonal antibody with anti-TF that shown this accompanying drawing can suppress to express in the born of the same parents of TF in the cell of TF active.Active in the born of the same parents of anti-TF Mab B inhibition TF, and anti--TF Mab A can not suppress the interior activity of the born of the same parents of TF.

The example of accompanying drawing 11. use-testings 12 screening antibody.Use the FFR-rFVIIa of 0.5nM and people anti--rate distribution of the thrombelastogram that TF antibody HuTF-31F2 obtains.

Further explain the present invention by the following examples, but, these embodiment are not used for limiting protection domain.No matter disclosed feature is separately and or all can constitutes with various forms in the mode of its arbitrary combination and to realize flesh and blood of the present invention in foregoing description and the following examples.

Embodiment

Embodiment 1

The preparation that people TF is had the people Mabs of immunologic opsonin

Reagent

People TF can be as Rao, and L.V.M. (ThrombosisResearch), separates from human brain described in the 51:373-384 1988 in " thrombosis research ".

(Dade Innovin Baxter) also can be used as people's thromboplastin reagent to the recombinant human TF of fatization.Prepare rat, rabbit, baboon and pig thromboplastin by brain tissue.45 ℃ of 0.9%NaCl that in brain tissue, add two volumes, and with manual glass homogenizer homogenate tissue.45 ℃ of down insulations 30 minutes and simultaneously once in a while after the jolting, with sample centrifugal 20 minutes with 2000xg.Decantation precipitation and five equilibrium supernatant liquor and be stored in-80 ℃ standby down.

Can be by recombinant human total length TF (American Diagnostica #4500) be reconstituted in the TF that obtains again fatization in the phosphatide vesicle (PC/PS 75/25).

The biotinylated people TF of following production: the concentration of vitamin H-NHS (positive succinimido vitamin H, Sigma H-1759) being dissolved into 1.7mg/ml at DMF (dimethyl formamide).To be dissolved in 0.1M NaHCO ₃The 1mg/ml people TF of damping fluid joins in 60 μ l vitamin H-NHS solution, and it was at room temperature reacted 4 hours.The reaction soln that will contain biotinylation TF is to PBS-damping fluid dialysed overnight.

Used FVIIa is as Thim, and L. etc. are at " biological chemistry " (Biochem) 27:7785-7793, the recombinant human FVIIa of preparation described in 1988.

STF: expression in escherichia coli, basically as Freskg  rd, P.-O. etc. are at " protein science " (Protein Sci.) 5, the recombinant human soluble TF of purifying described in the 1531-1540 (1996) _1-209

S2288: at H ₂Be dissolved into 17.24mg/ml (Chromogenix) among the O again

FX: the human plasma FX of purifying (HFX, Enzyme Research Laboratories Ltd.)

FXa: with Russel ' s Viper Venom activatory purifying human plasma FX (HFXa, EnzymeResearch Laboratories Ltd.)

Chromozyme X: at H ₂Be dissolved into 1.25mg/ml (Boehringer Mannheim) among the O

¹²⁵I-FVIIa: obtain by the operation of standard radio-labeling.

FFR-rFVIIa: the FVIIa that on avtive spot, is sealed by the D-Phe-L-Phe-L-Arg-chloromethyl ketone.

As Sorensen B.B. etc. at " journal of biological chemistry " (J.Biol.Chem.) 272:11863-11868, described in 1997 in preparation.

Immunization

Emulsification people TF in Freund's complete adjuvant.Give 40 μ g by subcutaneous injection to HuMab mouse or its hybrid (Medarex).The TF that uses similar injection system to be dissolved in incomplete Freund's adjuvant for mouse booster shots 20 μ g after 14 days and 28 days finally carries out multiple dosing with 14 days intervals.10 days blood samplings are also by the people TF specific antibody in TF ELISA (the test 1 and 2) test sera after the final shot.

Merge

Give from the people TF that tests the mouse booster shots 20 μ g that serologic test is positive among the 1-3 by intravenous injection, after 3 days, put to death animal.Extract spleen and make it be dispersed into unicellular suspension through sterile manner.

The fox myeloma cell is grown in the CD hybridoma substratum (Gibco 11279-023).

Merge splenocyte and myeloma cell (P3 * 63 Ag8.653, ATCC CRL-1580), and Sp2/0 (ATCC CRL-1581) myeloma cell line is made our syzygy (Kohler, G ﹠amp by the PEG-method; Milstein C. (1976), " European IMMUNOLOGY KEY WORDS INDEX (European J.Immunology), 6:511-19).Cell inoculation is incubated down on the microtitration flat board and at 37 ℃.In ensuing 2 weeks, change substratum three times.From every hole, take out from 100 μ l supernatant liquors of hybridoma and with TF ELISA (test 1 and 2) and test the TF specific antibody.

Embodiment 2: screening

Various tests used when being used for the screening of serum and culture supernatants and selecting antibody to seek specificity are described below:

Directly TF ELISA test (test 1):

At 4 ℃ of following people sTF that are dissolved in PBS with 1 μ g/ml the dull and stereotyped bag of Nunc immunity is spent the night.(contain 5mM CaCl with the sealing damping fluid ₂TBS with 2%BSA) sealing is dull and stereotyped also with TBS+0.05%Tween 20 washings, adds the supernatant liquor from hybridoma.After at room temperature being incubated 1 hour, washing is dull and stereotyped, and adds the Anti-Human IgG with horseradish peroxidase (HRPO) mark.After being incubated 1 hour again, washing dull and stereotyped and with TMB-substrate (Kem-EN-Tec) as colour developing as described in the manufacturers.On the ELISA-reader, measure the absorbancy at 450nm place.

TF ELISA test indirectly (test 2):

(Southern BiotechnologyAssociates, CAT-#2040-1) bag is dull and stereotyped and 4 ℃ of following incubated overnight by Nunc immunity to be dissolved in the anti-human IgG of goat of PBS with 0.5 μ g/ml.Dull and stereotyped with sealing damping fluid (TBS that contains 5mM CaCl2 and 2% BSA) sealing, and use TBS+5mM CaCl ₂+ 0.05% Tween 20 washings.Adding is from the culture supernatants of hybridoma, and at room temperature with flat board insulation 1 hour.After the washing, add concentration and be the biotinylation people s TF of 1 μ g/ml and be incubated 1 hour once more.After the washing, add streptavidin-HRPO solution of 100 μ l and be incubated 1 hour.As make dull and stereotyped colour developing with the TMB-substrate as described in testing 1.

FVIIa competition experiments (test 3):

Under 4 ℃ that the Nunc immunity is dull and stereotyped with people sTF (concentration is the PBS solution of 5 μ g/ml) incubated overnight.Washing is dull and stereotyped also with containing 5mM CaCl ₂Dull and stereotyped with the TBS damping fluid sealing of 2% BSA.Add Anti-Human TF Mabs and flat board is incubated 2 hours.Washing is dull and stereotyped, and (l μ g/ml is dissolved in and contains 5mM CaCl after this to add biotinylation people FVIIa ₂In the TBS damping fluid of 2% BSA), and with flat board insulation 1 hour.Washing is dull and stereotyped, after this adds the streptavidin of HRPO-mark and is incubated 45 minutes.Washing is dull and stereotyped once more, after this uses TMB-substrate (Kem-EN-Tec) as colour developing as described in the manufacturers.

The inhibition of FVIIa/sTF acid amides lytic activity (test 4):

Use soluble human TF (10nM), recombinant human FVIIa (10nM) and increase the restraining effect of Mabs (0.0122-50nM) the test Anti-Human TF Mabs of concentration the catalytic acid amides lytic activity of FVIIa-TF.At room temperature with the Anti-Human TF Mabs of different concns or FFR-rFVIIa and 10nM sTF and FVIIa at BSA damping fluid (the 50mM Hepes of pH7.4,100mM NaCl, 5mM CaCl ₂With 1mg/ml BSA) in the insulation 60 minutes, after this add substrate S2288 (1.2mM, Chromogenix).At the 405nm place with color reaction METHOD FOR CONTINUOUS DETERMINATION 30 minutes.The acid amides lytic activity is expressed as mOD/ minute.Can calculate the IC that Mabs suppresses FVIIa/TF acid amides lytic activity ₅₀Value.The IC of FFR-rFVIIa in this test ₅₀Value is 7 ± 3nM.

The inhibition that FXa produces (test 5).

At room temperature will be dissolved in fat TF (10pM), FVIIa (100pM) in the BSA damping fluid (referring to test 4) and anti--TF Mabs or FFR-RFVIIa (0-50nM) and be incubated 60 minutes, after this add FX (50nM).After 10 minutes, make this reaction terminating again by the stop buffer (the 50mM Hepes of pH7.4,100mM NaCl, 20mM EDTA) that adds 1/2 volume.By add substrate S2765 (0.6mM, Chromogenix) and measured the amount of the FXa of generation in 10 minutes in 405nm place METHOD FOR CONTINUOUS DETERMINATION absorbancy.Can calculate the FX activatory IC that Mab suppresses FVIIa/ fat TF-mediation ₅₀Value.The IC of FFR-rFVIIa in this test ₅₀Be worth is 51+/-26pM.

Biosensor test (test 6):

The standardized solution that makes Anti-Human TF Mab on the Biacore instrument comes test antibody by the chip that has at the immobilized antibody of human IgG.The sTF that is dissolved in 10mM hepes (pH7.4) that adds different concns subsequently, wherein said 10mM hepes contains 150mM NaCl, 10mMCaCl ₂With 0.0003% polysorbate 20.According to transmitter figure (sensorgrams), by comprehensive Biacore evaluation software calculating K _dValue.

TF-dependency setting test (test 7):

On ACL300 Research Blood coagulation instrument (ILS Laboratories), carry out this test.To be dissolved in Anti-Human TF Mabs diluent and the 25mM CaCl of 50mM imidazoles (pH7.4), 100mM NaCl, 0.1%BSA ₂According to 2: 5 mixed and join in the sample cup of Blood coagulation instrument.Will be from people, rat, rabbit, baboon or pig, the about 30 seconds cytozyme of setting time is put into reagent reservoir 2 with imidazole buffer dilution and in the sample that does not contain antibody, and people, rat, rabbit, baboon or porcine blood plasma is put into reagent reservoir 3.In analytic process, with antibody and the CaCl of 70 μ l ₂Mixture changes in the 25 μ l cytozyme reagent and pre-incubation 900 seconds, after this adds 60 μ l blood plasma, and measures setting time.The longest setting time is decided to be 400 seconds.Use each diluent of cytozyme as typical curve, be used for setting time is changed into the TF activity of comparing with the contrast that does not add anti--TFMabs or FFR-rFVIIa.The IC of FFR-rFVIIa in this test ₅₀Be worth is 4.4+/-0.4pM.

Mabs suppresses (test 8) to the catalytic FX activatory of FVIIa/ cell surface TF:

With monolayer cell, for example people lung fibroblast WI-38 (ATTC No.CCL-75), human bladder cancer cell line J82 (ATTC No.HTB-1), the human keratinized cell of expressing human TF is that CCD1102KerTr (ATCC no.CRL-2310), people spongioblast oncocyte U87 or MCF-7 MDA-MB231 originate as the TF in the catalytic FX activation of FVIIa/TF.(1mg/ml BSA and 5mM Ca have been replenished with buffer A (10mM Hepes (pH7.45), 150mM NaCl, 4mM KCl and 11mM glucose) and buffer B ²⁺Buffer A) with on 24-, 48-or the 96-hole flat board converge cell monolayer each the washing once.In cell, add FVIIa (1nM), FX (135nM) simultaneously and be dissolved in the Mab (or FFR-rFVIIa) of buffer B with different concns.Perhaps, cell with anti--TF Mabs or FFR-rFVIIa pre-incubation 15 minutes, is after this added rFVIIa and FX.Make FXa form 15 minutes down at 37 ℃.From every hole, take out 50 μ l aliquots and join in the 50 μ l stop buffers (having replenished the buffer A of 10mM EDTA and 1mg/ml BSA).By going to 50 μ l said mixtures in the microtitration plate well and in each hole, adding the amount that 25 μ l Chromozym X (final concentration 0.6mM) measure the FXa of generation.In 405nm place METHOD FOR CONTINUOUS DETERMINATION absorbancy, and use the FXa typical curve that the initial rate of color reaction is changed into FXa concentration.The IC of FFR-rFVIIa in this test ₅₀Value is 1.5nM.

Mab is right ¹²⁵I-FVIIa and cell surface TF bonded suppress (test 9):

Use the cell of expressing human TF, for example human fibroblasts WI-38 (ATTC No.CCL-75), human bladder cancer cell line J82 (ATTC No.HTB-1), human keratinized cell are that CCD1102KerTr (ATCC no.CRL-2310), people's glioblastoma clone U87 or MCF-7 MDA-MB231 carry out combination research.With buffer A (referring to test 8) and buffer B (referring to test 8) confluent monolayer on the tissue culture plate of 24-hole is respectively washed once.With described individual layer with 100 μ l cold buffer liquid B pre-incubations 2 minutes.The Mab (or FFR-rFVIIa) and the radiolabeled FVIIa (0.5nM that in cell, add different concns simultaneously ¹²⁵I-FVIIa) (final volume 200 μ l).Flat board is incubated 2 hours down at 4 ℃.When insulation finishes, remove unconjugated material and with ice-cold buffer B with cell washing 4 times, (200mM NaOH, 1% SDS and 10mM EDTA) makes its cracking with 300 μ l lysis buffers.Measure radioactivity with gamma counter (Cobra, Packard Instruments).Analyze binding data and use GraFit4 (Erithacus Software, Ltd., (U.K.) curve plotting.The IC of FFR-rFVIIa in this test ₅₀Value is 4nM.

Mab suppresses (test 10) to FVIIa/TF-inductive p44/42 MAPK activatory:

By to carrying out the amount that detection by quantitative (FujifilmLAS-1000) is measured phosphorylation p44/42 MAPK and/or Akt and/or p90RSK from the chemoluminescence of western blot analysis.Cell, for example CCD1102KerTr, NHEK P166, people's glioblastoma clone U87 or the MCF-7 MDA-MB231 of expressing human TF were cultivated 24 or 48 hours with 0-0.1% FCS in substratum, after this make the experiment of cell dormancy.On the same day of experiment, cell must have the degree of converging of 70-80%.By under 37 ℃ with cell pre-incubation 30 minutes in the substratum that does not contain serum, after this add 10-100nM FVIIa and be incubated 10 minutes and carry out this experiment with excessive Mab or FFR-rFVIIa.As the positive control of cell signaling, cell was handled 10 minutes with 10% FCS.With ice-cold PBS with cell washing 2 times, after this make cell at lysis buffer (the 20mM Tris that contains 0.1mM 4-(2-amino-ethyl) benzene sulfonyl fluorine (AEBSF) and 1mM benzamidine, 0.1% Triton X-100,1mM EDTA, 1mM EGTA, 50mM Sodium Fluoride, 10mM β-phospho-glycerol sodium, 5mM trisodium phosphate, 150mM NaCl, pH7.5.Face to use before and add: 1mM sodium orthovanadate, 5 μ g/ml leupeptins, 10 μ g/ml press down the enzyme peptide) middle cracking.Lysate is mixed with the SDS-sample buffer and last SDS-polyacrylamide gel.On each gel, load standard biological elementization protein labeling.By electroblotting will be on the SDS-polyacrylamide gel isolating protein transduction to nitrocellulose, and kinases p44/42 MAPK, Akt and p90RSK are manifested, and chemoluminescence is carried out quantitatively by Fujifilm LAS1000 by the electroblotting that uses phosphorylation specific antibody.

Epitope mapping test (test 11):

The preparation of soluble T F (sTF) variant

Use inverse PCR (QuikChange, Stratagene, La Jolla, CA, USA), with wild plasmid (Freskg  rd, P.-O. etc. " protein science " (Protein Sci.) 5,1531-1540,1996) make up sTF variant (I22C, W45C, K46C, Y94C, F140C, W158C, K201C) as template.As other place in the document (Freskg  rd, P.-O. etc. " protein science " (Protein Sci.) 5,1531-1540,1996) state, and through certain mode of revising at expression in escherichia coli and described wild-type of purifying and variant.(Biox, Sweden) technology is carried out lysis in the 10mM of pH7.5 Tris-HCl damping fluid, and it is suspended in the same buffer of having added 1mg DNA enzyme again by X-pressure.Under 4 ℃ with 11000 * g with this solution centrifugal 20 minutes, and make sex change among inclusion body 75ml 6M GuHCl, 0.5MNaCl, 20mM Tris-HCl, the pH8.0.After at room temperature being incubated 1 hour, dilute by denatured protein being added drop-wise in the 1L solution that contains 50mM Tris-HCl, 0.25M NaCl, pH8.0, simultaneously stir about carried out refolding in 2 hours gently.(Pharmacia, UJppsala Sweden) and as (1996) described FVIIa affinity chromatographies such as Freskg  rd carry out purifying to use Q-Sepharose Fast Flow.Verify proteinic homogeneity by SDS-PAGE.At A _280nmThe place measures and uses and calculate optical extinction coefficient 37440M ^-1Cm ^-1(Gill and von Hippel, 1989) determine concentration.

Wrap wild-type sTF and the variant (10 μ g/ml) that is dissolved among the TBS for MaxiSorp flat board (Nunc-Immuno), and seal with sealing damping fluid (TBS that contains 0.1%Tween 20 and 0.5%BSA).Flat board is washed with lavation buffer solution (TBS and 0.1%Tween 20).Applying the concentration that is dissolved in the sealing damping fluid is the Anti-Human TF Mabs of 1ng/ml and is incubated 1 hour.Use lavation buffer solution washing dull and stereotyped (6x) then.Use subsequently by the Anti-Human IgG that in sealing damping fluid dilutes at 1: 2000 through the HRP mark (Helica Biosystems, Inc), use TMBplus-substrate (Kem Tech Cat.4390A) detection antibodies.With final ELISA signal (OD _450-620) weigh combining of each antibody and all sTF variants.

Thrombelastography (test 12)

With people's cytozyme (for example, Innovin, Dade Behring finally dilutes 50,000x) and CaCl ₂(final concentration 16.7mM) and anti--TF Mabs mix and at room temperature are incubated 15 minutes.In RoTEG sample cup (Pentapharm), add people's whole blood (280 μ l) of citrate-stableization and, after this add 40 μ l cytozyme/CaCl 37 ℃ of following preheatings 5 minutes ₂/ anti--TF Mab mixture.Trace 1 hour in the enterprising promoting circulation of blood bolt of Ro-TEG instrument (Pentapharm) elasticity subsequently.Use CoagPro Software ^TM(MedScience, rhus Denmark) obtain velocity profile from thrombus figure (thrombograms).

Embodiment 3.

The test of people's tumour.The research end user is anti--and TF Mabs treatment is to the effect (test 13) of people's growth of tumor and transfer in the mouse model

Treatment:

Resist-TF Mabs 10mg/kg=0.1mg/10g by intravenously bolus injection administration of human; Volume injected is that every 10g mouse is used one of three kinds of treatment solution of 0.1ml:

A. vehicle Control

B.1mg/ml people FFR-rFVIIa

C.1mg/ml resist-TF Mabs

Model description:

1. the long and hepatic metastases of the former generation of colorectal carcinoma

Use the 7-8 Healthy female athymic mouse (nu/nu) in age in week.In order to destroy the residual immunotolerance of nude mice, implant preceding 2 days with 5Gy irradiation mouse (Vogel etc. 1997) carrying out people's tumour usually to people's Transplanted cells.Attack mouse (Li etc. " people's gene therapy " (Human Gene Therapy) 10:3045-3053,1999) by the tumour transplatation of carrying out the LS174T human colon cancer cell (ATCC CCL 188) of cultivation in the RPMI 1640 that contains 15% foetal calf serum (FCS) as described.Briefly, with trypsinase-EDTA collecting cell, washed twice and be suspended in again among the RPMI of heparin sodium aqua (1U/ml) that do not contained replenishing of serum.The 33106 LS174T cells of doing little otch then and will be dissolved in 50ml phosphate-buffered saline (PBS) under narcosis under the rib of the left side of mouse inject in the spleen.After 3-5 minute, connect the spleen blood vessel and take out spleen by operation.This process causes that (more than 95%) takes place in stable hepatic metastases.After transplanting, begin immediately with anti--TF Mabs treatment, and in the remaining research time limit, continue to carry out.The the 15th and the 30th day execution mouse of inoculated tumour cell, take out liver and also weigh, visible tumour brief summary on the liver surface is counted.In AFA (5% acetate, 75% ethanol, 2% formalin, 18% water), fixedly spend the night, be transferred in 100% ethanol liver sample and be embedded in the paraffin, prepare 5 μ m section be used for to shift brief summary carry out histology quantitatively, immunohistochemistry and apoptosis be quantitative.

Research 1-1:

Purpose: in order to check intravenously to anti--TF Mabs of nude mice bolus injection 10mg/kg macroscopic view growth and effect on liver metastasis to the LS174T colorectal carcinoma.

Mouse: 60 the 6 male NMRI of week homozygous nu/nu in age.

Group: mouse is randomized into 15 1 group 4 groups, and treats with solution A, B or C.

Stop: losing weight＞20% or occur putting to death animal under the objective sign of other serious toxicity.

Parameter: every day is with two quadrature reference record tumour sizes in the vegetative period process.Record body weight during beginning, and write down 2-3 time weekly.The execution back forms the metastatic tumor in the liver and measures.

II. growth of the primary of mammary cancer and lung shift

Cultivator breast cancer cell MDA-MB-231 (ATCC HTB26) in the improved Eagle substratum of Dulbecco (DMEM) that has replenished 10% foetal calf serum (FCS).With MDA-MB-231 cell (33106) through subcutaneous injection nude mice (7-8 week age female mice).Estimate the primary tumo(u)r growth as mentioned above and shift (Li etc. " people's gene therapy " (Human Gene Therapy) 12:515-526,2001)

Research II-1:

Purpose: for after checking intravenously to anti--TF Mabs of nude mice bolus injection 10mg/kg to the macroscopic view growth of MDA-MB-231 breast tumor and the effect of lung transfer.

Mouse: 60 the 6 male NMRI of week homozygous nu/nu in age.

Parameter: use two quadrature reference record tumour sizes every day in the vegetative period process.Record body weight during beginning, and write down 2-3 time weekly.The execution back forms the metastatic tumor in the lung and measures.

III. the former of glioma heterograft grows

Tumor cell line MG U373 is the polymorphic clone of people's glioblastoma, and it has height angiogenic activity, high vessel density and growth fast in nude mouse.According to standard step tumor inoculation is gone into flank (referring to being the appended scheme of experimental plan).Observe twice to the toxicity sign of mouse every day, and every day is with two quadrature parameter measurement tumours.

Tumour is implanted the homozygous nude mice flank of nu/nu place with NMRI background.Mouse is available from M﹠amp; B (Ry, Denmark) 7 the week age male mice.Animal is remained in the limit collarium border, and they are accepted aseptic food particles and can arbitrarily drink water.

Use the glioma model to carry out three kinds of different researchs:

Research III-1:

Purpose: for check intravenously to anti--TF Mabs of nude mice bolus injection 10mg/kg to the macroscopic view growth of U373 tumour and the influence of lung transfer.

Mouse: 60 the 6 male NMRI of week homozygous nu/nu in age.

Parameter: use two quadrature reference record tumour sizes every day in the vegetative period process.Record body weight during beginning, and write down 2-3 time weekly.

Research III-2:

Purpose: after the tumor growth of pretreat had been set up in check, intravenously gave anti--TF Mabs of nude mice bolus injection 10mg/kg influence to the macroscopic view growth of U373 tumour.

Mouse: 60 the 6 male NMRI of week homozygous nu/nu in age.

Group: mouse is randomized into 15 1 group 4 groups, and treats with solution A, B or C.When 6 continuous (every day) observed values show the Gompertzian growth, the treatment beginning.It is equivalent to 100-200mm ³

Stop: treatment continues to tumour and has grown to above about 1.0cm ³Overall dimension, promptly do not have diameter of tumor, or up to having set up the Gompertzian regrowth by 6 METHOD FOR CONTINUOUS DETERMINATION greater than 15mm.When test stops, from every treated animal, downcut tumour and be used for histology and immunochemistry evaluation.Losing weight＞20% or put to death animal when the objective sign of other serious toxicity occurring.

Parameter: use two quadrature reference record tumour sizes every day in the vegetative period process.Record body weight during beginning, and write down weekly 2 times.

Research III-3:

Purpose :-TF Mabs anti-in order to check is to the influence of nude mice encephalic U373 tumor growth.

Mouse: 60 the 6 male NMRI of week homozygous nu/nu in age.

Tumour: implant U373 through original position at the right side hemicerebrum according to standard step.

Stop: the mouse with chronic nervous lesion sign is implemented euthanasia.

Data: survival rate (promptly reaching the time of nervous lesion) is carried out quantitatively by the Kaplan-Meyer statistical analysis.

Embodiment 4 (test 14)

Knocking out the TF gene and inserting in the mouse (mTF-KO/hTF-KI mouse) of people TF gene, 0.5ml matrigel (matrigel) is filled in through the subcutaneous subcutaneous abdomen of putting into.In this matrigel, sneak into b-FGF (5ng), carry out quantitatively (blood vessel generation) by measuring the significant neovascularity that content of hemoglobin forms in to gel in 1 week back.Can single or non-repeatedly enteron aisle give described protein postevaluation people anti--the inhibition ability (content of hemoglobin suppresses %) of TF Mabs.

Embodiment 5 (test 15)

Distinguish the gene expression analysis test that prevents FVIIa and TF bonded antibody and prevent FX and TF bonded antibody

In the cDNA microarray analysis, having observed has three kinds of gene generation specificitys to raise in the BHK-TF cell of handling with FVIIa.These genes comprise: Fra-1, the gene of the antigen 1 that the Fos that promptly encodes regulates; Id2, the helix-loop-helix proteinoid member's that promptly encodes gene; Cyr61 with coding extracellular matrix signalling conductive protein.Design following test so that screening prevents anti--TF Mabs that FVIIa inductive Fra-1, Id2 or Cyr61 raise.

Cell cultures

Except as otherwise noted, reagent all is purchased the Technologies from GIBCO-BRL Life.

Make as Poulsen L.K. etc. at " journal of biological chemistry " (JBiol.Chem.) 273,6228-6232, the BHK-TF cell that produces described in 1998 are grown in the improved Eagle substratum of Dulbecco that contains 10% FCS, 100 IU/ml penicillin and 100 μ g/ml Streptomycin sulphates to obtaining 95-100% and converge, wash and in the substratum that does not contain FCS regrowth 16-18 hour.Washed cell and make its contact not contain FCS and contain the substratum of 100nM FVIIa once more.

In order to clone the fragment that is used for rna blot analysis, processing cell as described below.Cell is grown in contain in the improved Eagle substratum of Dulbecco of 10%FCS, 100 IU/ml penicillin and 100 μ g/ml Streptomycin sulphates to obtaining 95-100% and converges, wash and in the substratum that does not contain FCS regrowth 16-18 hour.Washed cell once more, and make its contact not contain FCS and contain the substratum 1 hour of 100nM FVIIa.CRL2091 cell (ATCC) is grown in contain in the improved Dulbecco substratum of Iscove of 10% FBS, 100 U/ml penicillin and 100 μ g/ml Streptomycin sulphates and converges to reaching 95-100%.Make cell carry out serum starvation 16-18 hour subsequently, and the substratum that contains 100nM FVIIa with not containing FBS was handled 6 hours.Mouse 3T3-L1 cell (ATCC) is maintained in the improved Eagle substratum of Dulbecco that has replenished 10% foetal calf serum, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates.Cell is grown to converge, and induced 1 hour with the substratum that contains 1 μ M dexamethasone (Sigma), 10 μ g/ml insulin humans (Novo Nordisk A/S) and 1 μ MBRL49653 (Novo Nordisk A/S).

The segmental clone who is used for rna blot analysis

Use superscript II test kit (Life Technolo-gies), according to the explanation of manufacturers, from the RNA of 3T3-L1 cell that separates personal dexamethasone, Regular Insulin and BRL49653 and handle 1 hour, clone Fra-1 by reverse transcription PCR.By reverse transcription PCR, clone Id2 and CYR61 from separating from respectively with 1 hour BHK-TF cell of FVIIa processing and RNA with 6 hours CRL2091 cell of FVIIa processing.The upstream and downstream primer is: Fra-1,5 '-GCGGCCGCCATGTACCGAGACTACGGGGAACCG-3 ' and 5 '-GCGGCCGCTCACAAAGCCAGGAGTGTAGG-3 '; Id2,5 '-CAGCATGAAAGCCTTCAGTC-3 ' and 5 '-CTCTGGTGATGCAGGCTGAC-3 '; Cyr61,5 '-CGTCACCCTTCTCCACTTGA-3 ' and 5 '-CTTGGTCTTGCTGCATTTCT-3 '.The PCR parameter is: by annealing 15 seconds 94 ℃ of following sex change 10 seconds, under 65 ℃ and extending a circulation of forming in 1.5 minutes down at 72 ℃; By annealing 15 seconds 94 ℃ of following sex change 10 seconds, under 64 ℃ and extending a circulation of forming in 1.5 minutes down at 72 ℃; By annealing 15 seconds 94 ℃ of following sex change 10 seconds, under 63 ℃ and extending a circulation of forming in 1.5 minutes down at 72 ℃; By annealing 15 seconds 94 ℃ of following sex change 10 seconds, under 62 ℃ and extending a circulation of forming in 1.5 minutes down at 72 ℃; By annealing 15 seconds 94 ℃ of following sex change 10 seconds, under 61 ℃ and extending a circulation of forming in 1.5 minutes down at 72 ℃; By annealing 15 seconds 94 ℃ of following sex change 10 seconds, under 60 ℃ and extending a circulation of forming in 1.5 minutes down at 72 ℃; By annealing 15 seconds 94 ℃ of following sex change 10 seconds, under 55 ℃ and extending 40 circulations forming in 1.5 minutes down at 72 ℃.All fragment clonings are gone into TOPO 2.1 (Invitrogen) and use the order-checking of Megabase sequenator.

Rna blot analysis

Use TriZol, from the BHK-TF cell that is incubated with FVIIa, FX, ASIS, 1F44A1 or TF8-5G9, separate total RNA according to the explanation of retailer.The RNA of 20 μ g is carried out size fractionation separate, transfer on the Hybond N+ film (Amersham) in the denaturant gel that contains 1% agarose, 20mM MOPS, 5mM NaOAc, 6% formaldehyde and 1mM EDTA by the kapillary trace, and crosslinked fixing by UV.Use [α- ³²P] dATP (Amersham), with the cDNA of Prime It test kit (Stratagene) label coding Fra-1, Id2 or Cyr61, and use Express Hyb (Clontech), according to the specification sheets hybridization of manufacturers, by the radioautograph display result.

Embodiment 6 (test 16)

The MAPK that is undertaken by Elk1 transcription factor/luciferase reporter gene (PathDetect) tests

The HeLa cell inoculation is converged to 40% in the T-80 flask, begin transfection after 1 day.Use 36 μ l FuGene (Roche) transfectional cell described in guide with 150ng pFA-ELK1 (Stratagene), 3 μ g pFR-Luc (Stratagene), 3 μ g people TF/pcDNA3 and 3 μ g murine protein enzyme activation acceptor 2/pcDNA3.1+.Used Versene on the 2nd day ^TM(Invitrogen) make the cell desorption, and be seeded in the black 96 holes observation dull and stereotyped (Packard) with the cell density of 20.000 cells/well.After cell and flat board adhered to again, the improved Eagle substratum of Dulbeccos (Invitrogen) that does not contain serum with 160 μ l/ holes was replaced above-mentioned substratum and is incubated 16 hours.

Cell is not contained one of substratum (reference substance), 20 μ l, 2.5 μ MFFR-rFVIIa (reference substance), 20 μ l, 2.5 μ M anti-TF Mab B or the anti-TF MabA of 20 μ l, the 2.5 μ M pre-incubation 1 hour of serum with 20 μ l.In the half hole, add 20 μ l, 0.5 μ M FVIIa, and substratum is joined in second half hole.After insulation 4 hours, make cell carry out the luciferase gene test.As described in manufacturers, in cell, add LucLite (Packard) reagent.On TopCountMicroplate Scintillation (Packard), read the luciferase expression level.

Claims

1. isolating people's antibody, this antibody and the epi-position generation immune response that is present on the people TF.

2. the described isolating people's antibody of claim 1, this antibody suppress combining of human blood coagulation factor VII a and people TF.

3. any described isolating people's antibody among the claim 1-2, this antibody is monoclonal antibody.

4. any described isolating people's antibody among the claim 1-3, this antibody is recombinant antibodies.

5. any described isolating people's antibody among the claim 1-4, wherein said antibody is the Fab fragment.

6. any described isolating people's antibody among the claim 1-4, wherein said antibody is F (ab) ₂Fragment.

7. any described isolating people's antibody among the claim 1-4, wherein said antibody is F (ab ') ₂Fragment.

8. any described isolating people's antibody among the claim 1-4, wherein said antibody is strand Fv fragment.

9. any described isolating people's antibody among the claim 1-8, wherein said antibody and people TF bonded K _d10 ^-15-10 ^-8In the scope of M.

10. any described isolating people's antibody among the claim 1-9, wherein said antibody and people TF bonded K _d10 ^-15-10 ^-10In the scope of M.

11. immunoreactive people's antibody can take place comprising the epi-position with being present on the people TF for the treatment of significant quantity in a pharmaceutical composition.

12. a pharmaceutical composition comprising the epi-position with being present on the people TF for the treatment of significant quantity immunoreactive people's antibody can take place, wherein said antibody is antibody any one among the claim 1-10.

13. a composition is comprising with the epi-position that is present on the people TF immunoreactive people's antibody taking place.

14. a composition, comprising with the epi-position that is present on the people TF immunoreactive people's antibody taking place, wherein said antibody is antibody any one among the claim 1-10.

15. the treatment of diseases method that philtrum is relevant with FVIIa/TF, this method comprise that immunoreactive people's antibody can take place the epi-position with being present on the people TF that described people is treated significant quantity.

16. the treatment of diseases method that philtrum is relevant with FVIIa/TF, this method comprise described people is treated any described antibody among the claim 1-10 of significant quantity.

17. the preparation method of people's antibody, this method comprises the following steps:

A) people's antibody of the anti-people TF of preparation;

Test antibody and screening suppress people's antibody of FXa generation, the IC of the people's antibody that is wherein screened in FXa generation test ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using 0.1nMFVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+10nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+5nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+0.1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value; Or

Test antibody and screening suppress people's antibody of TF-inductive FVIIa acid amides lytic activity, the IC of the people's antibody that is wherein screened in FVIIa/TF acid amides breaking test ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 10nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+40nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+20nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value; Or

18. the described method of claim 17, people's antibody of wherein said anti-people TF produces by the method that comprises the following steps:

A) to mammalian immune inoculation people TF;

B) separation is by the antibody of the Mammals generation of immunization.

19. the described method of claim 18, wherein said Mammals is a mouse.

20. any described method among the claim 17-19, this method comprises: test antibody and screening suppress people's antibody that grumeleuse forms, the IC of the people's antibody that is wherein screened in this test in the setting test of TF inductive ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is such as the IC that is lower than FFR-rFVIIa ₅₀Value+500pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+200pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+100pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+50pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+5pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value.

21. any described method among the claim 17-20, this method comprises: test antibody and screening suppress people's antibody of FXa generation, the IC of the people's antibody that is wherein screened in FXa generation test ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 0.1nMFVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+10nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+5nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+0.1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value.

22. any described method among the claim 17-21, this method comprises: test antibody and screening suppress people's antibody of TF-inductive FVIIa acid amides lytic activity, the IC of the people's antibody that is wherein screened in FVIIa/TF acid amides breaking test ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 10nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+40nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+20nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value.

23. any described method among the claim 17-22, this method are included in, and test antibody also screens the people's antibody that combines competition with FVIIa in the FVIIa competition experiments.

24. any described method among the claim 17-23, this method are included in the TF ELISA test that comprises TF test antibody and screening with people TF immunoreactive people's antibody takes place.

25. immune response can take place and suppress people's antibody that human blood coagulation factor VII a combines with people TF with the epi-position on being present in people TF, this antibody can obtain by the method that comprises the following steps:

A) people's antibody of the anti-people TF of preparation;

B) test antibody and screening suppress people's antibody that grumeleuse forms, the IC of the people's antibody that is wherein screened in this test in the setting test of TF inductive ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is such as the IC that is lower than FFR-rFVIIa ₅₀Value+500pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+200pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+100pM is such as the IC that is lower than FFR-rFVIIa ₅₀Value+50pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+5pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value; Or

Test antibody and screening suppress people's antibody of TF-inductive FVIIa acid amides lytic activity, the IC of the people's antibody that is wherein screened in FVIIa/TF acid amides breaking test ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using 10nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+40nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+20nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value; Or

Immunoreactive people's antibody can take place with people TF in test antibody and screening in the TF ELISA test that comprises TF.

26. the preparation method of people's antibody, this method comprises the following steps:

A) to mouse immune inoculation people TF;

C) isolation medium from contain the infinite multiplication cell that produces antibody to some extent;

D) immunoreactive people's antibody takes place in the people TF of test antibody and screening and solution form in the indirect TF ELISA test that comprises the TF that is in the solution form;

G) people's antibody of test antibody and screening inhibition FXa generation in FXa generation test, the IC of the people's antibody that is wherein screened ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+100nM (using in test 0.1nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+10nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+5nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+0.1nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value;

H) test antibody and screening suppress people's antibody that grumeleuse forms, the IC of the people's antibody that is wherein screened in this test in the setting test of TF inductive ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+1nM is such as the IC that is lower than FFR-rFVIIa ₅₀Value+500pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+200pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+100pM is such as the IC that is lower than FFR-rFVIIa ₅₀Value+50pM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+5pM is more preferably less than the IC of FFR-rFVIIa ₅₀Value;

I) in appropriate media, screen and cultivate the infinite multiplication cell that produces the antibody that is screened behind steps d-h;

27. the described method of claim 26, wherein said method further are included in the direct TF ELISA test that comprises immobilization TF test antibody and screening with immobilization people TF immunoreactive people's antibody takes place.

28. any described method among the claim 26-27, wherein said method further is included in FXa and produces in the test test antibody on the cell of expressing TF, and people's antibody of the generation of FXa on the cell of screening inhibition expression TF, the IC of the people's antibody that is wherein screened ₅₀Value is lower than the IC of FFR-rFVIIa ₅₀Value+500nM (using in test 1nM FVIIa) is such as the IC that is lower than FFR-rFVIIa ₅₀Value+100nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+50nM preferably is lower than the IC of FFR-rFVIIa ₅₀Value+10nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value+5nM is more preferably less than the IC of FFR-rFVIIa ₅₀Value.

29. any described method among the claim 26-28, wherein said method further are included in intact cell TF combines people TF expressed on the cell surface of intact cell with the FVIIa competition in conjunction with test antibody in the test and screening people's antibody.

30. any described method among the claim 26-29, wherein said method further are included in, and test antibody also screens the K that combines with people TF in the biosensor test _dValue be lower than 100nM, such as be lower than 10nM, preferably be lower than 5nM, preferably be lower than 1nM, more preferably less than people's antibody of 0.5nM.

31. any described method among the claim 26-30, wherein said method further comprise test antibody and screening inhibition FVIIa inductive MAPK signal conduction activatory people antibody in the MAPK signal conduction test.

32. further being included in test antibody in the epitope mapping test and screening, any described method among the claim 26-31, wherein said method with preferred epi-position on the TF immunoreactive people's antibody can take place.

33. the described method of claim 32, wherein said preferred epi-position comprises residue Trp45, Lys46 and Tyr94.

34. any described method among the claim 26-33, wherein said infinite multiplication cell is a hybridoma.

35. immune response can take place and suppress people's antibody that human blood coagulation factor VII a combines with people TF with the epi-position on being present in people TF, this antibody can obtain by the method that comprises the following steps:

A) to mouse immune inoculation people TF;

D) immunoreactive people's antibody can take place in the people TF of test antibody and screening and solution form in the indirect TF ELISA test that comprises the TF that is in the solution form;

36. immune response can take place and suppress people's antibody that human blood coagulation factor VII a combines with people TF with the epi-position on being present in people TF, it produces by method any among the claim 26-34.

37. immune response can take place with epi-position on being present in people TF and suppress combining of human blood coagulation factor VII a and people TF in a cell that produces people's antibody, wherein said people's antibody.

38. the described cell of claim 37, wherein said cell are isolating lymphoidocytes.

39. any described cell among the claim 37-38, wherein said cellular segregation is from mouse.

40. the described cell of claim 37, wherein said cell is a hybridoma.

41. the described cell of claim 40, wherein said hybridoma are by lymphoidocyte and the infinite multiplication cytogamy that makes generation antibody, provide the hybridoma that produces antibody to obtain.

42. any described cell among the claim 37-41, wherein said antibody can suppress combining of human blood coagulation factor VII a and people TF.

43. any described cell among the claim 37-42, immune response can take place with the three-dimensional surface that relates to Trp45, Lys46 and all these residues of Tyr94 in wherein said antibody.

44. any described cell among the claim 37-43, wherein said antibody is the Fab fragment.

45. any described cell among the claim 37-44, wherein said antibody is F (ab) ₂Fragment.

46. any described cell among the claim 37-44, wherein said antibody is F (ab ') ₂Fragment.

47. any described cell among the claim 37-44, wherein said antibody is the scFv fragment.

48. any described cell among the claim 37-47, the K that wherein said antibody combines with people TF _d10 ^-15-10 ^-8In the scope of M.

49. any described cell among the claim 37-48, the K that wherein said antibody combines with people TF _d10 ^-15-10 ^-10In the scope of M.