JP2000503528A - Novel factor VIIa inhibitors - Google Patents

Abstract

  (57) [Summary] The present invention provides a composition having a Factor VIIa active site inhibitory domain and a tissue factor domain for inhibiting FVIIa. The present invention also provides pharmaceutical compositions comprising the novel compounds and their use in diagnosis, treatment and prevention.

Description

DETAILED DESCRIPTION OF THE INVENTION                       Novel factor VIIa inhibitors Background of the Invention Field of the invention   The present invention relates to plasma factor VII / factor VIIa, Novel compounds that inhibit the activity associated with factor VIIa. In particular, the present invention Provided is a molecule having a factor VIIa active site inhibitory domain and a tissue factor domain I do. According to a preferred aspect, the two domains of the hybrid molecule of the invention are flexible. It is linked via a peptide linker domain. The present invention also relates to this novel compound And their use in diagnostic, therapeutic, and prophylactic methods We also provideDescription of related disclosures   Factor VIIa (FVIIa) depends on 50 kilodaltons (kDa) of vitamin K A two-chain plasma serine protease, which is a complex regulator of in vivo homeostasis Are involved in Factor VIIa is present in plasma at a concentration of about 0.5 μg / mL Proteolysis of one peptide bond of factor VII, a single-chain zymogen It is made. Activation of the conversion of zymogen factor VII into a double-stranded molecule Caused by cleavage of internal peptide bonds. Its cleavage site in human factor VII Are in Arg 152-Ile 153 (Hagen et al. (1986), Pr oc. Natl. Acad. Sci. USA, 83: 2412-2416; Thim et al. (1888), Biochem. 27: 7785-7793 page). In the presence of calcium ions, Factor VIIa has a high affinity for integral membrane proteins. It binds to tissue factor (TF), which is white matter. TF is a cofactor for factor VIIa Thus, the substrates Factor VII (FVII), Factor IX (FIX) and Enhances the proteolytic activation of Factor X (FX).   TF is a glycoprotein of 263 amino acid residues and an extracellular domain of 219 residues, Consists of one transmembrane domain and a short cytoplasmic domain (Morrisse y, J. et al. H. Et al., (1987), 50: 129. Extracellular domain of TF The protein consists of two fibronectin type III domains of about 105 amino acids each. FVIIa binding is completely mediated by the TF extracellular domain (Muller et al. (1994), Biochem. 33: 10864-10870; Gi bbs et al. (1994), Biochem. , Volume 33: 14003-140 10; Ruf et al. (1994), Biochem. , Volume 33: 1565 1572). The structure of the extracellular domain of TF has recently been determined by X-ray crystallography. (Harlos et al., (1994), Nature, 370: 662-). 666; Muller et al. (1994), Biochemistry, 3; 3: 10864).   The extracellular domain of TF is refined by alanine scanning mutagenesis Analyzed (Kelley et al., (1995), Biochemistry, 34: 10383-10392; Gobbs et al. (1994) supra; Ruf et al. (1994), supra). Amino acids 16-26 and 129- Residues in the region 147 bind to FVIIa and the blood coagulation function of this molecule Has contributed. Lys20, Trp45, Asp58, Tyr94, and P Each residue of he140 has a large free energy (ΔG) for binding to FVIIa. (1 kcal / mole) (Kelley et al., 1995 Year), supra).   Residues in the region of amino acids 157 to 168 are blood coagulation of TF-FVIIa (Kelley et al., (1995) supra; Ruf et al., (19) 1992); Biol. Chem. 267: 22206-22210) But not critical for FVII / FVIIa binding. No. 165 and 166 Lysine residues have been shown to be important for TF cofactor function, but FVIIa It is not involved in the formation of coalescence (Roy et al. (1991) J. Biol. Chem. 266: 22063; Ruf et al. (1992), J. Mol. Bi ol. Chem. 267: 6375). Replace these lysine residues with alanine Substitution results in a reduced rate of FX activation catalyzed by the TF-FVIIa complex (Ruf et al., (1992), supra). Both lysines 165-166 are residues of TF Located at the C-terminus of the fibronectin type III domain and based on mutagenesis results Surface opposite to the residues known to be important for FVIIa binding (Kelley et al. (1995), supra). Residues Trp158, Ly s159, Ser163, Gly164, Lys165, Lys166 and T One group of yr185 plays a certain role in the blood coagulation function of soluble TF, Not involved in FVIIa binding (Keeley et al., (1995) Out).   TF is constitutively expressed on blood cells separated from plasma by vascular endothelium (C Arson, S.M. D. And J. P. Brozna, (1993), Blood. Coag. Fibrinol. 4: 281-292). Endothelial cells and cells Expression of TF on the sphere can be an inflammatory cytokine or bacterial lipopolysaccharide. (Drake et al. (1989), J. Cel) l. Biol. 109: 389). TF exposed when tissue is damaged The extracellular domain forms a high affinity calcium-dependent complex with FVII. Once bound to TF, FVII is activated by peptide bond cleavage and To give protease FVIIa. Enzymes that catalyze this step in vivo are still uncertain Although not recognized, FXa, thrombin, TF-FVIIa, and And FIXa can catalyze this cleavage (Davie et al. (1991), vol. 30). : 10363-10370). FVIIa is its physiological substrate, FX and And FIX show only weak activity, whereas TF-FVIIa complex And FIX are rapidly activated.   TF-FVIIa complex constitutes the primary initiator of the extrinsic blood coagulation pathway (Carson, SD and JP Brozna, (1993), Bloo. d. Coag. Fibrinol. 4: 281-292; Davier, E . W. Et al., (1991), Biochemistry, 30: 10363. Rapport, S .; I. And L. V. M. Rao, (199 2 years), Arterioscler. Thromb. , Volume 12: 1111-11 21). The complex is composed of FX to Factor Xa (FXa), FIX to IX a Activation of factor (FIXa) and additionally FVII to FVIIa Initiates the extrinsic pathway. The action of TF-FVIIa is ultimately Induces the conversion of amino acids to thrombin and performs a number of biological functions (Badi mon, L .; Et al. (1991) Trends Cardiovasc. M ed. 1: 261-267). The most important function of thrombin is fibrino It converts gen into fibrin, the latter polymerizing to form a clot. TF-F The VIIa complex is a secondary factor that extends the physiological effects of the contact activation system. Even involved.   The involvement of this plasma protease system plays an important role in various clinical phenotypes However, these include arterial and venous thrombosis and septicemia. Disease, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulation (DIC), And various other disease states (Haskel, EJ et al., (199) 1 year), Circulation, 84: 821-827; Holst, J . Et al., (1993), Haemostasis, Vol. 23 (Supplement 1): 112- 117; Creasey, A .; A. Et al. (1993); C1in. I nvest. 91: 2850-2860; and Colman, R .; W. , (1989); Engl. J. Med. , 320: 1207-1209 Page; Bone, R .; C. (1992), Arch. Intern. Med. 152: 1381-1389). TF overexpression and / or Regular use has been linked to the pathophysiology of both thrombosis and sepsis (T Aylor et al. (1991) Circ. Shock) 33: 127; Warr et al. (1990) Blood 75: 1481; Poweras. He et al. (1994) Circ. Res. 74:56). TF is dynamic Expressed on the surface of cells in pulse sclerosis plaques (Wilcox et al., (19 89), Proc. Natl. Acad. Sci. USA, 86: 2839 page). In addition, TF has been implicated in tumor metastasis (Bromberg). Et al. (1995), Proc. Natl. Acad. Sci. USA, 92 Volume: 8205).   It has long been recognized that endogenous inhibitors to TF-FVIIa are present in serum (Schneider, CL, (1946), Am. J. Ph. ysiol. 149: 123). TF-FVIIa initiates blood clotting pathway Feedback inhibitors that prevent further zymogen substrate activation, histopathogenesis (Broze Jr., GJ). And others (1990), Biochemistry, 29: 7539-754. Page 6; Broze Jr. , G .; J. (1992), Semin. Hema tol. 29, 156-169). TFPI is involved in lipoproteins Also known as LACI or EPI, an anticoagulant and extrinsic pathway inhibitor ing. TFPI consists of an acidic amino-terminal region followed by three Kunitz-type domains and And a basic carboxyl terminal region. TFPI is TF-F in an FXa-dependent manner VIIa is believed to inhibit FXa by first binding FXa with a second Kunitz domain, Subsequently, FVIIa is bound at the first Kunitz domain (Girard, TJ. (1989) et al., Nature, 338: 518-520). FXa In the absence of TFPI, TFPI is a weak inhibitor of the TF-FVIIa complex (Gira rd, T .; J. , (1989), Science, 248: 1421. 1424).   Mutants of TFPI have also been produced that also inhibit TF-FVIIa activity. Kunit Mutants having the first two (residues 1-161) of the two domains have been produced and characterized (Hamamoto et al., (1993), J. Biol. Ch. em. 268: 8704-8710; Petersen et al. (1993). Year), J.M. Biol. Chem. 268: 13344-13351). Country WO 91/02753 and U.S. Pat. T retains protease inhibitory activity but has reduced binding affinity for heparin FPI variants have been described. One of the TFPI-FXa hybrid proteins is Normal plasma coagulation induced by TF-FVIIa was determined by an in vitro coagulation assay. It can be inhibited with significantly greater activity than TFPI alone (Girard and others). (1990), Nature, 248: 1421-1424).   TFPI and its variants were found to be post-thrombolytic arterial restenosis (Haskel, EJ. (1991), Circulation, 84: 821-827), Venous thrombosis (Holst, J. et al. (1993) Haemostasis) 23 (Supplement 1): 112-117) and as a result of septic shock. Disseminated intravascular coagulation (Creasey, AA, et al., (1993); Clin. Invest. 91: 2850-2860). It has been shown to affect homeostasis. However, TFPI is not It does not have all of the properties desired for anticoagulants used for therapy.   Recently, the serpin antithrombin III (ATIII) is present in heparin Under TF-FVIIa activity (Rao, LVM. Et al. (1993) Blood 81: 2600-2607; Laws. on, J.M. H. , (1993) et al. Biol. Chem. , Volume 268: 767-770; Broze Jr. , G .; J. , (1993), Bloo d, 82: 1679-1680; Mann, K. et al. G. FIG. , (1993), Blood, 82: 1680-1681). TF-FVII by TFPI The inhibition of a is reversible, whereas the inhibition by ATIII is essentially irreversible It is a target. In vivo TF-FVIIa inhibition between ATIII / heparin and TFPI No comparison of the relative importance of harm is known.   Bovine pancreatic trypsin inhibitor (BPTI), also called aprotinin, has an affinity Weak (Ki = 30 μM) recently competitively inhibits TF-FVIIa activity Proven (Chabbat, J. et al. (1993) Thromb. Re. s. 71: 205-215). In addition to this, BPTI is used for TF-derived blood However, blood coagulation time was determined by PT assay Approximately 75 μM was required for 1.4-fold extension (van-den-Be) sselaar, A .; M. H. P. Et al. (1993), Thromb. Ha emostas. 69: 298-299).   Recently, it is structurally similar to BPTI (Hynes, T. et al. (1990) iochemistry, Vol. 29: 10018-10020) Kunitz Domain of Amyloid β-Protein Precursor (APPI) Mutants-To select potent and specific active site inhibitors of factor VIIa Of large libraries for scaffolding in phage display (Dennis, MS and Lazarus, RA, (1994), J. Biol. Chem. 269: 22129-22136; Denni. s, M .; S. And Lazarus, R .; A. (1994); Biol. C hem. 269: 22137-22144). International publication WO95 / 238 No. 60 describes the generation of a Kunitz-type inhibitor for Factor VIIa.   The FVIIa inhibitor is a Kunitz serine protease inhibitor (eg, TF PI, BPTI, APPI). Kunitz-type serine protease inhibition Agents are a group of well-characterized proteins that contain Extensive including a characteristic three-dimensional fold containing a long binding loop that fits into the active site (Bode, W. and Huber, R., (1992)). Eur. J. Biochem. 204: 433-451). Kunitz type Serine protease inhibitors include, for example, serine proteases such as FVIIa Is a reversible inhibitor that binds slowly and strongly to the active site according to standard mechanisms. Binds and inhibits (Laskowski, Jr. M. and Kato, I., (19) 80), Ann. Rev .. Biochem. 49: 593-626) And is known.   The remaining Kunitz-type domain in the Kunitz-type domain / serine protease complex P preceding the peptide bond at which the side chain of group 15 is cleaved₁Meet the ranks. this P₁The residue indicates the position preceding the substrate or the inhibitor peptide bond to be cleaved, Schecter and Berger, (1967), Biochem. Biop hys. Res. Commun. 27, pp. 157-162.₁Conclusion Suitable for joint site. P₁Residues and P₁Cleavage to the 'residue is very slow, if any (Bode, W. and Huber, R., (1992), Eur. J. Bio. chem. 204: 433-451; Laskowski, M .; , Jr. And Kato, I .; (1980), Ann. Rev .. Biochem. , 49 Volume: 593-626).   Serine protease subsites and serine proteases in Kunitz-type domains The side chain (P_Five−P_Four') Numbers (Bode, W. and Huber, R., (1992), Eur. J. Bio. chem. 204: 433-451; Laskowski, M .; , Jr. And Kato, I .; (1980), Ann. Rev .. Biochem. , 49 Volume: 593-626)₁The interaction between the residue and the specificity pocket is It is the most important in energy and therefore represents the primary specificity determinant. P₁In place Amino acids generally govern the affinity of the inhibitor for the serine protease active site However, (Scott, CF et al., (1987), B1ood, Vol. 69: 1431-1436; Laskowski, M .; , Jr. And Kato, I .; , (1980) supra; Beckmann, J. et al. Et al. (1988), Eur. J. Biochem. 176: 675-682; Sinha, S .; In addition, (1991); Biol. Chem. 266: 21011-2101 3), residues outside this region (positions 11-14 and 16- of the secondary binding loop). Position 19 and residue 34) also have binding affinity and specificity for serine proteases It is known to play a certain role in sex (Kossiakoff, A.A. Et al., (1993), Biochem. Soc. Trans. , 21: 61 4- 618; Roberts, B .; L. Et al. (1992), Proc. N atl. Acad. Sci. ScL USA 89: 2429-2433). Kunit Crystal structure of the atypical domain is in the primary binding loop and contacts serine protease (Hynes, TR et al., (199) 0), supra; Bode, W .; And Huber, R .; , (1992), supra. Kossiakoff, A .; A. (1993), supra).   Tissue factor antagonists have also been demonstrated. Baboon anti-TF monoclonal antibody neutralization Prevent death in a sepsis model (Taylor et al., (1991); Circ. Shock, 33: 127), to determine endotoxin-induced DIC in rabbits. Weaker (Warr et al. (1990) Blood 75: 1481). And thrombogenesis in rabbits with arterial thrombosis model (Pawashe et al., (1994), Circ. Res. 74:56). Yes Lysine to alanine at amino acid residues 165 and 166 of soluble tissue factor. Binding to FVIIa with affinity comparable to wild-type tissue factor upon mutagenesis (Kelley et al., (1995), supra). Residue Lys165 of TF And Lys166 inhibit the inhibition of TF-FVIIa by the TFPI-FXa complex (Rao and Ruf, (1995), B. iochemistry, 34: 10867-10871). 165th and Complexes with TF Mutants Containing a Lys to Ala Substitution at Positions 166 and 166 For FVIIa, the FVIIa complexed with wild-type TF The rate of inhibition by TFPI-FXa is reduced as compared to FVI with functionality Tissue factor mutants that bind to Ia but have little blood coagulation (International publication WO94 / 28017). These molecules are TF-FVIIa Inactivates the cofactor activity of the tissue factor in the coalescence, but its molecule is FVIIa. There is no change in the ability to form a complex.                               Summary of the Invention   The present invention relates, for example, to FVII to FVIIa, FIX to FIXa or Catalytic conversion of FX to FXa and thereby the extrinsic pathway of blood coagulation Of TF-FVIIa mediated or related processes by blocking the first event of TF A compound that inhibits In addition to this, the compositions of the present invention may be FVII or Endogenous tissue by competing with endogenous tissue factor for binding to FVIIa Can neutralize the blood clotting effect of factors. Advantageously, the composition is a TF-FVIIa complex In a preferred embodiment, a strong inhibition can be achieved by providing a low-dose pharmaceutical formulation To The compounds of the present invention may be involved in TF-FVIIa-mediated or related processes. Useful in inhibiting and therapeutic methods.   The compositions of the present invention include an FVIIa active site inhibitory domain and a TF domain. In. According to a preferred aspect of the present invention, the TF domain is FVII / FVII a, but competes with endogenous TF for binding to FVIIa Acts as a cofactor for the conversion of IX to FIXa or FX to FXa The performance used is degraded. The FVIIa active site inhibitory domain of the composition of the present invention is FVIIa binds to the serine protease active site and catalyzes its zymogen substrate Prevent active activation. In a preferred embodiment, the active site inhibiting domain of the composition of the invention is It is a reversible active site serine protease inhibitor molecule.   According to certain preferred aspects of the invention, the composition further comprises an FVIIa active site Includes a linker domain that exists between the inhibitory domain and the TF domain. This resource Card is preferably (Gly)_Four-Includes Ser-linker module It is a hydrophilic flexible domain.   In one aspect, the composition of the invention is a polypeptide. In addition, the present invention Also included is an isolated nucleic acid, preferably a DNA, that encodes a polypeptide of interest. According to this aspect, the invention further relates to an expression operably linked to the DNA molecule. The control sequence, including the DNA molecule, and the control sequence was transformed in the vector Expression vectors, preferably plasmids, recognized by host cells, and the vectors thereof -Transformed host cells.   The composition of the present invention comprises a nucleic acid sequence encoding any of the amino acid sequences of the present invention. Isolated or synthesized to express the nucleic acid sequence in a suitable host. And an expression vector obtained by ligating the host with the present nucleic acid sequence. And culturing the host under conditions suitable for the expression of the nucleic acid sequence. A method comprising the steps of expressing in a host the protein encoded by the nucleic acid sequence May be manufactured. Preferably, the polypeptide is then isolated from the host cell culture. to recover. In this method, the ligation step further converts the nucleic acid into an appropriate secretion signal. The nucleic acid is operably linked to a suitable nucleic acid such that the host secretes the amino acid sequence. You may plan to link to the expression vector. This secretion signal is the leader Sequence, eg stII, lamB, herpes gD, lpp, alkaline phosphata Selected from the group consisting of May be selected, preferably stII.   The present invention further extends to therapeutic applications of the compounds described herein. Accordingly, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable additive. Includes products. Pharmaceutical compositions containing these molecules can cause vascular disease and inflammatory reactions Use in the treatment or prevention of diseases or disorders related to thrombotic or coagulopathy, including it can. These applications include, for example, mammals where inhibition of TF-FVIIa is indicated. Administering to the mammal a pharmaceutical composition in a pharmaceutically effective dose. It includes doing. Such indications include deep vein thrombosis, arterial thrombosis, postoperative blood Thrombosis, coronary artery bypass graft (CABG), percutaneous transluminal coronary angioplasty (PTCA), Includes seizures, tumor metastasis, inflammation, septic shock, hypotension, ARDS and DIC I do. The compositions of the present invention can also be used as adjuvants in thrombolytic therapy. Let's come.                             BRIEF DESCRIPTION OF THE FIGURES   Figure 1. Enzymes that modulate the coagulation, contact, fibrinolysis, inflammation and complement pathways And select a mediator and diagrammatically outline it. Activation of these pathways May lead to the clinical condition described.   FIG. 2 depicts an exemplary model of the composite. This compound has a TF domain, an active site A fusion protein having an inhibitory domain (APPI) and a linker domain. You. The TF portion of this molecule contains the structure of TF (Mul (1994), supra). Kunitz-type domain is APP I based on the structure determined by Hynes et al. (1990), supra. residue 22 polypeptide linkers add the C-terminus of the Kunitz domain to the N-terminus of TF It was designed in an extended conformation for binding. The distance between both ends is about 60 ° You. The C-terminus of TF is at the bottom of the molecule.   FIG. 3 shows a fusion gene for bacterial expression of a representative hybrid protein compound. Diagrammatically depicts the construction of. Only the fusion gene and promoter region of this plasmid Show. This fusion gene contains pst / encoding the TF7I-C Kunitz-type domain. BamHI fragment and pZTFAA and chemically synthesized oligonucleotide And by concatenating them.   FIG. 4 shows a representative sample of a sample obtained from various purification steps of a hybrid molecule. Draw a sketch of the polyacrylamide gel. This step involves freezing and thawing the cells, Giving osmotic shock to the peripheral cytoplasmic shock product (lane 1), In immunoaffinity chromatography using D3 monoclonal anti-tissue factor antibody Separating the TF-containing protein (lane 2), followed by size exclusion chromatography This is a step (lane 3) of removing high molecular weight molecular species by photography.   FIG. 5 shows a fusion protein having 1 to 6 linker modules (KDTFI to KDT). F6) depicts the effect of F6) on TF-FVIIa-dependent FX activation. Fusion protein Inhibition of the TF-FVIIa interaction was quantified by the FX activation assay. This assay consists of adding 100 pM FVIIa to 5 mM CaCl with 1 nM inhibitor._Two Incubate for 1 hour in TBS containing pH 7.5, then 10 nM membrane A 1 / 20-fold solution containing bound TF was added. Incubate for another hour After FX, FX was added to 190 nM. After an appropriate time, collect an appropriate amount, The reaction was stopped with an equal volume of 50 mM EDTA. Next, the activated FXa in the reaction stopped sample was SPECTROZYME^TM-Quantification using FXa as a chromogenic substrate.   FIG. 6 shows representative hybrid molecules KDTF5 (▼), soluble K165A, K1 66A-TF mutant (hTFAA) (■) and TF-FVIIa TF-FVII by Kunitz serine protease inhibitor TF7IC (●) Draw inhibition of a. Numerical values indicate that various inhibitions were obtained using twice-concentrated FVIIa and membrane TF. Measured in a modified FX activation assay using drug concentration. K_i* Measurement is rate fraction versus inhibitor This was done by fitting the concentration data to Equation 1 (below).   FIG. 7 shows that TF-FVIIa inhibitor was detected by prothrombin (PT) time assay. Draw the effect on the measured blood clotting time. Coagulation contains inhibitor citric acid Spiked human plasma with membrane bound TF (mTF) and CaCl_TwoMixed with a solution containing And started. The clotting time extension factor was determined by comparing the clotting time in the absence of the inhibitor (PBS vs. PBS). ) Divided by the clotting time of The clotting time of the latter is 53.2 ± 2.7 seconds. KDTF5 (▼), TF7IC (●), hTFAA (Ａ) And data for an equimolar mixture of TF7I-C and hTFAA (▲).   FIG. 8 shows the results obtained by the activated partial thromboplastin time (APTT) assay. 7 depicts the effect of a TF-FVIIa inhibitor on blood clotting time. Sample (citric acid Added human plasma and inhibitors) and activators (Intrumentation La) mixed with cephalin + activator APTT reagent (borateries) Incubate at 37 ° C. for 0 seconds, then CaCl 2_TwoTo measure the coagulation time. Specified. Multiple clotting times are absent in the presence of specified concentrations of inhibitor Defined as the quotient divided by the clotting time below (PBS control). Clotting time of the latter Was 63.7 ± 9.0 seconds. KDTF5 (▼), TF7IC (●), hT FAA (■) and equimolar mixture of TF7IC and hTFAA (Ａ) Show data.                         Detailed description of preferred embodiments Definition   Abbreviations used herein include: IXa for Factor IXa; X for Factor Xa Factor Ia as FXIa; Factor Xa as FXa; Tissue Factor as TF; Factor VII thymo Factor VIIa; FVIIa; factor VIIa; tissue factor-factor VIIa complex Combined with TF-FVIIa; FVII and / or FVIIa with FVII / FV IIa; BPTI as basic pancreatic trypsin inhibitor; Alzheimer's amyloid β- The Kunitz protease inhibition domain of the protein precursor inhibitor was replaced with APPI (Hyne s et al., (1990) supra); amino acid sequence Gly-Gly-Gly-Gl A polypeptide of 5 amino acids having y-Ser is represented by-(Gly)_Four-Ser and And G_FourS; TFPI for tissue factor pathway inhibitor; K for apparent equilibrium dissociation constant_i*;Professional Thrombin time is abbreviated as PT; activated partial thromboplastin time is abbreviated as APTT. I do.   TF-FVIIa-mediated or related processes or events or equivalently plasma The activity associated with FVIIa requires, according to the invention, the presence of TF-FVIIa. Event to be one of The general mechanism of blood clot formation is "Medical physiology", 13th edition, Language Review of Los Altos CA, pp. 411-414, (1987). writing. There are two pathways for blood coagulation, trons that induce platelet aggregation It requires both bin production and fibrin formation to stabilize platelet plugs. As outlined in FIG. 1, this pathway involves the presence of each separate proenzyme and procofactor. Includes several steps that require presence. This process involves fibrin cross-linking and thrombus formation Ends with Fibrinogen is converted to fibrin by the action of thrombin. Before that, thrombin is formed by proteolytic cleavage of prothrombin. This proteolysis is carried out by FXa binding to the activated platelet surface, Cleavage prothrombin in the presence of Va and calcium. TF-FVIIa Necessary for proteolytic activation of FX by the solid extrinsic pathway. Therefore, TF-FVIIa mediates or is associated with a process or activity associated with FVIIa From TF-FVIIa complex formation to fibrin platelet clot formation and first Any in the coagulation cascade, such as those requiring the presence of TF-FVIIa Including stages. For example, the TF-FVIIa complex converts FX to FXa and FIX to FIX. a and, in addition, activation of FVII to FVIIa by exogenous transduction Start the road. TF-FVIIa-mediated or related processes or FVIIa activity Are, for example, Roy, S .; (1991); Biol. Chem. , 266 Volume: 4665-4668 and O'Brien, D .; In addition, (1988), J. Clin. Invest. 82: 206-212. To factor Xa in the presence of factor VII and other necessary reagents It can be easily measured using standard assays such as those that convert.   TF-FVIIa-related disease or disorder is associated with chronic hemorrhage associated with fibrin formation Refers to a disease or disorder, for example, deep vascular thrombosis, arterial thrombosis, seizures, tumor transformation Vascular diseases such as transmigration, thrombolysis, arteriosclerosis and restenosis after angioplasty, even Inflammation, septic shock, venom, hypotension, adult respiratory distress syndrome (ARDS), Acute and chronic such as generalized intravascular coagulation (DIC) and other diseases Including signs. The TF-FVIIa-related disorder is, for example, a coagulation disease in vivo as described above. Is not limited to, for example, during a dialysis operation, hemofiltration or surgery Including blood moving continuously from the patient in an operation such as a blood bypass, for example, outside the body It also includes in vitro TF-FVIIa related processes such as blood coagulation due to blood circulation.   The terms “tissue factor protein” and “wild-type tissue factor” are naturally occurring mammalian tissue factors Polypeptide having an amino acid sequence corresponding to or amino acid of naturally occurring tissue factor Has an acid sequence and induces blood coagulation via interaction with plasma FVII / FVIIa Used to indicate a recombinant tissue factor that can be used. Naturally occurring TF Molecular species and eg rabbits, rats, pigs, non-human primates, horses, mice And tissue factors of other animal species such as sheep tissue factor. Mammalian tissue causes The amino acid sequence of the offspring protein is generally known or can be determined by standard techniques. Available. The numbers given to human sequences and amino acids are given by Morrissey, J. H. (1987), vol. 50, p. 129.   "HTFAA" and "K165A: K166AsTF" are the human TFs described above. Soluble tissue factor protein having an amino acid sequence from amino acid 1 to amino acid 219 A naturally occurring Lys amino acid at positions 165 and 166 thereof Indicates that the Ala residue was substituted.   Amino acid substitutions replace amino acids in wild-type tissue factor chemically or enzymatically or It means to replace with a group other than the wild-type amino acid by other appropriate means.   The terms “Kunitz-type serine protease inhibitor domain”, “Kunitz-type domain” "," Kunitz domain "," KD "and others are used interchangeably herein. Shows a polypeptide of about 58 amino acids, and a serine protease inhibitor BPT I (bovine pancreatic trypsin inhibitor; Creightton and Charles, (198 7 years), Cold Spring Harbor Symp. Quant. Bi ol. 52: 511-519) and conserved cysteine residues. And was first isolated in crystalline form in 1936 (Kunitz, M .; And Northhop, J .; H. (1936); Gen. Physiol. 19: 991-1007). Kunitz domain is an arrangement of cysteine residues, Three-dimensional folding, and sharing structural features. One or two proteins in a group Has been confirmed to contain three Kunitz domains. In this group; LACI (Lipoprotein-related coagulation inhibitors; also called TFPI; Broze, Jr. G . J. Et al., (1990), Biochemistry, 29: 7539- 754 6); APPI (Alzheimer's amyloid β-protein precursor; Hynes, T. R. , (1990), Biochemistry, 29: 10018- 10022); α-3 chain of human type VI collagen (see WO93 / 14119) ) And inter-α-trypsin inhibitors (Hochstrasser, KE). . (1985), Biol. Chem. Hoppe-Seyler, 366 Volume: 473).   The Kunitz domain contains six cysteines at specific intervals, which Exists as a disulfide bond (Bode, W. and Huber, R., (1) 992), Eur. J. Biochem. 204: 433-451). The three disulfide bridges stabilize the protein, resulting in the third dimension of the Kunitz domain. It is part of the cause of the folding property. 58 residue Kunitz serine protease BPT In I and APPI, cysteines are at positions 5, 14, 30, 38, 51 and 55 In the ranks. Removal of a single disulfide bridge does not result in significant structural changes (Eigenbrot, C. et al. (1990), Protein Eng., 3: 591-598.   The crystal structures of Kunitz domain serine protease inhibitors are BPTI and A PPI (Hynes, TR, et al. (1990), Biochem., Vol. 29) : 10018-10022). Antiparallel triple strand in the center Β-sheet and C-terminal α-helix form the core of this domain (Bode, W. and Huber, R., supra). This core domain segment Forms a scaffold that supports the exposed primary binding loop of correctly folded proteins ("Primary coupling loop" as defined herein) (Bode, W. and Huber, R. , Supra). The secondary binding loop, together with the primary binding loop, Involved in defining the interface between the cognate protease target (Rueh lman, A .; Et al. (1973); Mol. Biol. , 77:41 7-436).   The "primary binding loop" of the Kunitz domain (11-19 residues of APPI)             P_Five−P_Four−P_Three−P_Two−P₁−P₁'-P_Two'-P_Three'-P_Four' It is represented by The term "P₁Is the serine protein as defined herein above. It is used to indicate the position preceding the peptide bond to be cleaved by the inhibitor. As well And then "P₁'' To indicate the position after the cleaved peptide bond of this inhibitor use. Increased numbers are in front of the peptide cleavage bond (eg, P_TwoAnd P_Three) And backward (for example, P_Two'And P_Three') Indicates the sequential position. Residue number Is P₁It corresponds to that of BPTI so that the position is residue 15.   As used herein with reference to FVIIa, the term “active site” and others are catalytic sites And the binding site for the zymogen substrate,₁” Is the name of Scheter, I .; And Berger, A .; (1967), Bio chem. Biophys. Res. Commun. 27, 157-162 It is named on the page.   The term “amino acid” is used within the scope of the present invention for naturally occurring L-α-amino acids or residues. Means Commonly used terms and three letter abbreviations for each amino acid It is used in the specification (Lehninger, AL, "Biochemist." ry ", 2nd edition, pp. 71-92 (1975), Worth Publishe rs, New York).Mode of the present invention   The hybrid molecule of the present invention has at least two characteristic domains. Each of the molecules of the present invention has a Factor VIIa activity that binds to the active site of FVII / FVIIa. Protein-protein interactions at the sex site inhibitory domain and cofactor binding site Thus, it contains a tissue factor domain that binds to FVIIa / FVIIa. The present invention According to this, the active site inhibition domain is preferably located at the N-terminus of the tissue factor domain. Or through a flexible amino acid linker domain. 1. Tissue factor domain   The tissue factor domain of the composition of the present invention may be, for example, an amino acid of a mammalian tissue factor protein. By amino acid substitutions, insertions or deletions in the sequence, or by deglycosylation Tissue, such as those due to glycosylation mutations, including unglycosylated tissue factor molecules A variant of the factor protein, comprising at least factor VII / factor VIIa Tissue factor neutralizes the ability to induce blood clotting while retaining the ability to bind be able to. In certain aspects, the TF domain when in complex with FVIIa Has a reduced ability to catalyze the conversion of Factor X to Factor Xa products. There The tissue factor domain of the present invention may be, but is not limited to, a factor VII / Compete with wild-type tissue factor for the cofactor binding site of Factor VIIa, where Neutralizes or prevents wild-type tissue factor from acting as a cofactor in the blood coagulation cascade Believed to prevent.   The tissue factor domain of the present invention is characterized in that the existing tissue factor protein passes through the extrinsic coagulation pathway. Can inhibit or neutralize the induction of blood coagulation (Bach, R., (1988), CRC Crit. Rev .. Biochem. , 23:33 9-368). As the skilled expert will recognize, the term "neutralization" It is a relative term. Therefore, the biological activity of the tissue factor domain of the present invention is described. The term "neutralize" when used to refer to standard chromogen assays (Ro y, S. (1991); Biol. Chem. 266: 4665 4668; O'Brien, D .; Et al. (1988); Clin. In vest. 82: pp. 206-212). When added in molar excess, the presence of Factor X in the presence of Factor VII and other necessary reagents Tissue factor protein variants that inhibit the conversion of factor to factor Xa by at least 50% Means This tissue factor domain inhibits at least 50% in a 5-fold molar excess Those which inhibit at least 50% with a two-fold molar excess are more preferred. Will be. In a further preferred embodiment, the tissue factor domain of the invention is a wild-type tissue Factor X to factor Xa when present in a 1: 1 stoichiometric ratio with the factor protein Inhibits conversion by at least 50%. According to the present invention, the TF domain is limited to this. Although not a full-length phospholipid-associated tissue factor domain, And a transmembrane domain of wild-type tissue factor and And / or TF domains with all or part of the cytoplasmic domain deleted. Therefore, the biological activity of the TF domain of the present invention is measured by the chromogen assay. In doing so, the type of wild-type tissue factor is made to correspond to the specific test TF domain. Therefore, Contains all or part of the transmembrane domain of wild-type tissue factor TF domain may be all or part of transmembrane domain in similar phospholipid environment Test in a biological assay with the corresponding type of wild-type TF containing Transmembrane in the same way Soluble TF domain with the domain and cytoplasmic domain removed is a transmembrane domain And assay with the wild-type TF domain with the cytoplasmic domain removed.   A feature of the TF domain of the present invention is that the present protein binds to FVII / FVIIa. It is. Therefore, the TF domain of the present invention comprises wild-type TF protein and FVII / FVII. shares the residues required for the binding of TF to a. "Binding to FVII / FVIIa" Indicates that the TF domain of the present invention has a wild-type TF domain for binding at physiological concentrations. It has the ability to bind to FVII / FVIIa to the extent that it can compete with TF I do. Preferred among the TF domains are, for example, Kelley et al. (1995) Year), FVII / as determined by a standard binding assay as described above. About 10.0 picomoles (pM) and about 1 micromolar (μM) for FVIIa Have a KD between the two. More preferably, the TF domain is FVII / Between about 10 pM and about 10 nanomolar (nM) for FVIIa, most preferably Has a KD between about 10 pM and 1 nM.   Skilled experts will recognize residues in TF that contribute to FVIIa binding (Kelley et al. (1995), supra; Gibbs et al. (1994). Ruf et al., (1994), Biochemistry, 33: 1. Schullek et al. (1994) J. Mol. Biol. Chem. 269: 19399-19043; Muller et al., (19 1994), 33: 10864-10869). According to the present invention, the TF domain At least the residues necessary for binding to wild-type TF and the FVIIa / FVII. To share. Preferably, the tissue factor domain is between the wild-type tissue factor protein Share at least about 80%, more preferably about 85-95% sequence homology. I want to be.   TF domains include, but are not limited to, transmembrane domains and cytoplasm Includes full-length membrane-bound or phospholipid-associated tissue factor domain with both domains I do. The TF domain may also be a transmembrane domain of wild-type tissue factor and / or Tissue factor domains in which all or part of the cytoplasmic domain is deleted are also included. Follow The tissue factor domain of this molecule is It can also be produced as a soluble protein from which the protein moiety has been removed. Separately, TF protein The protein can also be produced as an integral membrane protein having a membrane anchor. Complete membrane anchor Domain-bearing tissue factor domains with phospholipid compositions such as liposomes Provided.   Preferred among the TF domains of the invention are the transmembrane domains of wild-type tissue factor. And a TF domain from which all or part of the cytoplasmic domain has been removed. The present invention According to this aspect of the invention, the TF domain is the N-terminal fibrone of wild-type tissue factor. It comprises at least a part of a Kuching type III domain. Preferably this TF domain Contains at least amino acids 1-102 of wild-type tissue factor. More preferably, The TF domains of the present invention are both fibronectin type III domains of wild-type tissue factor including. Preferably, according to this aspect of the invention, at least 1-2 of the wild-type TF The amino acid at position 19 is present.   The TF domain of the present invention retains the ability to bind FVII / FVIIa and furthermore A polypeptide capable of neutralizing the procoagulant activity of the wild-type tissue factor Including. For example, specific amino acid substitutions, insertions or deletions along the full length wild type TF Produces TF mutants with reduced ability to act as cofactors for FVIIa I do. Skilled experts recognize wild-type TF residues that contribute to TF's procoagulant function Will do. For example, the residue in the region of amino acids 157 to 168 is TF-F Contributes to the procoagulant function of VIIa (Kelley et al., (1995), supra) Ruf et al. (1992), supra) is important for FVII / FVIIa binding. is not. According to the present invention, all or some of these amino acids are selectively substituted. Or binding to FVII / FVIIa but wild-type tissue factor A TF domain capable of neutralizing the procoagulant activity of E. coli.   In a preferred embodiment, the wild-type tissue factor Trp158, Lys159, Ser 163, Gly164, Lys165, Lys166, and Tyr185 residues To provide the TF domain of the present invention by selectively substituting or deleting part or all of I do. Suitable substitutions are described in U.S. Pat. Includes substitutions by amino acids other than those with substantially positively charged side chains at pH I do. Exemplary substitutions include Trp158Phe, Lys159Ala, Ser16 3Ala, Lys165Ala, Lys166Ala, and Tyr185Al a or a part of a. In the most preferred aspect of the invention, the TF cofactor function Lysines at positions 165 and 166 that are important but do not inhibit FVIIa complex formation Residues (Roy et al. (1991), J. Biol. Chem., 266: 2). 2063; Ruf et al. (1992); Biol. Chem. , 267 Volume: 6375). Therefore, according to a preferred aspect of the present invention, By selectively substituting at least residues 165 and 166 of wild-type tissue factor The ability to bind FVII / FVIIa is retained, but as a cofactor as described above. To obtain molecules with reduced performance.   In a particular aspect, alanine substitution of these residues is preferred. However, TF- FX activation catalyzed by FVIIa complex (Ruf et al., (1992), supra) Any substitution that reduces the rate of Lysine at positions 165 and 166 Residues are those residues that have been determined by mutagenesis experiments to be important for FVIIa binding. (Kelley et al. (1995), supra) on the opposite molecular surface Located at the C-terminus of the fibronectin type III domain of TF.   Preferred tissue factor domains of the present invention are specifically incorporated by reference herein for their disclosure. Titled "Tissue Factor Mutants Useful for the Treatment of Myocardial Infarction and Coagulopathic Diseases" U.S. Pat. No. 5,346,991 describes this. This patent claims that endogenous tissue factor is blood The generation of a tissue factor mutant capable of inhibiting the ability to induce coagulation is described. These variants have one or both of the positively charged amino acid residues 165 and 166. Is substituted with an α-amino acid having no side chain that is substantially positively charged at physiological pH. It is a thing. These mutants include the cytoplasmic portion of wild-type tissue factor 244-263. The human tissue factor from which the residues and residues 220 to 243 of the transmembrane region have been removed. Including children. Any of these tissue factor mutants properly form the TF domain of the present invention Sometimes. International publication WO94 / 28017 also concludes FVII / FVIIa TF mutants that can be combined but have reduced procoagulant cofactor activity I have. The most suitable of the molecules described here correspond to the wild-type tissue factor protein. A tissue factor protein having the same amino acid sequence, and its molecule is a TF cofactor. At least one of the amino acids involved in the function is selectively substituted, deleted or moved Retains the ability to bind FVII / FVIIa but acts as the cofactor Gives molecules with reduced performance. 2. Active site domain   According to the present invention, the "active site inhibitory domain" of the composition comprises a factor VIIa activity It may be selected from any of several groups of inhibitors directed to the site. These active sites Inhibitors are broadly defined for the purposes of the present invention: i) reversibly bind the active site of FVIIa And an active site inhibitor cleaved by Factor VIIa; ii) Factor VIIa An active site inhibitor that binds reversibly but is not cleaved to the active site of An active site inhibitor that irreversibly binds to the factor VIIa active site; For a review of serine protease inhibitors, see "Proteinase Inhibitors (Prote inase Inhibitors "), (Studies in cell and tissue physiology) Papers, 12th Edition, Elsevier Science Publishers, New York, 1990) reference.   In the first group, it binds reversibly to the active site of factor VIIa, Kunitz-type and Casal-type serine proteins, active site inhibition domains that are cleaved The ase inhibitors are preferred, of which the Kunitz type is most preferred. Reversible activity Site-inhibiting domains may include, for example, ionic bonds, hydrophobic interactions or hydrogen bonds. Such non-covalent interactions bind to the active site of factor VIIa. Skill Experts suggest that Kunitz domains can bind slowly and tightly to serine proteases. We recognize that it is an inverse inhibitor.   The Kunitz domain active site inhibitory domain of the present invention includes any known mammalian Kunitz domain. , But also include Kunitz domain APPI (human Alzha). From Immer's disease amyloid β-protein precursor, Castro, M .; In addition, (199 0), FEBS Lett. 267: 207-212). Human TFPI (tissue factor protein inhibitor or LACI, Broze Jr .; G. FIG. J. Et al., (1990), Biochemistry, 29: 7539. ７５ 7546) TFPI-KDI (residues 22 to 79), TFPI-KD2 (residues 22 to 79). Groups 93-150) and TFPI-KD3 (residues 185-242); Ter-α-trypsin inhibitor (Vetr, H. et al. (1989), FEBS * Lett. 245: 137-140) ITI-KDI and ITI- KD2 (residues 22-79 and 78-135); collagen α3 (VI) chain precursor Body (Chu, ML, (1990), et al., EMBO J., 9: 385- 393) collagen α3 (VI) (residues 2899-2956); -Type protease inhibitor HKIB9 (Norris, K., Genbank Database, (December 31, 1993, release 39.0), January 1994 HKIB9 (7-60); BPTI (1-58), aprotinin Bovine basic pancreatic trypsin inhibitor (Creighton, TE and Charles); es, I. G. FIG. , (1987), Cold Spring Harbor S ymp. Quant. Biol. 52: 511-519). Preferred Kunitz-type serine proteases have sufficient affinity for FVIIa A molecule that binds and reversibly inhibits this molecule. This is a Kunitz-type domain FVIIa protein binding to FVIIa and measured by conventional assays (described below) It means that the medium action can be inhibited. Such a molecule is TF-FV The apparent dissociation constant for IIa (K₁*) Is about 100 nM or less, more Preferred Alternatively, the molecule has an apparent dissociation constant of about 10 nM or less. Kunitz The most preferred of the type domains are about 5 nM for TF-FVIIa or K less than that₁The one with *.   Apparent equilibrium dissociation constant (K₁*) Indicates Kunitz-type domain and FVIIa serine pro Observation of interaction with thease (Bode, W. and R. Huber) (19 92), Eur. J. Biochem. 204: 433-451; La. skowski, M .; Jr. And I. Kato, (1980), Annu. Re v. Biochem. 49: 593-626). The enzyme and the inhibitor form a reversible 1: 1 stoichiometric complex for the binding inhibitor. A method derived assuming formation (Bioth, J., (1974), Pro) teinase Inhibitors, pp. 463-469; Williams , J. et al. W. And Morrison, J. et al. F. (1979), Meth. Enz ymol. 63: 437-467). The data obtained is Equation 1: by nonlinear regression analysis [Where V_i/ V₀Is the activity rate (quotient of steady state inhibition rate divided by non-inhibition rate) [E₀] Is the total FVIIa concentration; and [I₀] Is the total inhibitor concentration] To fit.   A Kunitz-type domain suitable by way of example, but not by way of limitation, is amino It has a skeletal structure of 58 acids, and the 11th to 19th amino acids are serine proteases. Corresponds to the primary binding loop that matches the active site. Specific amino acids in the primary binding loop A Kunitz-type domain having a noic acid sequence exhibits favorable inhibition as described above. The above Thus, an exemplary Kunitz domain exhibiting favorable inhibition of Factor VIIa is at position P5. Pro; Gly at position P4; Pro, Val, Leu or Trp at position P3; P2 Cys at position; Arg, Lys or Met at position P1; Ala at position P1 '; Leu, Met or Ile; Met, Ile, Leu or Tyr at P3 'position; And a Kunitz-type domain having Leu, Lys or Arg at position P4 '. A preferred Kunitz-type domain according to the present invention is position 34 (P19 ') Phe, Val, Ile, Trp or Tyr; position 38 (P23 ') Cys; and position 39 (P24') His) is also characterized by Tyr or Gly.   An exemplary Kunitz-type active site inhibitory domain is described in International Publication WO 95/23860. And of structural formula I:Represented by   According to this equation, P5 to P4 ', P19', P23 'and P24' are Selected from₁Is amino acids 1-10 of APPI, VREVCSEQAE (SEQ ID NO: 1); amino acids 22 to 31 of TFPI-KD1, MHSFCAFKA D (SEQ ID NO: 2); amino acids 93-102 of TFPI-KD2, KPDFFCL EED (SEQ ID NO: 3); amino acids 185-194 of TFPI-KD3, GPSW CLTPAD (SEQ ID NO: 4); amino acids 22-31 of ITI-KD1, KEDS CQLGYS (SEQ ID NO: 5); amino acids 78-87 of ITI-KD2, TVAA CNLPIV (SEQ ID NO: 6); amino acids 1-10 of HKIB9, LPNVCAF PME (SEQ ID NO: 7); and amino acids 1-10 of BPTI, RPDFCLEP PY (SEQ ID NO: 8).   R_TwoAre amino acids 20-33 of APPI, RWYFDVTEGKCAPF (sequence No. 9); amino acids 41 to 54 of TFPI-KD1, RFFFNIFTRQCE EF (SEQ ID NO: 10); amino acids 112-125 of TFPI-KD2, RYFY NNQTKQCERF (SEQ ID NO: 11); amino acids 204 to TFPI-KD3 217, RFYYNSVIGKCRPF (SEQ ID NO: 12); Amino acids 41-54, RYFYNGTSMACETF (SEQ ID NO: 13); ITI- Amino acids 97 to 110 of KD2, LWAFDAVKGKCVLF (SEQ ID NO: 14 ); Amino acids 2918-2931 of collagen α3 (VI), KWYYDPNT KSCARF (SEQ ID NO: 15); amino acids 20-33 of HKIB9, RWFFN F ETGECELF (SEQ ID NO: 16); and amino acids 20-33 of BPTI, R It is selected from YFYNAKAGLCQTF (SEQ ID NO: 17).   R_ThreeIs selected from the group of YGG and YSG.   R_FourIs amino acids 40-58 of APPI, (Met52Ala) GNRNNNFD TEEYCAAVCGSA (SEQ ID NO: 18); amino acids 40-58 of APPI; GNRNFDTEEYCMAVCGSA (SEQ ID NO: 19); TFPI-KD1 Amino acids 61-79 of GNQNRFESLEECKKMCTRD (SEQ ID NO: 2 0); amino acids 132-150 of TFPI-KD2, GNMNNFETLEEC KNICEDG (SEQ ID NO: 21); amino acids 224-242 of TFPI-KD3 GNENFTSKQECLRACKKKG (SEQ ID NO: 22); ITI-KD1 Amino acids 61-79 of GNGNNFVTEKECLQTCRTV (SEQ ID NO: 2 3); amino acids 117 to 135 of ITI-KD2, GNGNKFYSEKECR EYCGVP (SEQ ID NO: 24); amino acids 2938 to of collagen α3 (VI) 2956, GNNEKFGSQKECEKVCAPV (SEQ ID NO: 25); HK1 Amino acids 40-58 of B9, GNSNNFLRKEKCEKFCKFT (SEQ ID NO: No. 26); and amino acids 40-58 of BPTI, AKRNNFKSAEDCM Selected from RTCGGA (SEQ ID NO: 27).   In a preferred Kunitz-type active site inhibitory domain,₁Is the array VREVCSE QAE (SEQ ID NO: 1), R_TwoIs the sequence RWYFDVTEGKCAPF (SEQ ID NO: 9 ); R_ThreeIs the sequence YGG; and R_FourIs the sequence GNNNFDTDEYCAAVCGS A (SEQ ID NO: 18) or GNRNNFDTEEYCMAVCGSA (SEQ ID NO: 19).   Therefore, suitable Kunitz-type active site inhibition domains include those in the following table:   Those skilled in the art will recognize that the Kunitz-type active site Understand that part or part may be used as the active site inhibitory domain of the present invention Will do. Kunitz binds reversibly with sufficient affinity to inhibit FVIIa Any portion of the domain is sufficient to be a Factor VIIa active site domain. You. Such molecules generally have at least the P of the Kunitz domain.₁And P₁'Remain And preferably the P of the primary binding loop of the Kunitz domain_FiveTo P_FourUp to contains.   The active site inhibitor of the present invention is an irreversible FVIIa serine protease inhibitor. There may be. Such irreversible active site inhibitory domains generally comprise Factor VIIa Forms a covalent bond with the active site of the child. For such irreversible inhibitors, common cell Protease inhibitors such as peptide chloromethyl ketone (William S. et al., (1989); Biol. Chem. 264: 7536-7 Pp. 540) or peptidylchloromethane; azapeptides; Guanidinobenzoate derivative and 3-alkoxy-4-chloroisocoumarin Acylating agents such as phenylmethylsulfonyl fluoride (PMS F), diisopropylfluorophosphate (DEP), tosylpropylchloro Methyl ketone (TPCK) and tosyl ridyl chloromethyl ketone (TLCK) Sulfonyl fluorides such as nitrophenylsulfonates and related compounds Heterocyclic protease inhibitors such as, for example, isocoumarin and coumarin Is included. 3. Linker   According to the present invention, the tissue factor domain preferably has a flexible phosphorous N-terminus. Inhibition of FVII / FVIIa active site of hybrid molecules via card mein Join the domain. The linker component of the hybrid molecule of the present invention is not necessarily It need not be involved in binding to FVII / FVIIa. Therefore, according to the present invention, Linker domain consists of FVII / FVIIa active site inhibitory domain and tissue factor domain. Any group in this molecule that spatially bridges with the main.   This linker domain may be of various lengths and specifications, but is not However, what is important according to the invention is the length of the linker domain, and its structure Absent. This linker domain connects the active site domain of the hybrid molecule to FVII / FVIIa fits into the serine protease active site groove and its tissue factor Those that bind the domain to the TF binding site on the FVII / FVIIa molecule are preferred. Good. Therefore, the length of the linker domain is the "function" of this hybrid molecule. Depends on the properties of the two domains.   Those skilled in the art will be aware of various combinations of known distances (Morrison And Boyd "Organic Chemistry", 3rd edition, Al lyn and Bacon, Boston, MA (1977)) It will be appreciated that it provides molecules of various lengths. For example, Linkard The ins may be polypeptides of various lengths. Amino acids of this polypeptide The composition determines the properties and length of the linker. In a preferred embodiment, the linker molecule is Constructs a flexible and hydrophilic polypeptide chain. As an example, this linker domain Is [(Gly)_Four-Ser] unit.   In a preferred aspect, the active site inhibition domain is a Kunitz serine protease inhibitor Agent, the TF domain is hTFAA, and the linker domain is about 30 Or a polypep containing between 5 and 35 amino acids extending further away It is a tide. More preferably, the linker has between 20 and 30 amino acids. Approximately 60 ° or more, including the acid. Most preferably this phosphorus Kerr is a polypeptide of about 22 to 27 amino acids and about 4 to 5 amino acids. [(Gly)_Four-Ser] unit. 4. Synthesis   The hybrid molecules of the invention can be prepared by various techniques well known in the art. May be combined. These include recombinant DNA technology, solid-phase synthesis, liquid-phase synthesis, Including synthetic techniques or combinations of these techniques. Selection of synthesis method is specific to the production It will depend on the molecule. For example, in one embodiment, a hybrid molecule of the invention Is not a complete “protein” in nature, but rather a combination of recombinant and liquid phase technologies. Synthesized.   For example, the invention includes both amino acid or protein portions and non-protein portions. In the synthesis of hybrid molecules, the protein portion of the molecule is generally modified using recombinant techniques such as: And then convert the non-protein part of the molecule to the protein part by solid or liquid phase methods. Combine to obtain this molecule.   A chemical bonding method is suitable for bonding the protein part and the non-protein part of the molecule. In this procedure, for example, proteins and non-proteins having one or more reactive groups Use conventional cross-linking reagents such as molecules that can react with one or more sites on white matter molecules. To use. Non-carcinogenic, non-toxic and substantially non-immunogenic in vivo and biocompatible Compatible crosslinkers are preferred. Typically, this crosslinker is a protein of the molecule Amino or sulfhydryl groups on the non-protein part Covalently bonded to a phenyl, amino, aldehyde or carboxylic acid group. Molecule For a review on how to cross-link, see Means and Feaney, (1990), Bioconjugate Chemistry, 1: 2-12. ing.   DNA encoding a protein for recombinant synthesis of the hybrid protein of the present invention There are a variety of techniques that can be employed to manufacture For example, amino of the resulting protein Deriving DNA based on naturally occurring DNA sequences encoding changes in acid sequences It is possible to The mutant DNAs are ligated together to form the hybrid of the present invention. Can be used to obtain the molecule. These techniques are simplified in the form of tissue factor A gene encoding a main and, for example, a Kunitz-type domain represented by structural formula I Obtaining a gene encoding an active site inhibitory domain such as Modified by recombinant techniques, for example as described below; A gene encoding a linker module with a length of Inserting the vector into a suitable host cell: culturing the host cell and Intended to cause expression of a bridging molecule; purify the molecule produced there are doing.   Somewhat more specific, the DNA sequence encoding the bifunctional molecule of the present invention. Sequences are obtained by synthetic construction of DNA sequences (Sambrook, J. et al., "Molecular Cloning", 2nd edition, Co ld Spring Spring Harbor Laboratory, N.W. Y. , (19 1989)).   For example, an expression vector encoding wild-type tissue factor is obtained and site-directed mutagenesis is performed. Induction (Kunkel et al., (1991), Meth. Enzymol., 20). 4: 125-139; Carter, P .; And others (1986), Nucl eic Acids Res. 13: 4331; Zoller, M .; J. Et al. (1982), Nucleic Acids Res. , Volume 10: 648 7), cassette mutagenesis (Wells, JA et al., (1985), Gene), 34: 315) or restriction selective mutagenesis (Wells, J. . A. Et al., (1986), Philos. Trans. R. Soc. Lon don Ser. A. 317: 415) and the tissue factor domain of this molecule. Get in. Next, the active site inhibitory domain is encoded using the mutant DNA. Can be inserted into an expression vector containing the DNA to be expressed. Then, in the same expression vector, If [(Gly)_Four-Ser] phosphorus of various lengths, such as one or more units A DNA sequence encoding the card domain can be inserted.   Oligonucleotide-mediated mutagenesis can be performed using the tissue factor variants and nicks of the invention. This method is suitable for producing a DNA encoding the active site inhibition domain. This technique is well known in the art and is described in Adelmann et al., (1983) Year), DNA) 2: 183. Briefly, wild-type tissue Factor or native or unconverted form of a Kunitz-type domain such as APPI DNA is added to the oligonucleotide encoding the desired mutation to the DNA template. Change by bridging. The template is unchanged or conventional A plasmid or bacteriophage single-stranded form containing the DNA sequence   Next, the DNAs encoding these mutants were replaced with DNs encoding the linker domain. Insert the A sequence across the appropriate plasmid or vector. Use this vector To transform host cells. Replicate sequences from species generally compatible with the host cell And a plasmid vector containing control sequences is used for those hosts. Usually this Vectors are replication sites and proteins that can enable phenotypic selection in transformed cells Has a sequence that encodes   For example, E. coli is a plasmid derived from one of E. coli, pBR322 (Ma ndel, M .; Et al. (1970); Mol. Biol. , 53:15 (P. 4). Plasmid pBR322 is ampicillin And a simple method for selection, containing the tetracycline resistance gene You. Other vectors include various promoters that are often important for expression, for example. Includes various features such as tar. For example, plasmids pKK223-3, pDR 720, and pPL-lambda are tac, trp) and P_L-Promoter Available and currently available (Pharmacia Biotechnology) Representative of expression vectors.   Other suitable vectors are vectors described herein using standard techniques. Can be built by combining the appropriate attributes of The proper attributes of this vector Is the promoter, ribosome binding site, mutant gene or gene fusion, Sequence, antibiotic resistance marker, copy number, and appropriate origin of replication.   In E. coli, the Kunitz domain is an unprocessed secreted protein (Castro, M. et al. (1990), FEBS Lett. 267: 207-212), cells Endogenously expressed protein (Altman, JD, et al., (1991), Protein -Eng. 4: 593-600) or fusion protein (Sinha, S. et al.). Or (1991); Biol. Chem. 266: 21011-21 Page 13; Laurizen, C.I. Et al., (1991), Prot. Exp ress. Purif. 2: 372-378; Auerswald, E .; A. Et al., (1988), Biol. Chem. Hoppe-Seyler, 369: 27-35).   The host cell can be prokaryotic or eukaryotic. Prokaryotes clone DNA sequences. Parental polypeptides, segment-substituted polypeptides, residue positions It is suitable for producing replacement polypeptides and polypeptide variants. For example E. coli K12 strain 294 (ATCC 31446) is E. coli B strain, E. coli X177. 6 strains (ATCC 31537), E. coli c600 and c600hf1, E. coli W3110 strain (F-, gamma, prototrophy / ATCC 27325), e.g. Bacillus, such as fungi, and, for example, Salmonella typhimurium or Other Enterobacteriaceae such as Latia marcescens and various pseudo It can be used in the same way as Monas species. Suitable prokaryotes are E. coli strain W3110 (ATCC 27325). When expressed in prokaryotes, the polypeptide typically contains N -Contains terminal methionine or formyl methionine and is not glycosylated. Of course, these examples are illustrative rather than limiting.   In addition to prokaryotes, for example, cells derived from yeast cultures or multicellular organisms Such eukaryotes may be used. In principle, all such cell cultures work It is possible. However, vertebrate cells are of most interest and vertebrate cells in culture Proliferation of animal cells (tissue culture) has become a reproducible procedure (see “Tissue culture (T issue Culture), Academic Press, Krus e and Patternson, eds. [1973]). Such useful host cell lines Examples of VERO and HeLa cells, Chinese hamster ovary (CHO) Cell lines, W138, 293, BHK, COS-7 and MDCK cell lines You. Yeast expression systems have been used to produce Kunitz domains (Wagn er, S .; L. Et al., (1992), Biochem. Biophys. Re s. Commun. 186: 1138-1145; Vedwick, T .; Et al. (1991); Indust. Microbiol. , 7:19 7-202). Especially yeast Pichia pastoris is Saccharo myces, cerevisiae, α, mating factor preprosigner Sequence and Pichia pastoris alcohol oxidase AOX1 Has been successfully used with a promoter and a termination sequence. Other yeast expression vectors Hosts commonly used to express proteins and heterologous proteins are also contemplated. 5. Composition   Hybrids of the Invention Comprising a TF Domain and an FVIIa Active Site Domain The molecule is typically provided in a composition suitable for the intended use. TF domain The hybrid molecules of the present invention include, for example, the hTFAA type described herein. It can be produced in a soluble form. According to this aspect of the invention, the tissue factor domain of the molecule is Produced without membrane anchor. Separately, the tissue factor domain is not It can also be manufactured as a full length membrane associated type with a car.   The hybrid molecule of the present invention may contain all or one of the transmembrane domains of wild-type tissue factor. It may include a TF domain including a part. According to the present invention, a membrane anchor domain A bifunctional molecule containing a TF domain containing a mild surfactant or phospholipid (P It is preferable to form a composition containing L). Membrane anchor or transmembrane domain A composition of the invention having a full-length TF domain containing a And are preferably formulated with a phospholipid. International publication WO94 / 28017 No. describes the preparation of phospholipid compositions containing TF domains that are suitable for the compositions of the present invention. are doing.   Suitable suitable for the pharmaceutical composition of the present invention are described in WO 94/28017. The preferred composition is a phospholipid composition that provides the highest stability and biological activity. this The phospholipid composition is preferably liposomal, as is generally well known in the art. Formulation to form a pharmaceutical composition. As described, the liposomes of the present invention Phospholipids suitable for use in the composition are saturated phospholipids having 12 to 20 carbon atoms. Or those containing unsaturated fatty acids. Suitable for use according to the invention Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerin (PG) and phosphatidylserine (P S). These phospholipids may be derived from any natural source, Phospholipids may themselves be composed of molecules having different fatty acids. A mixture of phospholipids consisting of phospholipids from different sources may be used. For example, PC, PG and PE may be made from egg yolk; PS is made from animal brain and spinal cord May be. These phospholipids may likewise be derived from synthetic sources. this Phospholipids are conveniently mixed in appropriate proportions and used to produce the compositions of the present invention. Produce a phospholipid mixture.   Methods for producing liposomes are generally well known and have been described. Liposo Illustrative examples of the method of producing the liposome include reverse addition of liposomes (see US Pat. Or including the mode described for incorporation of amphotericin B into lipid vesicles (See, for example, Lopez-Berstein, et al. (1985), J. In. fact. Dis. 151: 704-710; Lopez-Berens. tein, (1987), Antimicrob. Agents Chemo ther. 31: 675-678; Lopez-Berenstein et al. Or (1984); Infect. Dis. , 150: 278-283 And Mehta et al. (1984), Biochem. Biophys . Acta, 770: 230-234). Strong circulation time Conversion The liposomes prepared are prepared as described in U.S. Pat. No. 5,013,556. It may be made.   Thus, in one aspect, the invention relates to a hybrid molecule in the form of a liposome. Wherein the molecule has a TF domain portion and the lipid of the liposome is Membrane anchor domain appears to be inserted via bilayer lipids along with the bilayer bilayer Is intended.   Other suitable compositions of the present invention include those described above which comprise a pharmaceutically acceptable carrier. The nature of the carrier is, for example, usually a solid carrier or vascular for oral administration. Internal administration depends on the administration method such as a liquid salt solution carrier.   The compositions of the invention are pharmaceutically acceptable and compatible with the subject of the invention and the protein of the invention. Including components to be performed. These are generally suspensions, solutions and elixirs and specialties. Biology such as phosphate buffered saline, saline, Dulbecco's medium, etc. Target buffer. Aerosols may be used or starch, sugars, microcrystals Cellulose, diluent, granule, lubricant, binder, disintegrant, etc. (for oral administration) In the case of solid dosage forms, such as, for example, powders, capsules and tablets) May be used.   As used herein, the term "pharmaceutically acceptable" generally refers to a federal or state government. Approved by, or listed in, the United States Pharmacopeia, Listed in preparations for use with other generally recognized animals, especially humans Means what you have.   The formulation to be selected may be any of the various buffers described above or, for example, pharmaceutical grade mannitol, milk Sugar, starch, magnesium stearate, sodium saccharin cellulose, carbonic acid This can also be achieved using additives including magnesium and others. Of this composition “PEGylation” is a technique known in the art (eg, International Patent Publication WO92 / 92). U.S. Patent No. 5,122,614 to Enzon; and International Patents (See Publication WO 92/00748). Oral compositions In the form of solutions, suspensions, tablets, pills, capsules, sustained release agents, or powders. You may. 6. Treatment   The bifunctional molecule of the present invention is intended to prevent the biological effects of the TF-FVIIa complex. And can be used for treatment. Inhibition of TF-FVIIa is TF-FVIIa-dependent blood It is desirable when reducing coagulation is intended. These conditions include, but are not limited to: Examples include prevention of arterial rethrombosis associated with thrombolytic therapy. TF-FVIIa is deep Vascular thrombosis, arterial thrombosis, stroke, DIC, septic shock, cardiopulmonary bypass surgery, Important role in various clinical conditions, including adult respiratory distress syndrome, congenital angioedema It is suggested to play. Therefore, inhibitors of TF-FVIIa are And / or may play an important role in the regulation of thrombosis.   Thus, the present invention is directed to patients in need of a therapeutically effective amount of a hybrid molecule of the present invention. Method of preventing a TF-FVIIa-mediated event in a human comprising administering including. A therapeutically effective amount of a hybrid molecule of the present invention will achieve the desired effect. Is determined in advance. The amount to be used for treatment is the subject, route of administration and treatment It will vary depending on the condition. Therefore, the dose to be administered depends on the FVII present. / FVIIa binds to form an inactive complex to regulate blood coagulation in the subject to be treated This is a sufficient dose to lower.   Therapeutic efficacy is one or more of the symptoms associated with TF-FVIIa-dependent coagulation. It is measured by the above improvements. Such a therapeutically effective dose should be determined by a skilled professional. And depends on the age condition, condition, sex and condition of the subject to be treated. It becomes something. Appropriate dose ranges for systemic administration typically will be determined by the route of administration. Between about 1 μg / kg to about 100 mg / kg or more, depending on You. According to the present invention, a suitable therapeutic dose is about 1 μg / kg body weight and about 5 mg / kg. Is between weight. For example, appropriate clinical management conditions should be within the range specified for the intended treatment. Intravenous injection or infusion sufficient to maintain blood levels.   Pharmaceutical compositions comprising a polypeptide of the invention may be parenteral, topical, oral, or topical. (Eg, aerosol or transdermal), or any combination thereof It may be administered in any suitable manner. Appropriate clinical management requires first intravenous bolus injection and It includes one or more subsequent repeated administrations at intervals.   For example, in order to prevent re-formation of thrombotic occlusion during thrombolytic therapy, When the composition is administered in combination with a thrombolytic agent, a therapeutically effective dose of the thrombolytic agent is It is between about 80 and 100% of the normal dose range. Normal dose range of thrombolytic agent Is the daily dose used in treatment and is readily available to clinicians (see Physicians Desk Reference rences) ", 1994, 50th edition, Edward R. et al. Barnhart Publisher). A typical dose range depends on the thrombolytic agent used, 0.5 to about 5 mg / kg body weight for minogen activator (t-PA); 140,000 to 250,000,000 units / patient for putokinase; urokinase 500,000 to 625,000 units / patient; and anisoleated streptose Kinase-plasminogen activator complex (ASPAC) from 0.1 Will be about 10 units / kg body weight.   As used herein, a composition may comprise at least a molecule of the present invention and at least And a single dose form containing one thrombolytic agent. In terms of time Thus, the molecules of the invention are administered separately, but in separate administrations, eg, sequentially. It is meant to include those administered simultaneously by two administrations. These unions The composition and the composition dissolve or form occluded thrombi which cause the dissolution of the occluded thrombus Acts to prevent.   According to another aspect of the invention, for example, the hybrid molecule may be, for example, an artificial valve, Encountered in extracorporeal perfusion of blood through a prosthesis, stent, or catheter Can be used to prevent in vitro clotting. According to this aspect of the invention, Coating extracorporeal devices with the compositions of the present invention allows for clot formation based on the extrinsic activation pathway. Reduce danger.   The following examples are presented by way of illustration, but not by way of limitation. Disclosed herein All cited references expressly disclose the disclosure for reference.                                 Example material   Human factor VIIa, factor X, and factor Xa are Haematologic. It was purchased from Technologies (Essex Jct. VT). Membrane set Recombinant, full length (residues 1-263), human factor expressing human TF Embryonic kidney cell line (293) was prepared by sonication (Paborsky, L .; R. Et al. (1990), Protein Engineering, Volume 3: 547-553). Bovine trypsin, 4-methylumbelliferyl p-g Anidinobenzoate and CHAPS are available from Sigma Chemicals Purchased from. Bovine serum albumin (BSA), fraction V is from Calbioche m (La Jolla, CA). N^α-Benzoyl-L-arginine-p- Nitroanilide is available from Bachem California, Torrance, CA Purchased from. Human plasmin, S- 2302, S-2251 and S-236 6 was purchased from Kabi Vitrum (Sweden) and SPECTROZ YME^TMFXa is available from American Diagnostica (Greenwich, CT) and purchased from CHROMOZYM^TMt-PA is Boehring er Mannheim. All other reagents are commercially available It is the highest quality product that can be made.Example 1 Construction and Expression of Fusion Protein   The plasmid encoding the tissue factor-Kunitz domain fusion protein is from E. coli. Plasmid pZTFAA (Kell) designed for hTFAA expression by secretion of ey, R .; F. Et al., (1995), Biochemistry, 34: 1 0383-10392). As shown in FIG. 3, expression of pZTFAA The unit has a stII signal sequence that binds to the N-terminus of hTFAA and Is driven by the alkaline phosphatase promoter (phoA). pZ TFAA has elements necessary for replication of a single- or double-stranded plasmid. The coding sequence of the TF7I-C Kunitz domain was transferred to plasmid pA4G32-TF7. IC (Dennis, MS and Lazarus, RA, J. Biol. Chem. (1994), 269: 2219-22136) to Pst It was excised by restriction digestion with I and BamHI. ligated to the C-terminus of hTFAA For the construction of the gene encoding the fusion protein having the combined Kunitz domain, use Sa The cII restriction enzyme site was introduced into the pZTF by oligonucleotide-directed mutagenesis. AA (Kunkel, TA, et al., Methods in Enz). ymology, (1987), 154: 367-382). Obtained Rasmids are digested with SacII and SphI, followed by the Kunitz domain Ps Although ligated with the tI / BamHI fragment, the two oligonucleotide linkers Manufactured by Naru. One of the linkers has a SacII / PstI binding end, G_FourEncode one copy of the S module and read the N-terminus of the Kunitz domain correctly It was designed to fuse with hTFAA in the frame. The second linker is BamHI / Sphl binding end, providing a stop codon for construction. N-terminus of hTFAA Construction of a fusion protein gene having a Kunitz domain connected to pZTFA A cuts DNA at the junction between stII and the hTFAA segment. Cut. Plasmids cut with NsiI were digested with PstI / BamHI Linked to main fragment, oligonucleotide linker prepared by chemical synthesis did. One of the linkers has an NsiI / PstI binding end and a stII signal Join Kunitz domains in columns and frames. The second linker is BamHI / Nsi G with I bond end_FourEncode one S module, hTFAA N-terminus and frame Bind the Kunitz domain within. The stop codon is from pZTFAA. Multiple G of copy_FourThe fusion gene with S module is located in TFKD1 or KDTF1. Between the appropriate restriction sites (G_FourS)_nA chemically synthesized orifice that encodes a unit Constructed by inserting a go nucleotide. TFKD1 contains SacII / A PstI site was used, and a BamHI / NsiI site was used for KDTF1. fusion All genes were dideoxynucleotide sequencing (Sanger, F. et al., Proc. Natl. Acad. Sci. USA, (1977), 74: 5 463-5467).   Phagemid encoding the fusion protein, derived for expression from E. coli W3310 The transformed Escherichia coli 33B6 was transformed. Fermentation using overnight saturated culture (1%) The bacteria were inoculated into 10 L of the medium in the tank. Fermentation is described in the literature (Carter, P. et al., Bio / Technology, (1992), 10: 163-167). But at 30 ° C. instead of 37 ° C. of the stII signal sequence Therefore, the fusion protein was secreted into the cell periplasm. Centrifuge 32 hours after inoculation The cells were harvested and stored frozen at -20 ° C.Example 2 Purification of fusion protein   Extraction of fusion protein from E. coli cell paste, anti-TF monoclonal antibody (D3) Immunoaffinity chromatography using a column (Paborsky, L.) . R. In addition, Biochemistry, (1989), 28: 8072- 8077) as described for mutants of soluble tissue factor (Kel ley, R .; F. Et al., (1995), Biochemistry, Volume 34: 10383-10392). In some cases final phosphate buffered saline Use a Superdex 75 (Pharmacia) column equilibrated with water Introduce a size exclusion chromatography step to transform fusion proteins from high mass oligomers The monomer was purified. The purification process of the fusion protein was SDS-PAG as shown in FIG. Tracked with E. This procedure gave a highly purified fusion protein. Each purified Samples are run to verify that the protein mass matches the desired molecular composition. Sciex-API3 mass spectrometer equipped with a curated electron spray source For analysis. Horse myoglobulin multiply charged ion (MW = 16951 Da) The device was corrected using. The analysis results by mass spectrum are shown in Table I, It proves that the fusion protein has the expected molecular composition. Concentration of purified fusion protein The degree was measured by three methods (Table I): 1) detection of hTFAA by D3 antibody, 2) Measurement of Kunitz domain by titration of lipsin, and 3) measurement of total protein concentration Absorbance measurement to perform. All three methods gave similar concentrations. Mass spectrum data Considering these results, these results indicate that these fusion proteins are invariant Both Kunitz and TF domains function within proteins Prove that.   The fusion proteins tested are listed in Table I using the abbreviation KDTFn or TFKDn. Raise it. Where n is G_FourAn integer indicating the number of S linker units. In this symbol, TF indicates hTFAA. KD indicates the TF7I-c Kunitz domain. To TF The subsequent KD (TFKD1) has a KD domain via a peptide linker at its N-terminus. Shows the fusion protein binding to the C-terminus of hTFAA. KD followed by TF (KDT F1) binds the C-terminus of KD to the N-terminus of hTFAA via a peptide linker. 1 shows a fusion protein. Table I Characterization of the purified fusion protein.   The mass predicted from the fusion protein in native disulfide-bonded conformation Shown in two columns. The results of the mass spectrum analysis are shown in the third column. Columns 4-6 show each fusion protein. BIAcore for representative white matter products^TMD3 object immobilized on the flow cell Concentration determined based on binding to clonal antibody (structural / functional completion of TF domain) Completeness assay), trypsin inhibition (structural / functional completion of Kunitz domain) Assay for totality) and absorbance at 280 nm (total protein). ND = not measured.Example 3 Fusion Protein Inhibits TF-FVIIa-dependent Factor X Activation   Membrane tissue factor of fusion protein (mTF) -Factor VIIa (mTF-FVIIa) The relative potency of inhibiting the catalytic function of the complex was evaluated using the FX activation assay. In this assay, FX is added to a solution of mTF-FVIIa and the rate of FXa formation is measured. I do. Collect appropriate amount at various times and chelate calcium by adding EDTA To stop the reaction, and then determine the amount of FXa formed by the FXa-specific substrate SPECT. ROZYME^TM It is measured using FXa. SPECTROZYME^TM FXa FXa degradation does not require calcium. SPECTROZYME^TM FXa Hydrolysis is monitored by absorbance measurement at 405 nM. Using this speed FXa concentration may be calculated with reference to a standard curve constructed with purified FXa. FX activity The sexual assay was performed in a microtiter format and the change in absorbance was measured using Macintosh I with II software (Biometallics) With SLTEAR340AT plate reader controlled by Ici type computer Watched. KaleidaGraph 3rd Edition (Synergy Softwar e)) to perform a non-linear regression analysis.   For assays of activation, mTF, FVIIa and FX were added to a final concentration of 200 μL in a volume. Used at 500, 100 and 190 nM each. Inhibitor if added Was present at a final concentration of 1 nM. This solution was 50 mM Tris-HCl, pH 8 , 150 mM NaCl and 5 mM CaCl_TwoWas also contained. FVIIa storage Solution concentrations were determined by active site titration on quantified TF7I-C samples and FVIIa groups. CHROMOZYM as quality^TMDetermined by using t-PA. TF The concentration of 7IC was 4-methylumbelliferyl / p-guanidinobenzoate. Was determined accurately by titration using trypsin which had been titrated with active site (Ja meson, G .; W. Et al. (1973), Biochem. J. , 131 volumes : 107-117). 80 nM-trypsin plus an appropriate amount of diluted inhibitor 0 mM Tris, pH 8.0, 100 mM NaCl, 10 mM CaCl_Two, And 0.05% TRITON^TMIncubation for 1 hour at room temperature in X-100 5 mM-N-benzoyl-L-arginine-p-nitroanilide 20 μL was added to make a total volume of 150 μL. The change in absorbance at 405 nm was then monitored. Determine concentration assuming 1: 1 stoichiometry of inhibitor with trypsin or FVIIa did. Next, CH was added after adding a solution of FVIIa in which the active site of the mTF concentration was determined. ROMOZYM^TMDetermined from the increase in t-PA hydrolysis rate. FX and FXa Concentrations were provided by the manufacturer.   In the test for inhibiting the activity of the fusion protein, FVIIa was added to the fusion protein for 1 hour at 37 ° C. After incubation, mTF was added. After adding mTF, incubate at 37 ° C. After another hour of incubation, the substrate FX was added. The reaction time is Start at time of addition and collect 25 μL volumes at various times with equal volumes of 50 mM EDTA. Mixed. The formed FXa was washed with a factor Xa buffer (10X = 0.2M-HEPES). pH 7.4, 1.5 M-NaCl, 0.25 M-EDTA, 1% PEG-800 0) to a final concentration of 1 ×, followed by 0.5 mM SPECTROZYME^TMF It was measured by adding Xa. The final volume at each time point was 200 μL, SPECTROZYME^TMFXa hydrolysis rate is monitored by 405nm absorbance change Report in mOD / min.   Figure 1 shows the effect of 1 nM-inhibitor on mTF-FVIIa-catalyzed FX activation It is shown in FIG. FXa production rate of TF7IC or hTFAA alone at 1 nM concentration Is equivalent to the uninhibited control. Fusion proteins KDTF1 and TFKD1 also have FX activity It hardly affected the conversion rate. In contrast, KDTF2 slightly inhibits FX activation did. Fusion proteins with longer linkers have an increased degree of inhibition and KDTF5 Complete inhibition was observed with the fusion protein. KDTF4 seems to be equivalent to KDTF5 However, KDTF6 and KDTF7 were slightly weaker inhibitors. These de Connects the Kunitz domain and the hTFAA domain with a linker of appropriate length. Shows that a potent inhibitor can be obtained. The best phosphorus in this series Kerr corresponds to a polypeptide chain length of 45-60 ° in the extended conformation G_FourIt has 4 or 5 S modules.Example 4 Measurement of equilibrium dissociation constant   FX activation apparent equilibrium dissociation constant (K_i*) Represents hTFAA, TF7-C and It was measured for KDTF5. These experiments were performed at different concentrations of FVIIa and mTF, respectively. Except that the concentrations were set to 200 pM and 1 nM and the inhibitor concentration was changed, Performed as described. The standard curve is SPEC using purified FXa (Hematech). TROZYME^TMFXa is hydrolyzed, and the hydrolysis rate observed at each measurement time point is The concentration was converted to the concentration of the generated FXa and constructed. Then analyze these data to find the minimum The initial rate of FXa generation was calculated for each inhibitor concentration by square regression. Each initial speed Was compared with the non-inhibition rate to obtain the rate of change in FX activation rate at each inhibitor concentration. These values are plotted in FIG. HTF using Equation 1 for nonlinear regression analysis 160 ± 19 nM, 3.8 for AA, TF7IC and KDTF5, respectively K of ± 0.5 nM and 2 ± 6 pM_i* Value obtained. Phosphorus estimated to be about 60cm in length Fusion of hTFAA and TF7-C to car increases inhibitor titer by at least 100-fold It is clear that it has been increased.Equation 1 :   [Eq.₀] Is the enzyme concentration. [I₀] Is the inhibitor concentration. V_iIs [ I₀] Is the initial rate of FXa generation in the presence of V₀Is in the absence of the inhibitor It is initial speed]Example 5 Blood Coagulation Assay   Coagulation time was measured using an ACL300 Research Coagulation analyzer. Measured. For the prothrombin time (PT) assay, incubation The time was set to 120 seconds and the acquisition time to 600 seconds. 29 expressing full length TF Membrane obtained from 3 cells (Paborsky, LR, et al., (1989), Bio chemistry, 28: 8072-8077) at 25 mM CaCl 2._Two And had been mixed. At least combined citrated normal human plasma and inhibitors Was also assayed after 5 minutes incubation. Incubation at 37 ° C for 2 minutes The sample (plasma and inhibitor) and reagents (CaCl_Two/ TF) Dynamically mixed. The clotting time was determined by optical evaluation. In a total volume of 160 μL Final concentrations of 0.028 to 5.6 μM of inhibitor, 0.5 nM of mTF, CaC l_TwoWas 12.5 mM and plasma was 50%.   In the activated partial thromboplastin time (APTT) assay, an activation time of 70 Seconds and acquisition time were set to 360 seconds. Citrated normal human plasma and inhibition The assay was performed after the combined incubation of the agents for at least 5 minutes. Sample (blood Serum and inhibitors) and activators (Instrumentation Labo) ratories, Cephalin + Activator APTT reagent) Is added automatically by pipette and incubated at 37 ° C. for 70 seconds, then CaCl_TwoWas added and the clotting time was determined by optical evaluation. In a total volume of 200 μL Final concentrations are 0.028-5.6 μM of inhibitor, 10% of activator, CaCl_Two Was 10.0 mM and plasma was 50.0%. Results-Fusion protein KDTF5 showed very strong coagulation inhibition in PT assay as shown in FIG. showed that. KDTF5 is more potent than TF7I-C or hTFAA alone, Inhibition was at a lower dose than that observed with the mixture of 7IC and hTFAA. KD TF5 showed a doubling of clotting time at an inhibitor concentration of about 800 nM. TF7I-C and For an equimolar mixture of hTFAA, a 2-fold extension was shown at 1.9 μM. hTFAA alone Showed a 2-fold increase in clotting time at 4 μM-concentration, whereas TF7I-C The concentration did not show a 2-fold extension. This data indicates that KDTF5 has a TF-dependent blood coagulation Solid, potent inhibitors that are more potent than non-covalent mixtures of these two inhibitors Indicates that it has a titer.   As shown in FIG. 8, KDTF5 is the previously observed APTT of TF7I-C alone. Effect on blood coagulation time by assay (Dennis, MS and Lazaru) s, R.S. A. J. Biol. Chem. 269: 22129-22136 P. [1994]). KDTF5 and TF7IC and hTFA The non-covalent mixture with A together with TF7I- Slightly stronger than C alone. KDTF5 at an inhibitor concentration of about 200 nM or All TF7I-C / hTFAA mixtures showed a two-fold increase in clotting time. Concentration 4 00 nM TF7I-C was required for doubling the clotting time. At the concentration tested hTFAA alone had no effect on blood clotting time as measured by the APTT assay.Example 6 Measurement of antithrombotic titer using rabbit deep inner wound model   Male New Zealand White Rabbits (~ 4kg) IM with Ketamine / Xylazine Anesthetize by injection to the surgical anesthesia phase. Fix the rabbit in the supine position on the restraint board, 3 Heat to 7 ° C and shave the neck and inner thigh. Teflon catheter into ear marginal vein And the femoral artery for drug delivery and sampling, respectively. Blood test before procedure Samples are collected and subjected to coagulation tests (APTT and PT). Bleeding time in the hind limb claw skin Evaluate from the cut wound made in An incision is made in the cervical region and the left common carotid artery and its branches are surgically Exposure to Ultrasound flow probe (Transonics ™) with common carotid artery -Attach to the common carotid artery approximately 5 cm caudal to the internal carotid bifurcation. Basula with stable blood flow After the drug has arrived, the drug (saline or test compound) is infused into the marginal ear vein. A contracted embolectomy catheter (Fogarty ™, 3F) was then placed on the tongue branch. It is inserted through the incision into the lumen of the common carotid artery. Temporarily stop blood flow through the arteries, And loosely fix it to the incision with a 2-0 silk tie. catheter The blood flow is resumed after the fixation is performed. Deflate balloon to 2mm of blood flow probe And inflate with saline until resistance of the vessel wall is felt. catheter Is retracted at a constant speed to the first branch point, and then contracted.   This procedure is repeated several times in each experimental animal, after which the catheter is removed. Catete The balloon operation from initial insertion to removal takes about 3 to 5 minutes and is 1.5 Give a 2 cm injury area. A blood sample is collected for 40 minutes to perform PT measurement, Evaluate nail dermal bleeding time and monitor blood flow through the carotid artery. Patency time can be measured It is defined as the total length of time during which the effective blood flow is detected in the artery (maximum value = 40 minutes). Open Percentage indicates the percentage of test animals showing carotid artery blood flow ≧ 5 minutes.   After the experiment is completed, the rabbit is euthanized, and the carotid artery is removed and cut open. Thrombus is present If so, remove and blot and weigh.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C07K 14/745 C07K 14/81 14/81 A61K 37/02 (C12N 1/21 C12R 1:19) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES FI, GB, GE, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, UZ, VN (72) Inventor Lee, Joffrey F. United States of America 1170 Mason Drive, Pacifica, California

Claims (1)

[Claims] 1. A fusion protein comprising: a) a Factor VIIa ("FVIIa") active site inhibitory domain; b) a linker domain; and c) a tissue factor ("TF") domain. 2. 2. The fusion protein of claim 1, wherein said FVIIa active site inhibitory domain is a reversible serine protease inhibitor. 3. 2. The fusion protein of claim 1, wherein the FVIIa active site inhibitory domain is an irreversible serine protease inhibitor. 4. 3. The fusion protein of claim 2, wherein the reversible serine protease inhibitor comprises a Kunitz domain ("KD"). 5. 5. The fusion protein of claim _4, wherein the linker domain comprises a-(Gly) _4- Ser-linker module. 6. 6. The fusion protein of claim 5, wherein the linker domain comprises four-(Gly) _4- Ser-linker modules. 7. The fusion protein of claim 5, wherein the linker domain comprises five-(Gly) _4- Ser-linker modules. 8. Formula: KD-[(Gly) _4- Ser] _X -TF wherein KD is a Kunitz-type active site inhibitor of factor VIIa; X is an integer between 1 and 10; and TF is Is a tissue factor domain]. 9. 9. The fusion protein of claim 8, wherein the tissue factor variant is hTFAA. 10. 10. The fusion protein of claim 9, wherein the KD is TF7IC. 11. 11. The fusion protein according to claim 10, wherein X is 4. 12. 11. The fusion protein according to claim 10, wherein X is 5. 13. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the fusion protein of claim 1. 14． A method for inhibiting the procoagulant effect of human FVIIa, comprising contacting the fusion protein of claim 1 with FVIIa in an amount sufficient to inhibit the procoagulant effect of TF-Factor VIIa (FVIIa). 15. 14. A method of inhibiting the procoagulant effect of human tissue factor-factor VIIa (TF-FVIIa) in a mammal, comprising administering to the mammal a therapeutically effective amount of the composition of claim 13. 16. 16. The method of claim 15, further comprising administering the composition in combination with a thrombolytic agent. 17． 16. The method of claim 15, further comprising administering the composition in combination with an anticoagulant. 18. 14. A method of treating a mammal indicated for inhibiting Factor VI Ia, comprising administering to the mammal a pharmaceutically effective amount of the composition of claim 13. 19. An isolated DNA molecule encoding the fusion protein of claim 8. 20. 20. The DNA molecule of claim 19, further comprising an expression control sequence operably linked to said DNA molecule. 21. An expression vector comprising the DNA molecule of claim 20, wherein the host cell transformed with the vector recognizes the regulatory sequence. 22. 22. The vector of claim 21 which is a plasmid. 23. A host cell transformed with the plasmid of claim 22. 24. 24. A method for expressing a DNA molecule encoding a serine protease inhibitor in a host cell, comprising culturing the host cell of claim 23 under conditions suitable for expressing the inhibitor. 25. 25. The method of claim 24, further comprising recovering the inhibitor from the culture medium.