CA2161772A1 - Fibrin-targeted inhibitors of thrombin - Google Patents

Abstract

A chimeric molecule that contains a fibrin-binding portion of an antibody covalently linked to an inhibitor of thrombin, which molecule is administered to inhibit thrombus formation and growth.

Description

~ WO94/2~491 2 t 6 ~ 7 7 2 PCT~S94/~81 FIBRIN-TARGETED INHIBITORS OF THROMBIN
Field of the Invention This invention relates to the inhibition of blood 5 clot formation.
Back~round of the Invention Clot formation resulting in the blockage of a major coronary artery can lead to myocardial infarction, one of the most common causes of death in industrialized 10 societies. Enzymatic recanalization of an occluded coronary artery with a drug such as streptokinase or tissue plasminogen activator (tpa) has been shown to reduce mortality significantly in patients suffering from acute myocardial infarction (Gruppo Italiano per lo 15 Studio della Streptoch;n~si nell'Infarto Miocardico (GISSI) Lancet 2:871-874, 1987; Wilcox RG, Lancet 2:525-530, 1988; ISIS-2 Collaborative Study Group, Lancet

2:349-360, 1988). However, limitations of thrombolytic therapy include a very high rate of reocclusion (Gold et 20 al., Circulation 73:347-352, 1986) and the relative resistance of arterial clots to lysis (Jang et al., Circulation 79:920-928, 1989). Acute thrombotic reocclusion is also a frequent and major setback after otherwise successful percutaneous transluminal coronary 25 angioplasty (PTCA) (Detre et al., Circulation 80:421-428, 1989), and constitutes a formidable problem after the placement of intracoronary stents (Roubin et al., Circulation 85:916-927, 1992). Adjunctive therapy with currently available antiplatelet (ISIS-2 supra) and 30 anticoagulant (Hsia et al., N Engl J Med 323:1433-1437, 1990) agents has shown some benefit in overcoming these difficulties and also in reducing restenosis (Hanke et al., Circulation 85:1548-1556, 1992).
Thrombin is an enzyme in blood that plays a 35 central role in blood clot development by catalyzing the WO94/25491 PCT~S94/0~81 formation of fibrin from fibrinogen and, perhaps more importantly, as the most potent activator of platelets.
The anti-coagulant, heparin, has been shown to be of only limited efficacy in antagonizing the action of thrombin 5 in experimental studies. A number of direct inhibitors of thrombin have been shown to be effective in preventing platelet-dependent arterial thrombosis and rethrombosis after thrombolytic reperfusion in experimental animals (Haskel, Circulation 83:1048-1056, 1991; Jang et al., 10 Circ. Res. 67:1552-1561, 1990; Heras et al., Circulation 82:1476-lg84, 1990; Kelly et al., Blood 77:1006-1012, 1991; Sitko et al., Circulation 85:805-815, 1992). One of these thrombin antagonists is hirudin, an anti-coagulant derived from the leech, Hirudo medicinalis (Dodt et al., FEBS 165:180-184, 1984).
Plasminogen activators, such as streptokinase and urokinase, catalyze the conversion of plasminogen to its active fibrinolytic form, plasmin. These reagents activate circulating as well as fibrin-bound plasminogen 20 and thus not only affect the lysis of fibrin in the thrombus, but also promote fibrinolysis elsewhere in the body. Tissue-type plasminogen activator has somewhat improved the specificity problem, but therapeutic administration of both fibrinolytic agents and thrombin 25 inhibitors is still a problem because of their lack of selectivity and potential to cause generalized hemorrhaging.
Summary of the Invention The experiments described herein demonstrate that 30 fibrin-specific antibody-targeted hirudin is more potent in inhibiting thrombus growth than hirudin alone because the local concentration of hirudin is increased by antibody binding to fibrin at the site of the thrombus.
Clinical applications include localized inhibition of 35 thrombosis as contrasted with systemic anti-coagulation.

W094/25491 PCT~S94/0~81 216~77~

Anti-thrombin targeting of thrombin inhibitors can be especially useful in highly thrombogenic situations such as coronary stent implantation and can be used as an adjunctive therapy with highly selective thrombolytic 5 agents.
Targeting of the thrombin antagonist has been accomplished by conjugating the antagonist to a fibrin-specific antibody, such as the monoclonal antibody termed 59D8. The epitope to which the 59D8 antibody binds 10 becomes available only after thrombin cleaves fibrinopeptide B. It has been shown that 59D8 does not cross-react with uncleaved fibrinogen. Thus, only at sites where thrombin activity has become manifest will the anti-thrombin-conjugate locally accumulate and 15 inhibit further thrombin action. This selectivity of inhibition, triggered by the very molecule that needs to be inhibited, permits a substantial reduction in the total amount of anti-thrombin activity that needs to be administered. Because inhibitor concentration would be 20 concentrated at the thrombus, the plasma concentration of inhibitor required for anti-coagulation would be lower, thus resulting in a reduction in the potential for generalized hemorrhaging.
The invention features a chimeric protein 25 containing an antagonist of thrombin activity linked to a fibrin-binding domain of an antibody. Preferably, the chimeric protein contains a thrombin antagonist linked to a fibrin-specific antibody, fibrin-specific Fab' fragment, or Fv fragment. The fibrin-binding domain of 30 the antibody preferably does not bind to the fibrin precursor, fibrinogen. More preferably, the fibrin-binding domain is specific for the new amino terminus of the fibrin ~ chain that becomes exposed after thrombin has cleaved fibrinopeptide B. An example of such a 35 fibrin-binding domain is that of monoclonal antibody W094/25491 PCT~S94/0~81 - ` 21 61 772 59D8. The portion of the chimera that inhibits thrombin activity is preferably hirudin or an active fragment or derivative of hirudin.
By the term "active fragment" is meant a peptide 5 unconjugated to an antibody which has the ability to inhibit the action of thrombin with at least 50% of the thrombin inhibitory activity of the native hirudin as measured in the S-2238 chromogenic assay, described below.
The term "fragment", as applied to a polypeptide, denotes a peptide of the whole protein that can be proteolytically cleaved from the native protein, recombinantly expressed, or synthetically made, and is at least 6 contiguous residues. In this invention, a 15 fragment is usually about 20 contiguous residues, preferably at least 40 contiguous residues, more preferably at least 50 contiguous residues, and most preferably at least 60 contiguous residues in length.
Such peptides can be generated by methods known to those 20 skilled in the art, including proteolytic cleavage of the protein, de novo synthesis of the fragment, or genetic engineering. Peptides can be made up of D- or L-amino acids or a mixture of both. Particularly preferred would be a peptide with a D-amino acid as the amino- or 25 carboxyl-terminal amino acid, in order to inhibit proteolytic degradation of the chimeric protein. Also within the invention are chemical derivatives of the above peptides. Chemical derivatives are defined as peptides to which one or more negatively charged side 30 groups have been added. Derivatization methods, which are well known in the art, include but are not limited to sulfation, methyl sulfonation, phosphonation and carbonation of the tyrosine hydroxyl group; or sulfonation, phosphonation and carbonation of the 35 tyrosine benzoyl meta carbon. Preferably, either or both WO94/25491 PCT~S94/0~81 termini of the chimeric protein would be protected from proteolytic degradation by the presence of a st~n~rd protective group such as an aldehyde. Also useful are amino-terminal blocking groups such as t-5 butyloxycarbonyl, acetyl, theyl, succinyl,methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4,-dinitrophenyl.
The components of the chimeric protein are joined using a covalent bond, such as a disulfide bond. In another embodiment, the chimeric protein is produced recombinantly, with the two components of the chimera joined by a peptide bond.
The therapeutic method of the invention specifies the administration of the chimeric protein in a pharmaceutically suitable vehicle. The invention further specifies introduction of the chimera into the circulatory system of an animal. In another embodiment, 20 the chimeric proteins are introduced into or contacted with blood that has been removed from the individual, followed by the return of treated blood to the animal.
The invention also includes a mixture of the chimeric protein of the invention with another chimeric 25 protein that contains a fibrin-binding domain of an antibody linked to a fibrinolytic agent, e.g., as disclosed in U.S. Patent 5,116,613, herein incorporated by reference. The fibrin-specific antibody portion of the second chimera may be identical to or different from 30 that of the first chimera. The thrombin antagonist is preferably hirudin, whereas the fibrinolytic agent is preferably streptokinase, staphylokinase, urokinase, or tissue-type plasminogen activator. The mixture of proteins is introduced into the circulatory system of an 35 animal to dissolve and prevent further formation of WO94/25491 PCT~S94/0~81 21 61 772 ~

aberrant blood clots. In another embodiment, the mixture is introduced into blood that has been removed from the animal for treatment, and subsequently returned to the animal.
The invention also features a nucleic acid encoding the chimeric protein and a cell that contains the nucleic acid and expresses the protein, as well as the therapeutic administration of the nucleic acid to an individual to prevent coagulation of blood. Such cells lO may be useful, for example, in a therapeutic method of introducing into an animal (e.g., a human patient), cells that have been transfected with and express the chimeric protein.
Also included in the invention is a therapeutic 5 method of preventing and dissolving blood clots that specifies the administration of the nucleic acid encoding the ch; m~ric protein and the nucleic acid encoding a second Ch; mPriC protein that contains a fibrin-binding domain linked to a fibrinolytic agent. More preferably, 20 the therapeutic method specifies the administration of two populations of cells, one of which contains and expresses nucleic acid encoding the chimeric protein of the invention, and a second population of cells that produces a chimeric protein that contains a fibrin-25 binding domain linked to a fibrinolytic agent.
The invention includes a nucleic acid encoding arecombinant chimeric protein containing an antagonist of thrombin activity linked to a fibrin-specific antibody and a cell expressing the recombinant chimeric protein.
30 Also within the invention is a method of administering into the blood of a patient a nucleic acid encoding such a chimeric protein, either alone or together with a nucleic acid encoding a chimeric protein that contains a fibrin-binding domain linked to a fibrinolytic agent, as 35 well as a method of separate or simultaneous WO94/2~491 PCT~S94/0~81 administration of cells expressing each of the recombinant proteins.
Brief Description of the Drawinqs Fig. lA and Fig. lB are photographs of hirudin-5 59D8-Fab' conjugate and its parent molecules separated using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12.5% gel) and stained with Coomassie-blue; electrophoresis was performed under non-reducing (Fig. lA) and reducing (Fig. lB) conditions.
10 Lane 1, molecular weight standards; lane 2, 59D8-Fab';
lane 3, final preparation of hirudin-59D8-Fab' conjugate;
lane 4, hirudin. The molecular weight standards were phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean 15 trypsin inhibitor (20.1 kDa), and lactalbumin (14.4 kDa).
Fig. 2A and Fig. 2B are graphs depicting thrombin inhibition as a function of binding to an immobilized target molecule, ~-peptide. Hirudin-dependent inhibition 20 of thrombin-mediated cleavage of chromogenic substrate, S-2238, was recorded at 405 nm and expressed as a percentage of inhibition of total thrombin added. Fig.
2A: hirudin-59D8-IgG conjugate (---) versus hirudin (o-O). Fig. 2B: hirudin-59D8-Fab' conjugate (---) versus 25 hirudin (o-o). Each point represents mean of three independent experiments.
Fig. 3A and Fig. 3B are graphs depicting thrombin inhibition (% of total) plotted against applied concentration of thrombin inhibitor in the fibrin-30 Sepharose assay. Each data point represents the mean ofthree independent experiments. Thrombin inhibitors in Fig. 3A are hirudin-59D8-IgG conjugate (---) and hirudin (o-o), and in Fig. 3B, hirudin-59D8-Fab' conjugate (---) and hirudin (o-o).

W094/25491 PCT~S94/0~81 2 1 6 1 772 ~

Fig. ~A ~nd Fig. 4B are graphs illustrating fibrin deposition on the surface of a plasma clot. Fig. ~A
shows a time course of fibrin deposition by hirudin or hirudin-59D8-IgG conjugate, on the surface of a clot 5 suspended in a fibrinogen solution: hirudin at 80 ATU/ml (1~-~), 8 ATU/ml (o-o), 2.4 ATU/ml (o-o) and hirudin-59D8-IgG
conjugate at 7.4 ATU/ml (---), 1.2 ATU/ml (---). Fig. ~B
is a graph depicting inhibition of fibrin formation on 10 the surface of a clot suspended in a fibrinogen solution as a function of thrombin inhibition by hirudin (o-o) or hirudin-59D8-IgG conjugate (---) at 120 minutes. Each point in Fig. 4B represents the mean of three replicates.
Fig. 5A a~ Fig. 5B are graphs showing fibrin 15 deposition in fibrinogen solution. Fig. 5A is a graph showing a time course of fibrin deposition on the surface of a clot suspended in a fibrinogen solution by hirudin or hirudin-59D8-Fab' conjugate: hirudin at 100 ATU/ml (0-~), 30 ATU/ml (o-o), 10 ATU/ml (o-o) and hirudin-59D8-20 Fab' conjugate at 9.25 ATU/ml (---), 3 ATU/ml (---), 0.1 ATU/ml (---). Fig. 5B is a graph depicting fibrin deposition on the surface of a clot suspended in a fibrinogen solution as a function of thrombin inhibition by hirudin (o-o) or hirudin-59D8-Fab' conjugate (---) at 25 180 minutes. Each point in Fig. 5B represents the mean of three replicates.
Fig. 6A and Fig. 6B are graphs showing fibrin deposition in human plasma. Fig. 6A is a graph showing the time course of fibrin deposition on the surface of a 30 clot suspended in human plasma by hirudin or hirudin-59D8-Fab' conjugate: hirudin at 100 ATU/ml (o-o), 10 ATU/ml (o-o), hirudin-59D8-Fab' conjugate at 10 ATU/ml (---), 1 ATU/ml (---) and NaCl control (x-x). Fig. 6B is a graph showing inhibition of fibrin formation on the 35 surface of a clot suspended in human plasma as a function W094/25491 21 ~ t 7 7 2 PCT~S94/0~81 of thrombin inhibition by hirudin (o-o) or hirudin-59D8-Fab' conjugate (---) at 120 minutes. Each point in Fig.
6B represents the mean of three replicates.
Fig. 7 is a graph showing a time course of fibrin 5 deposition on the surface of a clot suspended in human plasma for hirudin and a 1:3 molar mixture of uncoupled hirudin and 59D8 Fab': hirudin at 44 ATU/ml (O-0) and 10 ATU/ml (o-o); mixture at 43 ATU/ml (O-~) and 9 ATU/ml (C-C); and NaCl control (x-x).
Detailed Description - In accordance with the present invention, specific targeting of anti-thrombotic agents to the surface of a clot was studied. The thrombin antagonist, hirudin, which is capable of inhibiting fibrin-bound thrombin, was 15 conjugated to the anti-fibrin antibody 59D8 and the fibrin-specific Fab'-fragment of 59D8. 59D8 binds selectively to the new amino-terminus of the fibrin ~-chain that becomes exposed after thrombin cleaves fibrinopeptide B (Hui et al., Science 222:1129-1131, 20 1983), an early event in clot formation. Targeting of hirudin in this manner results in a dramatic reduction of fibrin deposition at the very site of thrombin action and clot growth.
Re~gents The chromogenic substrate, S-2238 (H-D
phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline dihydrochloride), was obtained from Chromogenix, Mo~ 1, Sweden. N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP) was purchased from Pierce Chemical (Rockford, Illinois), and 125I-labelled fibrinogen was obtained from Amersham (Arlington Heights, Illinois).
Fresh frozen plasma was purchased from the University of Heidelberg blood bank and the German Red Cross. All other chemicals were purchased from Sigma Chemical, St.
35 Louis, M0.

WO94/25491 PCT~S94/0~81 2161772 ~

~iru~in Recombinant Hirudin having the sequence of the native protein (Dodt et al., FEBS 1104 165:180-184, 1984, herein incorporated by reference) (SEQ ID NO:1) [r-5 Hirudin LU 52369, specific activity: 17,000 anti-thrombin units (ATU)/mg] was obtained from Xnoll AG, Ludwigshafen, Germany. Fragments and derivatives of hirudin with anti-thrombin and anti-coagulant activity are known in the art, such as Hirudin PA (SEQ ID NO:2) 10 (Dodt, J. et al., U.S. Patent 4,767,742); a polypeptide defined by the sequence X-AA3-[AA4-AA62]-AA63 Z, wher 3 is a conservative amino acid residue other than tyrosine that is not susceptible to electrophilic chemical modification, AA4-AA62 is amino acids 4-62 of the native 15 hirudin sequence (Dodt et al. FEBS 1104 165:180-184, 1984) (SEQ ID NO:3), AA63 is a tyrosine residue or a tyrosine residue modification so as to contain an electron-withdrawing substituent in the 3-, or

3-, 5-positions of the phenyl ring, X is hydrogen or an 20 N-terminal extension sequence corresponding to some or all of the native hirudin sequence, and Z is a hydroxyl group or a C-terminal extension corresponding to some or all of the native hirudin sequence (Winant, R.C. et al., U.S. Patent 5,118,790); peptides characterized by the 25 sequence Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-X
(SEQ ID NO:4) and D-retro forms thereof, where X can be COOH, Leu or Leu-Gln; or the sequence Y-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Z (SEQ ID NO:5) and D-retro forms thereof, where Y can be NH2, an amino protecting group, 30 at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp (SEQ ID NO:6), or at least the C-terminal portion of the amino acid sequence: Val-Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pro-Glu-Ser-His-Asn-Asn-Gly-Asp (SEQ ID NO:7), and Z can be COOH, Leu, or Leu-Gln; and WO94/25491 PCT~S94/Q~81 the tyrosine residue is characterized by the presence of a negatively charged side group (Maranganore, J.M., European Patent Application 333356). Each of the above references is herein incorporated by reference.
5 Thrombin Inhibitors Other thrombin inhibitors are known in the art, such as antithrombin III (Sheffield et al., Blood 79:2330-2339, 1992); ~,~' monochlormethylene diadenosine 5'5'''-P1P4-tetraphosphate (Kim et al., Proc. Natl. Acad.
10 Sci. USA 89:11056-11058,1992); boroarginine peptides, such as Ac-(D)Phe-Pro-boroArg-OH, Boc-(D)Phe-Pro-boroArg-CloH16, H-(D)Phe-Pro-boroArg-OH, and H-(D)Phe-Pro-boroArg-C1oH16 (Kettner et al., J. Biol. Chem. 265:18289-18297, 1990); synthetic peptides, such as D-Phe-Pro-Arg (CSAP), [Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(OSO3)-Leu (SEQ ID NO:8), the sulfated C-terminal dodecapeptide of hirudin] (ESAP), and tD-Phe-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu] (SEQ ID NO:9) (BAP) (Kelly et al., Proc. Natl. Acad. Sci. USA 89:6040-20 6044, 1992); 3,4,-dihydro-3-benzyl-6-chloromethylcoumarin (Mor et al., Biochim Biophys Acta 1038:119-124, 1990); D-phenylalanyl-prolyl-arginine chloromethyl ketone-treated ~-thrombin (PPACK-IIa) (Schmaier et al., Thromb. Res.
67:479-489, 1992); tripeptide inhibitors, such as D-Phe-25 Pro-Arg-H (ALD) and D-Phe-Pro-Arg-CH2Cl (CMK) (Bagdy et al., Thromb. Res. 67:221-231, 1992); benzamidine-based inhibitors such as N~ -naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide (NAPAP) (Sturzebecher et al., Biol. Chem.
30 Hoppe-Seyler 373:491-496, 1992); and arginine-based inhibitors such as (2R,4R)-4-methyl-1-[N~-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulphonyl)-L-arginyl]-2-piperidine carboxylic acid (MQPA) (Bode et al., Eur. J.
Biochem. 193:175-182, 1990); thrombin inhibitors 35 incorporating a scissile peptide bond, such as N~-2161772 ~

acetyl[D-Phe45, Arg~(COCH2)47, Gly48]desulfo hirudin~5~65 (P79) (DiMaio et al., FEBS Lett. 282:47-52, 1991); and ketomethylene pseudopeptides, such as [Ac-(D)-phe45~pro46~Arg~(cocH2)co47~48~Gly4s]Hirudin45 65 5 (Hirutonin-1), [Ac--(D)--Phe45,Pro46,Arg9~[COCH2]CH2CO47]Hirudin45 65 (Hirutonin-2), [Ac-(D)-Phe45,Pro46,Arg~Y[COCH2]CH2CH2C047~48]Hirudin45~65 (Hirutonin-3), and 10 [Ac-(D)-Phe45,Pro46,Arg~[COCH2]CH2CH2CH2C047r48]Hirudin45~65 (Hirutonin-4) (DiMaio et al., J. Med. Chem. 35:3331-3341, 1992). The above references pertaining to thrombin inhibitors are herein incorporated by reference.
Fibrin-specific ~ntibodies Monoclonal anti-fibrin antibody 59D8 (ATCC
Acc-~c~ion No. HB 8546) was prepared and purified as previously described (Matsueda et al., U.S. Patent

4,916,070, herein-incorporated by reference). The method for the production of other fibrin-specific monoclonal 20 antibodies lacking fibrinogen cross-reactivity is described in detail in Matsueda et al., U.S. Patent 4,927,916, herein incorporated by reference.
Ex~mple 1: Chemically conjugated ~irudi~-59D8 IgG ~nd Hirudin-59D8 F~b' 2 5 Prepar~tion of 59D8-IgG conjugate The disulfide-linked hirudin-IgG conjugates were prepared by reacting an N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP) derivative of hirudin with 30 2-iminothiolane-substituted anti-fibrin monoclonal antibody 59D8. 8 mg hirudin were dissolved in 2 ml 50 mM
sodium carbonate buffer, pH 8.5. Subsequently, 2 ml of n-propanol were added. SPDP was added in 3-fold molar excess (20 mM in absolute ethanol) dropwise to the 3 5 hirudin solution. This mixture was stirred gently at WO94/25491 PCT~S94/0~81 2`161772 room temperature for 3 min and then applied to a Sephadex G-25 column (1.6 X 90 cm) that had been equilibrated with PBS (O.1 M NaH2PO4, O.1 M NaCl, pH: 7.4). Peak protein fractions were pooled and analyzed for 2-pyridyldisulfide

5 content (Carlsson et al., Biochem J. 173:723-737, 1978) and typically showed 0.6-1.5 residues per hirudin molecule. The SPDP substitution level was kept intentionally low to limit loss of hirudin activity and to avoid formation of higher molecular-weight aggregates.
lO Specific activity of SPDP-modified hirudin was on average 53% that of unmodified hirudin.
Other thrombin antagonists can be modified with reagents, such as SPDP or succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), to add 15 a single terminal thiol-reactive group. Other antagonists modified in this manner can then be conjugated to 2-iminothiolane-substituted anti-fibrin antibody or fibrin-specific Fab' or Fv.
Anti-fibrin monoclonal antibody 59D8 was raised 20 against ~-peptide, Gly-His-Arg-Pro-Leu-Asp-Lys (SEQ ID
NO:lO). Monoclonal antibody 59D8 (4 mg/ml in 0.14 M
NaCl, 3.7 mM Na2PO4, 1 mM KCl, pH 7.4) was mixed in equal amounts (v/v) with a 1,OOO-fold molar excess of 2-iminothiolane (in 25 mM sodium borate, pH 9.3). The 25 reaction was allowed to continue for 25 min at room temperature with gentle stirring. Excess iminothiolane was removed by gel filtration on Sephadex G-25 that had been pre-equilibrated with PBS, pH 6.6. A lO-fold molar excess of the SPDP-modified hirudin was then mixed with 30 the iminothiolated antibody, with gentle stirring at room temperature for 5 hours. The reaction was stopped by the addition of lO-fold molar excess (compared to the hirudin concentration) of iodoacetamide in O.1 M Na2HPO4, pH 8Ø

W094/25491 . 2 1 6 1 7 7 2 PCT~S94/0~81 Preparation of 5sD8-Fab~ conjugate The (Fab') 2 of antibody 59D8 was obtained by pepsin cleavage according to standard methods. It was then affinity purified on a ~-peptide Sepharose affinity 5 column, made by coupling a synthetic ~-peptide, Gly-His-Arg-Pro-Leu-Asp-Lys-(Cys) (SEQ ID NO:11), to lysine-Sepharose via the cysteine residue, using maleimidobenzoylsuccinimide ester. The eluate of this column (0.2 M glycine, pH 2.8) was dialyzed against PBS
10 and found to contain only (Fab')2 and traces of Fab' or Fab, as assessed by SDS-PAGE. Reduction of the 59D8 (Fab') 2 was carried out at room temperature for 18 hours in 1 mM 2-mercaptoethylamine, 1 mM EDTA, 10 mM sodium arsenite, followed by the addition of solid Ellman's 15 reagent (SIGMA Chemical Company, St. Louis, M0) to a concentration of 5 mM. After 3 h at room temperature, excess reagent was removed by gel filtration on a Sephadex G-25 column (30 X 2 cm) and equilibrated with 0.1 M sodium phosphate pH 6.8. In this protected form, 20 59D8 Fab' remains structurally and functionally intact at 4C under sterile conditions for at least 1 year. The thiol form of 59D8 Fab' was easily regenerated by treatment with 10 mM 2-mercaptoethylamine for 30 minutes at room temperature, followed by gel filtration.
Chimeric proteins can also be made by linking fibrin-specific Fv fragments to thrombin antagonists. Fv fragments can be made according to methods known in the art (Anthony et al., Mol. Immunol. 29:1237-1247, 1992;
Huston et al., Proc. Natl. Acad. Sci USA 85:5879-5883, 30 1988) and then modified to introduce a sulfhydryl group by 2-iminothiolane substitution as described above.
The thiol form of 59D8 Fab' (at 1 mg/ml) was mixed with SPDP-modified hirudin (1 mg/ml) and allowed to react for 16 hours at room temperature. To minimize the amount 35 of uncoupled 59D8 Fab' in the final preparation, hirudin WO94/25491 PCT~S94/0~81 was present in the mixture in a 10-fold molar excess.
The reaction was stopped by addition of an excess of iodoacetamide, as described above.
The thiol form of 59D8 Fv or other fibrin-specific 5 Fab' or Fv fragments can be conjugated to hirudin as described above.
Purifi¢ation of 59D8 conjugates The hirudin-59D8-IgG conjugate and the hirudin-59D8-Fab' conjugate were both partially purified from the 10 reaction mixture by affinity chromatography on a ~-peptide- Sepharose column. The affinity column retains the conjugated as well as unconjugated 59D8-IgG or 59D8-Fab', but not uncoupled hirudin. The eluate (0.2 M
glycine, pH 2.8) from this column was dialyzed into PBS
15 and stored under sterile conditions at 4C.
Alternatively, uncoupled hirudin may be removed from the reaction mixture by gel-filtration on Sephadex G-100.
Electrophoresis and densitometric gCAnn; ng SDS-PAGE was performed according to the method of 20 Laemmli (Laemmli et al., Nature 227:680-685, 1970), and proteins were visualized by staining with Coomassie brilliant blue G-250 (Neuhoff et al., Electrophoresis 9:255-262, 1988). For relative protein quantitation, the gels were scanned at 633 nm with an LKB ultrascan XL
25 laser-densitometer. Quantitation was made on unreduced gels which provide separate bands for hirudin-conjugated 59D8-IgG and unconjugated 59D8-IgG or hirudin-conjugated 59D9-Fab' and unconjugated Fab'.
Protein dQtermin~tion Protein concentrations were assayed according to Lowry (with bovine serum albumin used as a st~n~rd) (Lowry, J. Biol Chem 193:265-275, 1951), or by measuring optical density at 280 nm.
Qu~ntitation of hirudi~ activity (S-2238 assay) W094/25491 PCT~S94/0~81 216~772 ~

The S-2238 assay measures thrombin activity and its inhibition by anti-thrombins. To 100 ~l of sample hirudin or hirudin-59D8 conjugate in assay buffer (20 mM
sodium dihydrogen carbonate, 0.15 M NaCl, 0.1% BSA, pH
5 7.4), 20 ~l of thrombin solution (2.5 U/ml water) were added and incubated at room temperature for 10 min. Then 50 ~l (0.833 mg/ml) of chromogenic substrate S-2238 (H-D
phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline io dihydrochloride) were added. After exactly 5 min of incubation, the reaction was stopped by addition of 50 ~l 20% acetic acid, and the test result was read at 405 nm.
Quantitation was obtained by comparing inhibition of thrombin activity in the test sample with that achieved 15 by different known concentrations of unconjugated hirudin. A linear relationship was achieved with certainty for anti-thrombin concentrations between 0 and 0.6 U/ml hirudin-activity, and samples with higher activity were diluted appropriately.
20 ~-Pepti~e ass~y 96-well microtiter plates were coated overnight at 4C with ~-peptide. 100 ~l coating buffer (20 mM Tris, 50 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.4) containing 0.005 mg ~-peptide were applied to each well. After 25 coating, the microtiter plate was first washed 5 times with coating buffer supplemented with 0.05% Tween 20 and then three times with water. Free binding sites were blocked overnight at 4C by the addition of 250 ~l bovine serum albumin (2% w/v in coating buffer) per well. The 30 washing step was repeated and defined amounts (by the S-2238 assay) of either hirudin or hirudin-59D8 conjugate (hirudin-59D8 IgG or, alternatively, hirudin-59D8 Fab' in 0.1 M NaCl, 0.1 M NaP04, pH 7.4) were applied in 100 ~l ~f buffer per well and incubated for 2 h at room 35 temperature. Samples were run in triplicate.

WO94/25491 PCT~S94/0~81 After washing 7 times with modified coating buffer (20 mM Tris, 0.5 M NaCl, 1 mM CaCl2, 1 mM MgCl2, 0.2%
Tween 20, pH 7.4) and three times with water, 100 ~l of assay buffer (20 mM NaHP04, 0.15 M NaCl, 0.1% BSA, pH
o 5 7.4) were applied per well and 20 ~l (1.25 U/ml) of thrombin solution were allowed to incubate for 1 h at room temperature. Then 50 ~l of chromogenic substrate (S-2238 at 0.83 mg/ml) were applied and after 20 minutes, the reaction was stopped by addition of 50 ~l of 20%
10 acetic acid, and the resulting absorption read at 405 nm.
Hirudin activity was detected as inhibition of thrombin activity and was expressed as % inhibition of total thrombin activity applied.
Fibrin-8epharose ass~y This assay is as described by Bode et al., J.
Biol. Chem. 262:10819-10823, 1987, herein incorporated by reference, with the following modifications. For the purposes of assaying targeted anti-thrombin activity, trace labeling of fibrin-monomer is not necessary. 200 20 ~l fibrin-Sepharose were incubated for 2.5 h at room temperature with defined amounts (by the S-2238 assay) of hirudin or hirudin-59D8-conjugate (hirudin-59D8-IgG or, alternatively, hirudin-59D8-Fab') in 100 ~l of buffer.
After washing the fibrin-Sepharose three times with 3 ml 25 0.15 M NaCl, 200 ~l S-2238 assay buffer and 40 ~l (0.05 U) thrombin solution (1.25 U/ml) were added and incubated for 1 h at room temperature. Subsequently lOo ~l of chromogenic substrate S-2238 were added, and the reaction was stopped after 25 min by addition of 100 ~l 20% acetic 30 acid. After centrifugation (5 min, 1,000 x g), absorption of the supernatant was read at 405 nm.
Hirudin activity was again detected as inhibition of thrombin activity and was expressed as % of total thrombin activity applied.

W094/2~491 PCT~S94/0~81 ~ 21 61 772 Inhibitio~ of thrombu~ growth in ~ fibrinogen solution The method of Runge (Runge et al., Biochemistry 27:1153-1157, 1988, herein incorporated by reference) was followed, with the following modifications. Fresh-frozen 5 plasma obtained from 5 donors was pooled, aliquoted and refrozen. Before each experiment, plasma was ultracentrifuged at 30,000 rpm for 60 min in a Beckmann SW 40 rotor in order to obtain platelet-free plasma (platelet count less than 1,000 platelets/~l).
10 Immediately before each experiment, the anti-thrombin activities of hirudin and hirudin conjugate were calibrated using the S-2238 assay (i.e., the inhibition of thrombin peptidase activity by hirudin and its conjugates was determined, and appropriate dilutions were 15 made so that the inhibition of thrombin peptidase activity was identical for each sample). Platelet-free plasma clots were made by adding to plasma, in the following sequence: l25I-labelled human fibrinogen (20,000 cpm/ml plasma), 0.5 M CaC12 (final concentration 20 0.05 M) and thrombin (0.5 NIH units/ml plasma).
Immediately after the addition of thrombin, the solution was drawn into silastic tubing (i.d. 4 mm) and allowed to clot for 2 h at 37C. The tubing was then cut into 1.8 cm sections yielding clots of approximately 0.2 ml. The 25 clots were removed from the tubing, and each was placed in a plastic vial and washed three times with 2 ml 0.15 M
NaCl. The radioactivity level of each was then determined and only clots within 5% of the mean value were used for experiments.
After counting, clots were resuspended in 100 ~1 TBS (0.1 M Tris, 0.1 M NaCl, pH 7.4). Experiments were initiated by the addition of 100 ~1 hirudin or conjugate solution contA; n; ng defined amounts of anti-thrombin activity. Then, 200 ~1 of fibrinogen solution (8 mg/ml) 35 trace labelled with 125I-fibrinogen (25,0000 cpm/ml) were WO94/25491 PCT~S94/~81 added to the clot. The final fibrinogen concentration in the test assay was thus in the physiological range (4 mg/ml). At predetermined intervals, clots were removed from the test tube, washed three times with 2 ml 5 0.15 M NaCl and assayed for radioactivity. Clots incubated without thrombin inhibitor (NaCl-controls) often could not be quantitatively removed from the incubation vial because of complete clotting of the assay mixture.
10 Inhibition of thrombuQ growth in human plasma These assays were executed as described above.
Clots were incubated with 200 ~1 of platelet-free plasma (instead of 200 ~1 of fibrinogen solution) and trace labelled with 125I-fibrinogen (250,000 cpm/ml).
15 Stati~tical Analysis The dose-response curves in Figs. 2 and 3 were compared (hirudin-59D8 conjugate versus unconjugated hirudin control within each assay) by fitting each curve with the antilogit function:
eB2 ( ln ( ATU ) ln(ATUO) ) % Thrombin inhibition (ATU) = Bl .
+ eB2 ( ln ( ATU ) ln(ATUO) ) 25 where B1 is the ultimate thrombin inhibition at infinite concentration of inhibitor, B2 is the rate constant for the increase in inhibition with increasing concentration (ATU) of inhibitor, and ATUo is the concentration of inhibitor (in ATU/ml) at which 1/2 the ultimate (B1) 30 inhibition is achieved. For each curve the three parameters B1, B2 and ATUo and the variances for each parameter were estimated by using the FIT FUNCTION
procedure of the RS/1 Data Analysis Software (BBN
Software Products, Cambridge, MA). Corresponding 35 parameters (B1, B2 and ATUo) for each curve were compared by the t test.

W094/25491 PCT~S94/~81 2161772 ~

The time-course curves describing fibrin deposition in Figs. 4, 5, and 6 were compared by batching fibrin values for each curve across all three time points and then, after confirming by one way analysis of 5 variance (ANOVA ONEWAY procedure, RS/1 software) that significant differences existed among some of the curves, performing t tests on the mean fibrin deposition values (in ~g) for each curve. This procedure served as a means of protecting against detecting excessive differences due 10 to multiple comparisons.
The dose-response curves in Figs. 4, 5, and 6 were compared by first performing linear regression analysis of fibrin deposition versus dose of inhibitor for each inhibitor separately and then comparing the 15 slopes and intercepts of the regression lines by the t test. The FIT FUNCTION procedure (RS/1 software) was used to estimate the variances of the linear regression parameters.
The time-course curves in Fig. 7 were 20 sufficiently linear to permit analysis of covariance, with time as a covariate. This analysis tested the hypothesis that the time course of fibrin deposition was identical among the various inhibitors (with and without the NaCl control). The analysis of covariance was 25 followed by pairwise planned comparisons (linear contrasts) of the various inhibitor curves (P4V
procedure, BMPD Statistical Software, University of California Press, Berkeley and Los Angeles, 1990).
All error estimates cited are standard 30 deviations of the estimates. Potency differences are expressed by the ratios of the estimated ATUo parameters.

WO94/25491 PCT~S94/0~81 Ch~ra¢terization of hirudin-59D8-IgG and hirudin-59D8-F~b' conjug~t~s The nature of the disulfide-linked conjugates obtained by coupling SPDP-modified hirudin either to 5 iminothiolane-modified antibody or to Fab' was defined in part by the coupling conditions and by the purification procedure.
The biological activity of hirudin is sensitive to SPDP-modification, because intact, unmodified lysine-l0 47 is essential for its anti-thrombin effects.
Therefore, basic reaction conditions, which lead to preferential modification of the amino-terminus of the protein, were chosen. Furthermore, SPDP was used at a low concentration in order to introduce not more than one 15 reactive group into the hirudin molecule. Even when prepared under the above conditions, however, SPDP-hirudin was on average only 53% as active as unmodified hirudin in the S-2238 chromogenic assay for thrombin inhibition.
The conjugate was partially affinity purified on a ~-peptide-Sepharose column. The eluate contained the desired hirudin-59D8-IgG or hirudin-59D8-Fab' conjugate as well as unconjugated 59D8-IgG or 59D8-Fab'.
In order to minimize the amount of uncoupled IgG or Fab', 25 a l0-fOld molar excess of modified hirudin was used in the reaction mixture.
Both the parent molecules and the conjugates were analyzed by SDS-PAGE under reducing and non-reducing conditions (Fig. lA and Fig. lB). When electrophoresed 30 under non-reducing conditions (Fig. lA), the hirudin-59D8-Fab' conjugate (lane 3) was visualized at 57 kDa. A
l:l molar ratio of hirudin and 59D8-Fab' was to be expected because each molecule contained only one reactive sulfhydryl group. However, the chemistry and 35 the procedure used to purify hirudin-59D8-Fab' conjugate WO94/25491 PCT~S94t~81 - 2161772 ~

did not completely remove unconjugated 59D8-Fab', which migrates at 50 kDa (compare lane 2: 59D8-Fab'). When electrophoresed under reducing conditions (Fig. lB), the disulfide bond linking hirudin to 59D8-Fab' was broken 5 and the two chains of 59D8-Fab' could be visualized tlane 2 for 59D8-Fab' and lane 3 for hirudin-59D8-Fab').
Hirudin stained poorly under these conditions.
Densitometric ~uantitation of the bands revealed an approximately 1:1 molar ratio of hirudin-10 59D8-Fab' conjugate and uncoupled 59D8-Fab' in the final preparation used for the assays described below.
Thrombin inhibition in the absence of fibrin (the target molacule) The thrombin inhibitory activity of hirudin was 15 compared with that of hirudin-59D8-IgG conjugate or hirudin-59D8-Fab' conjugate in the absence of the target molecule, fibrin, by measuring inhibition of thrombin peptidase activity. Thrombin-dependent cleavage of the chromogenic substrate, S-2238, was recorded at 405 nm.
20 The inhibitory activities of hirudin and hirudin-59D8-IgG
conjugate or hirudin-59D8-Fab' conjugate were made comparable at all concentrations tested and at all time points recorded after an appropriate calibration procedure. In subsequent experiments, appropriate 25 dilutions of hirudin-59D8 IgG, and hirudin-59D8 Fab' were used so that inhibition of thrombin peptidase activity was identical for each experimental point.
Thrombin inhibition as a function of hirudin binding to ~-peptide Thrombin inhibitory activities of hirudin and hirudin-59D8-IgG conjugate or hirudin-59D8-Fab' conjugate were compared as a function of binding to immobilized ~-peptide, the antigen against which the antibody had been raised. This assay was designed to ~;m; ze the 35 functional differences among the tested molecules. After W094/25491 PCT~S94/0~81 thrombin inhibitory activity of each species had first been assessed in the absence of ~-peptide, samples with identical thrombin inhibitory activity were then assayed with ~-peptide as a target molecule. Fig. 2A shows that ~ 5 the hirudin-59D8 IgG conjugate was twelve-fold more potent than hirudin in an assay that depends on binding to the ~-peptide (B1=55+2% versus 4.3+0.5%, p<0.0001).
Hirudin-59D8 Fab' was as potent as the IgG conjugate (Fig. 2B). At 32.5+3.5%, ultimate inhibition by the Fab' 10 conjugate was significantly greater than that by unconjugated hirudin (0%; p<0.0001). This difference indicates that a single antigen binding site of the antibody is sufficient for effective targeting. Both conjugates performed significantly better than hirudin.
15 The reason for this large difference is the specific binding of the conjugates to the immobilized target molecule through the 59D8-IgG or 59D8-Fab' antigen-combining site, whereas unconjugated hirudin, which is incapable of specific binding to the target molecule, is 20 washed out.
Thrombin inhibition as a function of hirudin b; n~; n~ to immobilized fibrin Hirudin, hirudin-59D8-IgG conjugate and hirudin-59D8-Fab' conjugate were compared in the fibrin-25 Sepharose assay as follows: after thrombin inhibitoryactivity of each species had first been assessed in the absence of fibrin, samples with identical thrombin inhibitory activity were then assayed with fibrin as a target molecule. Fig. 3 shows that at the highest level 30 of thrombin inhibition, hirudin-59D8 IgG was llO0-fold more potent (p<0.0001) and hirudin-59D8 Fab' was 1900-fold more potent (p<0.0001) than unconjugated hirudin.
800 units of hirudin were required to achieve 32.3%
thrombin inhibition, whereas only 1 unit of hirudin-59D8-35 IgG conjugate was required to obtain 39.9% inhibition and W094/25491 21 61 7 7 2 PCT~S94/0~81 0.5 units of hirudin-59D8-Fab' conjugate required to achieve 37.2% inhibition of thrombin.
Inhibition of thrombus growth in fibrinogen solution The inhibition of fibrin formation on the 5 surface of a plasma clot suspended in Tris-buffer containing fibrinogen at physiological concentration is shown in Fig. 4. Complete inhibition of fibrin formation for 3 h was achieved using 7.4 ATU/ml of hirudin-59D8-IgG
conjugate, compared to 80 ATU/ml hirudin (Fig. 4A).
10 Conjugate at 7.4 ATU/ml allowed significantly less fibrin deposition than hirudin at 8 ATU/ml (233 ~g less, p=0.0002) and hirudin at 2.4 ATU/ml (252 ~g less, p=0.0001). Conjugate at 1.2 ATU/ml allowed significantly more fibrin deposition than hirudin at 80 ATU/ml (113 ~g 15 more, p=0.01).
When fibrin deposition was plotted against inhibition of thrombin peptidase activity, hirudin-59D8 IgG conjugate was about 10 times more effective than hirudin (Fig. 4B). The intercept fibrin deposition for 20 hirudin-59D8 IgG was significantly lower than that for unconjugated hirudin (p=0.003). The slopes were the same.
Results for hirudin-59D8 Fab' were similar (Fig. 5). Fibrin deposition for hirudin-59D8 Fab' at 25 9.25 ATU/ml was 21 ~g lower than that for hirudin at 100 ATU/ml (p=0.001), 71 ~g lower than that for hirudin at 30 ATU/ml (p=0.0001), and 129 ~g lower than that for hirudin at 10 ATU/ml (p=0.0001) (Fig. 5A). Also, fibrin deposition for hirudin-59D8 Fab' at 3 ATU/ml was 30 significantly lower, by 74 ~g, than that for hirudin at 10 ATU/ml (p=0.01). All other fibrin deposition curves were indistinguishable. For the dose-response curves (Fig. 5B), the intercept fibrin deposition for hirudin-59D8 Fab' significantly lower than that for unconjugated 35 hirudin (p=0.003); the slopes were equal.

W094/25491 PCT~S9410~81 Inhibition of thrombus growth in human pl~sma:
The enhanced potency of hirudin-59D8-Fab' conjugate was also demonstrated in human plasma. Fig. 6A
shows that fibrin deposition was 84 ~g lower for 5 conjugate at 10 ATU/ml than for hirudin at 10 ATU/ml (p=0.0001), and 101 ~g lower than for NaCl control (p=0.0001), whereas deposition was indistinguishable from that for hirudin at 100 ATU/ml. Fibrin deposition for the conjugate at 1 ATU/ml was 90 ~g higher than that for 10 hirudin at 100 ATU/ml (p=0.0001) and was indistinguishable from that for hirudin at 10 ATU/ml or that for the NaCl control. Fibrin deposition for hirudin at 100 ATU/ml was 112 ~g lower than that for the NaCl control (p=0.0001). With a 17 ~g lower mean fibrin 15 deposition over time, hirudin at 10 ATU/ml was no different than the NaCl control.
For the dose-response cures in Fig. 6B, the intercept fibrin deposition for hirudin-59D8 Fab' was significantly lower than that for hirudin. These results 20 in plasma were similar to those in fibrinogen solution (compare Figs. 5B and 6B); in both systems, the fibrin-targeted hirudin conjugate is 10 times more potent than hirudin .
Comparison of Eirudin with a Mixture of Eirudin and 25 Antibody To exclude any influence of uncoupled antibody or Fab' on the activity of hirudin, an equimolar mixture of the two components was compared with hirudin alone.
Fig. 7 shows that the mixture of components was not 30 significantly more potent than hirudin alone. The fibrin-deposition curves for hirudin at 44 ATU/ml and the mixture at 43 ATU/ml were indistinguishable. Hirudin at 10 ATU/ml and the mixture at 9 ATU/ml were also indistinguishable. All curves depicting inhibitor 35 activity were significantly different from the NaCl W094/25491 2 1 6 1 7 7 2 PCT~S94/0~81 ~

control curve (p<0.00005). Fibrin deposition for hirudin at 44 ATU/ml was lower than that for the mixture at 9 ATU/ml (p=0.003). Deposition for the mixture at 43 ATU/ml was lower than that for hirudin at 10 ATU/mll (p=0.01).
The hirudin-58D8-Fab' conjugate was shown to have a molecular mass of 57 kDa, corresponding to a 1:1 molar ratio of hirudin and 59D8-Fab'. Densitometric quantitation revealed that for each mole of conjugate, 10 one mole of uncoupled 59D8-Fab' was present in the final preparation. Compared to uncoupled hirudin in a quantitative assay measuring (as reflected by thrombin inhibition) an anti-thrombin's ability to bind to ~-peptide (as a surrogate for the amino-terminus of the 15 fibrin beta-chain) or to immobilized fibrin, the hirudin-59D8-Fab' conjugate delivered more anti-thrombin activity than did hirudin in the presence of the target molecule, fibrin. Hirudin-59D8-Fab' conjugate proved to be more potent than hirudin or a mixture of hirudin and 59D8-Fab' 20 in the prevention of thrombus growth either in a fibrinogen solution or in human plasma.
The increased local concentration of hirudin and the superior inhibition of thrombus growth by the hirudin-59D8-Fab' conjugate is attributed to antibody 25 targeting of the chimeric molecule. Enhanced thrombin inhibition could only be demonstrated with the conjugate;
an uncoupled mixture of hirudin and 59D8-Fab' was indistinguishable from hirudin. Thus, the enhancement of anti-thrombin activity demonstrated for the conjugate 30 cannot be attributed to residual free 59D8-Fab' in the final conjugate preparation.
Importantly, there was no apparent difference between hirudin-59D8-Fab' conjugate and hirudin-59D8-IgG
conjugate. Both were e~ually superior to uncoupled 35 hirudin, indicating that one antibody binding site is ~ WO94/25491 2 1 6 1 7 7 2 PCT~S94/0~81 enough to achieve the full targeting effect. This is particularly important with respect to the therapeutic use of a genetically engineered hybrid molecule consisting of 59D8-Fab' and hirudin.
5 Ex~mple 2: Recombinant hybrid antithrombotic reAgents Recombinant Hirudin-59D8 antibody The construction of the recombinant hirudin-antibody hybrid expression vector was accomplished using the vector pSV2gpt, containing the pUC 12 polylinker 10 inserted into the EcoR1-PstI site. An XbaI fragment containing the mouse y-2b heavy chain constant region was inserted into the polylinker. Constructs containing antibody 59D8 sequence have the cloned heavy chain rearrangement of 59D8 inserted into a unique EcoRl site 15 5' to the constant region sequence. In the original construct, portions of the y-2b constant were removed and replaced with a sequence encoding plasminogen activator as described in Runge et al., Proc. Natl. Acad. Sci. USA
88:10337-10341, 1991, herein incorporated by reference.
20 To make a hirudin-antibody hybrid, the plasminogen activator sequence was replaced with a cDNA sequence encoding hirudin (Harvey et al., Proc. Natl. Acad. Sci.
USA 83:1084-1088, 1986), using recombinant cloning methods well known in the art.
Some constructs include the factor Xa recognition sequence (5' ATA GAA GGA CGA AGC 3') (SEQ ID
N0:12) between the 59D8 heavy chain and the hirudin cDNA
to allow proteolytic cleavage by Factor Xa of the recombinant protein into its two functional units to 30 study their respective activities. The vectors were transfected into 59D8 heavy chain loss variant hybridoma cells by electroporation for expression of the recombinant hirudin-antibody hybrid and selected for expression of the recombinant hybrid protein.

W094/25491 PCT~S94/0~81 Hybrid molecules can be purified from the culture media by two steps of affinity chromatography.
Culture supernatant can be chromatographed on a ~-peptide-Sepharose column. The eluate from the ~-peptide-5 Sepharose column, containing recombinant hybrid moleculesin which the 59D8 portion is functional, can then be chromatographed using a thrombin-Sepharose column. This purification scheme ensures that both moieties of the hybrid molecule possess their correct function.
10 ~xample 3: Pharmaceutical use of chemically conjugated or recombinant targeted ~nti-thrombin protein reagents For the treatment of patients in thrombogenic situations, including but not limited to coronary stent implantation and PTCA, chimeric proteins of the 15 invention, generated by chemical conjugation or expression of recombinant hybrid proteins, can be administered in a pharmaceutically acceptable carrier such as physiological saline, in a manner similar to those presently used for the administration of 20 streptok;n~e or tpa. The chimeric proteins can be administered intraperitoneally, intramuscularly, subcutaneously, or intravenously. It is expected that the preferred route of administration is intravenous, with a dosage of approximately 0.001-0.5 mg/kg of body weight 25 per day; the chimera of the invention can be advantageously administered locally within the blood vessel at the site of expected clot formation.
The invention also includes an ex vivo method of therapy. This method of the invention would be of 30 particular benefit in situations in which the blood of a patient is removed for filtering (e.g., kidney dialysis) or gas exchange procedures, or when the patient requires blood transfusions. For treatment of blood extracorporeally, blood can be removed from the 35 individual using methods known to those skilled in the ~ WO94/25491 2 t 6 t 7 7 2 PCT~S94/0~81 art, such as venous puncture. The chimeric protein in a physiologically acceptable carrier can then be mixed with the blood, and subsequently returned to the individual using methods known to those skilled in the art, such as 5 intravenous drip.
In some cases, it can be useful to dissolve existing thrombotic occlusions in addition to preventing the formation of new clots. Administration of the chimeric protein of the invention with either (1) a 10 fibrinolytic agent such as streptokinase, staphylok;~se, urokin~e, or tpa, or (2) a second chimeric protein containing a fibrin-binding domain linked to a fibrinolytic agent, would then be appropriate. The production of a chimeric protein containing a fibrin-15 binding domain linked to a fibrinolytic agent has beendescribed (Haber et al., U.S. Patent 5,116,613, herein incorporated by reference). These agents can be administered to a patient sequentially or simultaneously as described above.
20 Ex~mple ~: Pharmaceutical u~e of recombinant hybrid DNA
reagents Patients in thrombogenic situations may be treated by administering the nucleic acid of the invention, such that the expression and secretion of the 25 chimeric protein takes place in the cells of the patient, such as blood cells. The nucleic acid of the invention may be introduced into target cells of a patient by standard vectors and/or gene delivery systems. Suitable gene delivery systems include liposomes, receptor-30 mediated delivery systems, naked DNA, and viral vectorssuch as herpes viruses, retroviruses, and adenoviruses, among others.
r The invention also includes cells transfected with the DNA of the invention. Standard methods for 35 transfecting cells with isolated nucleic acid are well WO94/25491 PCT~S94/0~81 known to those skilled in the art of molecular biology.
Preferably, the cells are blood cells, such as antibody-producing B cells, and they express a chimeric protein of the invention encoded by the nucleic acid of the 5 invention.
A therapeutic composition is provided which includes a pharmaceutically acceptable carrier and a therapeutically effective amount of a nucleic acid, wherein the nucleic acid includes a promoter operatively 10 linked to a sequence encoding the recombinant chimeric protein of the invention, to generate high-level expression of the chimeric protein in cells transfected with the nucleic acid. The therapeutic composition may also include a gene delivery system as described above.
15 Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal: e.g., physiological saline. A
therapeutically effective amount is an amount of the nucleic acid of the invention which is capable of 20 producing a medically desirable result in a treated animal.
As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the 25 particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages for the hybrid nucleic acid molecules of the invention will vary, but a preferred dosage for intravenous administration is 30 approximately from 106 to 1022 copies of the nucleic acid molecule.

Other embodiments:
For prevention of thrombotic occlusion post-operatively, a bio-polymer delivery system designed for -~ WO94/25491 2 1 6 1 7 7 2 PCT~S94/04881 the slow release of the chimeric protein of the invention may be implanted in close proximity to blood vessels that have been injured, such as those involved in coronary bypass surgery or coronary stent implantation. Such bio-5 polymer delivery systems are well known in the art (see, e.g., Folkman et al., U.S. Patent 4,164,560, herein incorporated by reference). The stent itself can be made of a bio-polymer that has been impregnated with the chimeric protein of the invention, and which therefore 10 mediates slow local release of the protein to prevent restenosis of the blood vessel.
Other embodiments are within the following claims.

WO 94125491 , PCT/US94/04881 2161772 ~

S~CUENCB LISTING
( I ) r~D~T- INFORMATION:
(i) APPTICANT: Edgar Haber Chri~toph Bode Marschall S. Runge ($~) TI T-~ OF l~vkn~lONs FIBRIN-TARGETED INHIBITORS OF
THROMBIN
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~A~, An~ S~ Fish & Richardson ~B~ 8TREET: 225 Franklin Street ,C~ CITY: Boston ID, STATE: Mas~ chll ~ettu E~ Cuu-~Y: U.S.A.
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(~) 8EQUENCB ~r~TCTTc8s A'l LENGTHs 65 Bl TYPEs amino acid (C~ 8T~n~DNESSs ~DJ TOPOLOaYs Linear (xi) SEQUENCE ~sCDTPTIoNs SEQ ID NO: l:

Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cy~
lu Gly Ser Asn Val Cys Gly Gln Gly Asn Lys Cys Ile Leu Gly Ser Asp Gly Glu Lys Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu Gln (2) INFORMATION FOR 8EQUENCE IDENTIFICATION NUMBER: 2:
(i) 8EQUINCE C~ C~TSTICS:
~A) LENaTHs 66 ~B) TYPE: amino acid ) STRANDFDNESS:
~D) TOPO~OGY: Linear (~i) SEQUENCE D~rDTPTIoN: SEQ ID NO: 2:

Ile Thr Tyr Thr A~p Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys lu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys Ile Leu Gly Ser Gln Gly Lys Asp Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Gln Gly Asp Phe Glu Pro Ile Pro Glu Asp Ala Tyr Asp Glu WO 94/25491 ~ 2 1 6 1 7 7 2 PCT/US94/04881 ~

( 2 ) INFOR~IATION FOR 8EQUENCE IDENTIFICATION NWBER: 3:
( i ) SBQUENCB r~l7~PRVTSTICS s ~AJ LENGTEEs 58 IBJ TYPBs amino acid C I ST~I~mRDNBSS s ~DJ TOPOLOaYs Linear ( a:$ ) SEQUBNCE DR-CrD TPTION s SEQ ID NO : 3:
~p Cy~ Thr Glu Ser Gly Gln A~n Leu Cy~ Leu CYQ Glu Gly Ser Asn al Cys Gly Gln Gly Asn Lys Cys Ile Leu Gly Ser Asp Gly Glu Lys sn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly A~p Phe Glu Glu Ile Pro Glu Glu (2) INFORMATION FOR S15QUBNCB ID13NTIFICATION NWBER: 4:
($) SEQUE:NCB rW~PTS~ICS:
~A- L~5NGT~ s 12 B~l TYPBs amino acid CJ 8~ Rl-N SSs ~DJ TOPOLOGYs Linear (~c$) SBQulsNC15 ~R~DTPTIONs SEQ ID NO: 4:

Asn Gly A~p Phe Glu Glu Ile Pro Glu Glu Tyr Xaa (2) INFORMATION FOR Sl5QU15NCI~ IDBNTIFICATION NWBERs 5:
($) SEQIIBNCB ~ TSTICS:
~A) LBNGT~: 8 , B) TYPB: amino acid ~C) ST~ RDNBSSs ~D) TOPOLOGYs LLnear ~c$) SBQUBNCB DRfsr~TpTIoNs SEQ ID NO: 5:

Phe Glu Glu Ile Pro Glu Glu Tyr WO 94/25491 2 1 6 t 7 7 2 PCT/US94/04881 (2) INFOP~MATION FOR 8EQUENCF ID~NTIFICATION NUMBBR: 6:
(i) 8EQUENCE ~rPDTSTICS:
~Aj LENGT~: 16 IBI TYPE: amino acid I C I STPUNDEDN SS:
~DJ TOPOLOaY: Linear (xi) SEQUENC~ ~C~DTPTION: SEQ ID NO: 6:

Val Thr Gly Glu Gly Thr Pro Lys Pro Gln Ser His A~n Asp Gly A~p (2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 7:
(i) 8EQUENCE rn~DTS~ICS:
(A) LENGTH: 16 (B) TYPE: amino acLd (C) STR~n~nNEss:
(D) TOPOLOaY: Linear (xi) SEQVENCE D~CCDTPTION: SEQ ID NO: 7:

Val Thr Gly Glu Gly Thr Pro Asn Pro Glu Ser His A~n Asn Gly Asp (2) INFORMATION FOR SEQUENC~ IDENTIFICATION NUMB~R: 8:
(i) SEQUENCE ~U~-D-~r~DTS~ICS:
~A`l LENGT~: 12 ~Bl TYPE: amino acid ~CJ STRANDEDNFSS:
~D~ TopoLoay: Linear (xi) SEQUENCE n~rDTPTION: SEQ ID NO: 8:

AQn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu . - 36 -(2) INFORMATION FOR 8EQUENCB __ r~TTFI QTION NUMBER: 9s (i) SEQUEN Q C~Rn~r~TSTICSs ~Aj T-~NGTH: 20 (B~l TYP~: amino acid ,C~ STRANDLDNESSs ~D~ TOPOLOGY: Linear (xi) SEQUENCE D~-cr~TPTIoN: SEQ ID NO: 9:

Phe Pro Arg Pro Gly Gly Gly Gly Aqn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu (2) INFORMATION FOR SEQUEN Q IDENTIFI Q TION NUMBER: 10:
(i) SEQUEN Q ~r~TSTICS:
~A) LENGT~: 7 ~B) TYPEs amino acid ,C) STRANDEDNESS:
~D) TOPOLOGY: Linear (~) SEQULNCE nF~TPTION: SEQ ID NO: 10:

Gly Hi~ Arg Pro Leu Aqp Lys l 5 (2) INFORMATION FOR SEQUENCE I~ l~l Q TION NUMBER: ll:
(i) SEQUENCE C~U~rF~TSTICs:
(A) LENGTH: 8 (B) TYPEs amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: Linear (~i) SEQUEN Q D~r~TPTION: SEQ ID NO: ll:

Gly Hi~ Arg Pro Leu Asp Lyq Cy~

~ wo 94/254gl 2 1 6 1 7 7 2 PCT/US94/04881 (2) INFOR~ATION FOR SEQUEN OE IDENTIFICATION NUMBER: 12:
(~) 8EQUEN OE ~rPT8TIC8t A'I LENaT~s 15 ~BI ~YPEs nucleic acid ~C, 8TRANDEDNe88s ~ingle ~DJ TOPOLOaYs linear (x~) 8EQUeNCE -DTPTIONs SEQ ID NO: 12:

Ile Glu Gly Arg Ser What is cl A ~ ~ d is:

Claims (20)

1. A chimeric molecule comprising a fibrin-binding portion of an fibrin-binding antibody linked by a covalent bond to an inhibitor of thrombin.

2. The molecule of claim 1, wherein said antibody is a monoclonal antibody specific for the amino terminus of the fibrin .beta. chain.

3. The molecule of claim 1, wherein said antibody is monoclonal antibody 59D8 (ATCC Accession No HB 8546).

4. The molecule of claim 1, wherein said inhibitor of thrombin is hirudin.

5. The molecule of claim 1, wherein said inhibitor of thrombin is a thrombin-inhibiting fragment of hirudin.

6. The molecule of claim 5, wherein said fragment has been chemically modified to contain a sulfate, carboxylate, sulfonate, phosphonate, carbonate, methyl sulfonate or methyl phosphonate group.

7. The molecule of claim 1, wherein said inhibitor of thrombin is antithrombin III; .beta.,.beta.' monochlormethylene diadenosine 5'5'''-p1p4-tetraphosphate; Ac-(D)Phe-Pro-boroArg-OH; Boc-(D)Phe-Pro-boroArg-C10H16; H-(D)Phe-Pro-boroArg-OH; H-(D)Phe-Pro-boroArg-C10H16; D-Phe-Pro-Arg;
[Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(OS)3)-Leu;
[D-Phe-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu]; 2,3,-dialkylindoles 3,4,-dihydro-3-benzyl-6-chloromethylcoumarin; D-phenylalanyl-prolyl-arginine chloromethyl ketone-treated .alpha.-thrombin; D-Phe-Pro-Arg-H; D-Phe-Pro-Arg-CH2Cl; N.alpha.-(.beta.-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide; (2R,4R)-4-methyl-1-[N.alpha.-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulphonyl)-L-arginyl]-2-piperidine carboxylic acid; N.alpha.-acetyl[D-Phe45, Arg?(COCH2)47, Gly48]desulfo hirudin45-65; Hirutonin-1; Hirutonin-2;
Hirutonin-3; or Hirutonin-4.

8. The molecule of claim 1, wherein said binding portion is a fibrin-specific Fab' fragment.

9. The molecule of claim 1, wherein said binding portion is a fibrin-specific Fv fragment.

10. The molecule of claim 1, wherein said molecule comprises a fibrin-specific antibody linked by a covalent bond to an inhibitor of thrombin.

11. A pharmaceutical composition useful for inhibiting thrombin activity, comprising the molecule of claim 1 in a pharmaceutically acceptable carrier.

12. A method of preventing coagulation of blood, comprising introducing into said blood the molecule of claim 1.

13. A mixture comprising (a) the molecule of claim 1, and (b) a chimeric molecule comprising a fibrin-binding portion of a second antibody linked by a second covalent bond to a thrombolytic agent.

14. The method of claim 12, further comprising introducing into said blood a chimeric molecule comprising a fibrin-binding portion of a second antibody linked by a second covalent bond to a thrombolytic agent.

15. The mixture of claim 13, wherein said first antibody is identical to said second antibody.

16. The mixture of claim 13, wherein said thrombolytic agent is streptokinase, staphylokinase, urokinase, or tissue-type plasminogen activator.

17. A nucleic acid encoding the molecule of claim 1, said molecule being a protein.

18. A cell expressing the molecule of claim 1.

19. A method of preventing coagulation of blood, comprising introducing into said blood the nucleic acid of claim 17.

20. A method of preventing coagulation of blood comprising introducing into said blood the cell of claim 18.