AU5145893A

AU5145893A - Clot directed anticoagulant, process for making same and methods of use

Info

Publication number: AU5145893A
Application number: AU51458/93A
Authority: AU
Inventors: Peter Gregory Bundesen; Paul Eisenberg; Carmel Judith Hillyard; Dennis Brian Rylatt
Original assignee: Agen Ltd
Current assignee: Agen Ltd
Priority date: 1992-10-12
Filing date: 1993-10-12
Publication date: 1994-05-09

Description

CLOT DIRECTED ANTICOAGULANT, PROCESS FOR MAKING

SAME AND METHODS OF USE

TECHNICAL FIELD

This invention relates to a clot-targeting, anticoagulant molecule, a clot- targeting, anticoagulant binding molecule, compositions for treating a clot in a mammal, methods of treating a clot in a mammal, and kits for treating a clot in a mammal.

BACKGROUND ART

Blood coagulation is the result of a complex series of events ultimately leading to the proteolytic conversion of circulating soluble fibrinogen to insoluble fibrin. Fibrin formation is a normal physiological response to vascular tissue injury of various aetiology. If the thrombotic process is overwhelming or persistent, partial or total vascular occlusion may result. The clinical consequences of thrombosis include: coronary artery disease (e.g. myocardial ischaemia), cerebral ischaemia (e.g. transient cerebral ischaemic attacks), or peripheral vascular disease (e.g. deep venous thrombosis, peripheral artery occlusion). Atherosclerosis and its complications are the prime cause of death in western society.

The atherosclerotic process is associated with extracellular lipid uptake and accumulation within fibroblasts and smooth muscle cells of lipid, resulting in the pathological lesion referred to as an atherosclerotic plaque or atheroma . The factors that promote the development of atherosclerotic plaques are unknown; however, plasma hyperlipaemia, diabetes, hypertension, and cigarette smoking are known risk factors. The natural history of thrombosis is dependent on a balance between procoagulant activity at the site of the thrombus and fibrinolytic activity, which induces dissolution of the thrombus. Clot-associated procoagulant activity is supported by the recruitment of circulating coagulation factors and their activation on the surface of the clot. There is a need for an agent which can prevent clots and which is effective in reducing mortality associated with thrombotic disease, and which is not also associated with untoward bleeding side effects.

OBJECTS OF INVENTION An object of this invention is to provide a clot-targeting, anticoagulant molecule.

Other objects are to provide a clot-targeting, anticoagulant binding molecule, compositions for treating a clot in a mammal, methods of treating a

SUBSTITUTE SHEET clot in a mammal, and kits for treating a clot in a mammal.

DISCLOSURE OF INVENTION

The inventors realised that it is factor Xa associated with or entrapped within the clot which induces prothrombin activation, thereby increasing local thrombin concentrations, which in turn provides the focus for extension of the thrombus particularly when this enzyme is subsequently exposed by the process of clot dissolution it could easily initiate clot reformation depending on the local balance of fibrinolytic activators and inhibitors. In other words, whole-blood clots, particularly when platelet rich, induce activation of the coagulation system by at least two phenomena: one is ascribable to the activity of thrombin bound to fibrin, and the other to clot-associated Xa activity. Both clot-associated thrombin and Factor Xa appear to be relatively protected from inhibition by anti-thrombin III or heparin-anti-thrombin III. Factor Xa activity associated with whole-blood clots induces thrombin formation de novo, even when clot-bound thrombin is inhibited. Thus the aim of the present invention is to provide a high concentration of an anticoagulant agent at the surface of a clot. Clot-targeted anticoagulants provide a novel approach for preventing the local progression of clotting while not inhibiting circulating clotting activity. This novel approach has particular applicability to the treatment of coronary thrombosis after angioplasty or thrombolysis, conditions in which the high doses of conventional anticoagulants required to prevent reformation of the coronary clot result in a high incidence of bleeding complications.

According to a first embodiment of this invention there is provided a clot-targeting, anticoagulant molecule comprising a clot-targeting binding molecule coupled to an anticoagulant.

The clot-targeting, anticoagulant molecule may further comprise at least one thrombolytic coupled to the clot binding molecule or may further comprise a thrombolytic coupled to the anticoagulant.

The clot-targeting binding molecule may be one capable of coupling to another coagulant molecule in vivo.

According to another embodiment of this invention there is provided a clot-targeting binding molecule which is capable of coupling to a coagulant molecule in vivo as well as a composition comprising such a molecule together with an acceptable carrier, diluent, excipient and/or adjuvant. The clot-targeting, anticoagulant molecule of the first embodiment may comprise one or more clot-targeting binding molecules coupled to one or more anticoagulants.

SUBSTITUTE SHEET According to a second embodiment of this invention there is provided a composition for treatment of a clot in a mammal, comprising a clot-targeting, anticoagulant molecule of the first embodiment together with an acceptable carrier, diluent and/or excipient.

As used herein treatment of a clot includes treatment to maintain the size of a clot in mammal, treatment to reduce a clot in mammal and a treatment to prevent reformation of a clot in mammal.

According to a third embodiment of this invention there is provided a method of treating a clot in a mammal comprising administering to a mammal requiring such treatment an effective amount of a clot-targeting, anticoagulant molecule of the first embodiment or a composition of the second embodiment.

The method of the third embodiment may further include administering to the mammal an anticoagulant molecule.

According to a fourth embodiment of this invention there is provided a method of treating a clot in a mammal, comprising administering to a mammal requiring such treatment an effective clot treating amount of a clot-targeting binding molecule and an anticoagulant capable of being bound by the clot- targeting, binding molecule in vivo.

In the method of the fourth embodiment the clot-targeting binding molecule may be administered simultaneously or before or after the administration of the anticoagulant capable of being bound by the clot-targeting, anticoagulant molecule. The idea for this embodiment is that the clot-targeting molecule targets and binds to a clot in the mammal and then binds to the anticoagulant capable of being bound to it which prior to now is circulating in the system of the mammal after having been administered to the mammal. Alternatively, the clot-targeting molecule binds to the anticoagulant capable of being bound to it and then targets and binds to a clot in the mammal.

According to a fifth embodiment of this invention there is provided a kit for treating a mammal requiring treatment selected from the group consisting of a treatment to treat a clot therein, a treatment to reduce a clot therein and a treatment to prevent reformation of a clot therein, comprising (a) a clot- targeting, binding molecule; and (b) an anticoagulant capable of being bound by the clot-targeting, binding molecule in vitro and/or in vivo.

The kit of the fifth embodiment may further comprise an acceptable carrier, diluent and/or excipient.

For the treatment of clots, a composition of the second embodiment may be administered parenterally, e.g. by injection and by intra-arterial

I SUBSTITUTE SHEET infusion, rectally or by inhalation spray. The compositions can also be delivered locally by drug delivery catheter devices, in order to increase the dose. It is also possible to administer the compositions orally provided the compositions contain materials such as liposomes which substantially prevent the anticoagulant molecule from being decomposed in the digestive tract.

Alternatively, the anticoagulant molecule may be coupled to a carrier which enables it to pass through to the blood stream without substantially decomposing in the digestive tract.

Advantageously the mammal is a bovine, human, ovine, equine, caprine, Leporine, feline or canine vertebrate.

More typically, the mammal is a human, the composition is a pharmaceutical composition and the carrier, diluent, excipient and/or adjuvant are pharmaceutically acceptable.

Where the mammal is not human, the composition is typically a veterinary composition and the carrier, diluent, excipient and/or adjuvant are veterinarily acceptable.

For parenteral administration, the clot-targeting, anticoagulant molecule or its salt may be prepared in sterile aqueous or oleaginous solution or suspension. Suitable non-toxic parenterally acceptable diluents or solvents ⁱnclude water, Ringer's solution, isotonic salt solution, 1,3-butanediol, ethanol propylene glycol or polyethylene glycols in mixtures with water. Aqueous solutions or suspensions may further comprise one or more buffering agents Suⁱtable buffering agents include sodium acetate, sodium citrate, sodium borate or sodium tartrate, for example.

The dosage form of the clot-targeting, anticoagulant molecule will compπse from 0.01% to 99% by weight of the active substance. Usually dosage forms according to the invention will comprise from 0.1% to about 20%, more typically 0.5% to 10% by weight of the active substance.

Compositions of the second embodiment may be prepared by means known in the art for the preparation of pharmaceutical compositions including blendⁱng, grinding, homogenising, suspending, dissolving, emulsifying dⁱspersing and mixing of the clot-targeting, anticoagulant molecule together with the selected excipient(s), carrier(s), adjuvant(s) and/or diluent(s).

In the method for the treatment of clots in accordance with the third embodiment of the invention, a molecule of the first embodiment or a composition of the second embodiment will usually be administered by ⁱn^jectⁱon. A suitable treatment may comprise the administration of a single dose

or multiple doses of the clot-targeting, anticoagulant molecule of the first embodiment, or of a composition of the second embodiment. An alternatⁱve suitable treatment may comprise the administration of a single dose or multⁱple doses of the clot-targeting, anticoagulant binding molecule of the first embodiment or a composition of the second embodiment, followed by or preceded by or simultaneously administrating at least one anticoagulant. Usually, the treatment will consist X administering from one to five doses daⁱly of the clot-targeting, anticoagulant molecule for a period of from one day to several days. A further treatment^ may be given during the lifetime of the patient. Most usually, the treatment will consist of the administratⁱon of the clot-targeting, anticoagulant molecule of the first embodiment or a composⁱtⁱon of the second embodiment for a period of from one day to several days.

The administered dosage of the clot-targeting, anticoagulant molecule of the first embodiment or a composition of the second embodiment can vary and depends on several factors, such as the condition of the patient. Dosages wⁱll range from O.OOOlmg to 200 mg per kg. Usually, the dose of the actⁱve substance will be from O.OOlmg to lOmg per kg of body weight, more typⁱcally 0.1 mg - 10 mg per kg of body weight. In the case of a bolus infusion the dose of the active substance is typically 0. lmg to 50mg per kg of body weight.

Examples of molecules with affinity for components of clots and whⁱch can target and bind to clots, include fibrin targeting and binding proteins e.g. plasmin and plasminogen, antibodies (see Z. H. Oster and P. Som, J. Nucl Med., 1990; 175:79-85, the contents of which are incorporated herein by cross reference) and fragments that bind to cross-linked fibrin and fibrin monomer, or neo-epitopes of fibrin in a clot (e.g. fragment Ei which is a plasmic degradatⁱon product of human cross-linked fibrin, see for example S.A. Olexa, and A.Z. Budzynski, Proc. Nail. Acad. Sci USA, 1980; 77:1374-1378, and L.C. Knⁱght et al The Journal of Nuclear Medicine, 1992; 33: 710-715; modified fragment Ei, see L.C. Knight et al., Biochim. Biophys Acta, 1987; 924: 45-53; fragment EX 2, see L.C. Knight et al., Biochim. Biophys Acta, 1987; 924:45-53, antif_ibrin antibodies including antifibrin monoclonal antibody 59D8, antifibrin 59D8 monoclonal antibody Fab' and F(ab')₂ fragments, see L.C. Knight et al. Radiology 1989; 173: 163-169, antifibrin monoclonal antibody C22A, antifibnn C22A monoclonal antibody Fab' and F(ab')₂ fragments, antifⁱbrin murine monoclonal antibody , antifibrin murine monoclonal antibody Fab' and F(ab')₂ fragments, see S.F. Rosebrough et al., Radiology 1985; 156: 515-517, fibπn specif_ic monoclonal antibody MoAb, fibrin specific monoclonal antibody MoAb

I SUBSTITUTE SHEET Fab' and F(ab')₂ fragments, see S. F. Rosebrough et al. Radiology 1987; 162: 575-577, antifibrin monoclonal antibody GC4, antifibrin GC4 monoclonal antibody Fab' and F(ab')₂ fragments, see S. F. Rosebrough et al. 1990; 31: 1048 the contents of which are incorporated herein by cross reference, antifibrin monoclonal antibody T2GIs, antifibrin T2GIs monoclonal antibody Fab' and F(ab')₂ fragments, see L.C. Knight, A.H. Maurer, LA. Ammar et al., Radiology, 1989; 173:163-169, L.C. Knight, A.H. Maurer, LA. Ammar et al., J. Nucl. Med. 1988; 29:494-502, S.S.L. Harwig, J.F. Harwig, R.E. Coleman and M.J. Welch, Thromb. Res., 1975; 6: 375-386, S. DeNardo, H. Bogren and G. DeNardo, Am. J. Roentgenol. 1985; 145: 1045-1052, A. Alavi et al. Radiology, 1990; 175:79-85, P. deFaucal et al. J. Nucl. Med. 1991; 32:785- 791, the contents of all of which are incorporated herein by cross reference). Note, the term "epitope" is used herein to refer to a binding site on or in a target clot which is specifically recognized by a clot binding molecule, and is not limited to binding sites recognized by antibodies. Other fibrin targeting and binding molecules, include D-dimer monoclonal antibody DD 3B6/22, fibrin specific antibodies such as fibrin antifibrin antibody MA 59D8, bifunctional antibodies which bind to fibrin or platelets or other clot components. Examples of molecules with affinity for platelets and which can bind to platelets, include platelet binding antibodies, or platelet binding molecules, bifunctional antibodies which bind to activated platelets, activated platelet binding antibodies (e.g. MAb 7E3, MAb 50H.19, MAb PADGEM and MAb P256, see S. F. Rosebrough et al. 1990; 31: 1048 the contents of which are incorporated herein by cross reference), activated platelet binding molecules and an antigen-binding fragment of a clot-targeting antibody, (e.g. MAb P256, F(ab')₂, and Fab' fragments of MAb P256, see for example A.W.J. Stuttle, J.M. Ritter, A.M. Peters and J.P. Lavender, Nuclear Medicine Communications, 9, 813-815 (1988) the contents of which are incorporated herein by cross reference). The clot targeting and binding molecule (CBM) may be any substance having a preferential affinity for a clot component including monoclonal or polyclonal antibodies, enzymes, or other binding proteins or substances (or binding fragments thereof). Where the clot component is an antigen, the CBM is usually an antibody, molecular recognition unit, or an antigen-binding fragment of an antibody, such as a F(ab')₂, Fab', Fv or VH fragment. Antibodies which recognize at least one of the constituents of clots may be prepared by conventional techniques using clots, or the purified constituents thereof, as immunogens. These antibodies may be monoclonal or polyclonal in nature. Either the intact antibody, or specific

antigen-binding fragments thereof, may be used as a CBM. The antibody or antibody fragment may be polyvalent, divalent or univalent.

A variety of anticoagulants including thrombin inhibitors can be included into the molecules of the invention, such as hirudin and hirudin analogues, C- terminal hirudin peptides and analogues, tick anticoagulant peptide (TAP), heparin, coumarin, short peptide inhibitors of thrombin such as small peptide- like inhibitors such as^* PPACK (d-phe-pro-arg-chloromethylketone), DuP 714 (ac-d-phe-pro-boroarginine), agratropin (2R, 4R)-4- methyl -1- [N2 -(3-methyl- 1,2,3,4,-tetrahydro -8-quinolinesulphonyl)-L- arginyl]-2- piperidinecarboxylic acid monohydrate, tick anticoagulant peptide or extrinsic pathway inhibitor or tissue pathway inhibitor. Examples of anticoagulants may be found in U.S. Patent Nos. 4,399,065, 4,703,036 and 5,114,922 and references therein, the contents of which are incorporated herein by cross reference. Fibrinogen receptor antagonists which are suitable anticoagulants for use in the present invention include cyclo(S,S)-N^α-acetyl-Cys-(N^α-methyl)Arg-Gly-Asp-Pen NH₂ Ali et al., EP 0341 915, cyclo(S,S)-(2-mercapto) benzoyl-(N^α-methyl)Arg-Gly- Asp-(2-mercapto), EP Application No. 90311537.6, peptides or peptide like compounds disclosed in Pierschbacher et al., WO 89/05150 (US/88/04403); Marguerie, EP 0 275 748; Adams et al., U.S Patent 4,857,508; Zimmerman et al., U.S. Patent 4,683,291; Nutt et al., EP 0 410 537; Nutt et al., EP 0 410 539; Nutt et al., EP 0 410 541; Nutt et al, EP 0 410 767; Nutt et al; EP 0 410 833; Nutt et al., EP 0 422 937; Nutt et al, EP 0 410 540; Nutt et al., EP 0 422 938; Alig et al., EP 0 372 486 Ohba et al., WO 90/02751 (PCT/JP89/00926); Scarborough et al., WO 90/1562 (PCT/US90/03417); Klein et al., U.S. Patent 4,952,562; Ali et al., PCT US 90/06514; Alig. et al., EP 0 381 033; and Alig et al., EP 0 384 362; the contents of all of which are incorporated herein by cross reference as well as and the cyclic peptides.

Ac-C s-(N e)Arg-Gly-Asp-Pen-NH2 Ac-Cys-Asn-Dtc-Amf-Gly-Asp-Cys-OH I I .or I I

Dtc=4,4^,Dimethylthiazolidine-5-carboxylic acid Amf=para - aminomethylphenylalanine

Generally, the anticoagulant, included in the molecules of the invention, acts as an antithrombin agent and/or inhibitor of earlier steps in the coagulation cascade such as factor Xa (for example TAP), factor Va, factor X, factor IXa, factor Villa, factor Vila, the prothrombinase complex, vitamin-K-dependent

SUBSTITUTE SHEcT j gamma carboxylation of prothrombin, factor X, factor VII, factor V, factor VIII, or factor IX, thereby limiting local procoagulant processes at the site of thrombosis. In other words, anticoagulants include factor Xa inhibitor including TAP, factor Va inhibitor, factor Villa inhibitor, factor Vila inhibitor, factor IXa inhibitor, prothrombinase complex inhibitor, vitamin-K-dependent gamma carboxylation of prothrombin inhibitor, factor X inhibitor, factor VII inhibitor, factor V inhibitor, factor Vffl inhibitor, and factor IX inhibitor.

Thrombolytic agents such as urokinase, scuPA streptokinase or tPA may also be coupled to the clot binding molecule of the invention to prevent arterial wall intimal proliferation. Anti growth factors (e.g. anti-PDGF and other targeted growth factors e.g. FGF's to induce reendothelialization) may also be coupled to the clot binding molecule of the invention or the targeting of growth factors via use compounds that attack protease sensitive bonds, to prevent rebuild-up of a clot. The clot-targeting, anticoagulant molecule of the invention may be a single molecule with clot binding and anticoagulant moieties, or a complex of two or more molecules. As used throughout the specification the term "molecule" will be used to cover both moieties and molecules, and "conjugate" to cover both a single hybrid molecule with clot binding and anticoagulant moieties and two conjugated molecules one of which has clot binding behaviour and the other of which has anticoagulant behaviour. In one embodiment, the conjugate is obtained by coupling a CBM to an anticoagulant (ACM). In a further embodiment the conjugate is obtained by coupling a CBM to an ACM and a thrombolytic (TMB) and/or thrombolytic binding molecule (TBM). The CBM, the ACM and optionally the TMB and/or TBM may be coupled together directly or indirectly (e.g. via a linker molecule), and by covalent or non- covalent means (or a combination thereof).

The clot-targeting, anticoagulant binding molecule of the invention may be a single molecule with clot binding and anticoagulant binding moieties, or a complex of two or more molecules. In one embodiment, the conjugate is obtained by coupling a CBM to an anticoagulant binding molecule (ACBM). In a further embodiment the conjugate is obtained by coupling a CBM to an ACBM and a TMB and/or TBM. The CBM, the ACBM and optionally the TMB and/or TBM may be coupled together directly or indirectly (e.g. via a linker molecule), and by covalent or non-covalent means (or a combination thereof).

Of particular importance in the context of the present invention are clot-

SUBSTITUTE SHEET targeting, anticoagulant molecule such as [anticoagulant-antibody to clot], [anticoagulant-antibody to clot-thrombolytic agent], [anticoagulant-thrombolytic- antibody to clot] and [thrombolytic-anticoagulant-antibody to clot] where the term antibody is understood to encompass clot targeting and binding monoclonal or polyclonal antibodies and clot targeting and binding fragments thereof in particular, F(ab') , Fab', Fv or VH fragments, especially D-dimer including MAb DD-3B6/22, F(ab')₂ fragments of MAb DD-3B6/22, Fab' fragments of MAb DD-3B6/22, all the fibrin targeting monoclonal antibodies disclosed in U.S. Patent No. 4,758,524 (the contents of all of which is incorporated herein by cross reference), monoclonal antibody DD-1D2/48, F(ab')₂ fragments of MAb DD-1D2/48, Fab' fragments of MAb DD-1D2/48, monoclonal antibody DD-1C3/108, F(ab')₂ fragments of MAb DD-1C3/108, Fab' fragments of MAb DD-1C3/108, and all the other fibrin targeting and binding antibodies disclosed elsewhere in this specification, and any other monoclonal either human or mouse or other mammal which has specificity for fibrin or D dimer but not fibrinogen or fragments thereof, fibrin specific antibodies such as fibrin antifibrin antibody MA 59D8, monoclonal antibody DD-1C3/108, bifunctional antibodies which target and bind to fibrin or target and bind to platelets, particularly activated platelets or other clot components or a clot antigen-binding fragment of an antibody, such as a F(ab')₂, Fab', Fv or VH fragment. The antibody or antibody fragment may be polyvalent, divalent or univalent. MAb DD-3B6/22 is commercially available from Agen Biomedical Limited, 11 Durbell Street, Acacia Ridge, Qld 4110 and Agen Inc., 20 Waterview Boulevard, Parsippany, New Jersey, U.S.A. 07054. MAb DD-1D2/48 and MAb DD1C3/108 are also available from these sources.

Below are listed reagents and references disclosing some of the covalent coupling methods known in the art. Covalent Coupling Reagents

1. SPDP (N-Succinimidyl-3,2-(pyridyldithio) propionate) Neurath et al., 1981, J. Virol. Meth. 3:155-165.

2. MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester) Kitagawa et al., 1976, J. Biochem. 79:223-236.

3. SIAB (N-succinimidyl-4-iodoacetylaminobenzoate) Weltman et al., 1983, Bio. Techniques 1: 148-152. 4. Other cross linking agents include cross-linking agents include 4,4'- dithiobisphenylazide, dithiobis-(succinimidylpropionate), 2- iminothiolane, dimethyl-3,3'-dithiobispropionimidate.2HCl,

SUBSTITUTE SHEET 10

disuccinimidyl tartrate, 3,3'-dithiobis-(sulfosuccinimidylpropionate⁾, eth_yl-4-azidophenyl-l,4-dithiobut_yrimidate.HCl, N-succinimιdyl-(4- azidophenyl)-l,3'-dithio-propionate, N-4-azido_Phenylthio)phthalιmιde, sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-l ,3 '- dithiopropionate, sulfosuccinimid_yl-(4-azidophen_yldithio)-propιonate, bis-[2-(succinimid_ylox_ycarbon_ylox_y)-eth_yl]sulfone an^d

_Sulfosuccinimid_yl-2-(p-azido_Salic_ylamido)-eth_yl-l,3'dithiopropionate.

Selective Bifunctional Reagents

P-isothioc_yanatobenzo_ylchloride (U.S. Patent 4,680,338)

Bifunctional Reagents

1. BSOCOES - Bis[2-(succinimidoox_ycarbon_ylox_y)eth l]sulfone

Zarling et al., 1980, J. Immunol. 124:913-920.

2. BS - Bis(sulfosuccinimid_yl)suberate Staros, 1982, Biochemist^ 21:3950-3955.

Other Reagents

1. Glutaraldeh_yde

Avrameas, 1969, Immunochem. 6: 43.

2. Periodate Oxidation Nakane et al. , 1974, J. Histochem. C_ytochem. 22 : 1084-1091

3. Carbodiimide

Khorana, H.G., 1953 The Chemistiy of Carbodiimides, Chem. Rev.

53:145-166 and references therein. 4 Disulphide Exchange

All the above references concerning coupling techniques are incorporated herein b_y cross reference.

The coupling may also be noncovalent, for example, b_y (a) attachⁱng biotin to one and avidin (or streptavidin) to the other), (b) attaching an antⁱ antibod_y to one, which then binds the other, (c) attaching Protein A to one, which then binds the Fc portion of the other, or (d) attaching a sugar to one and a corresponding lectin to the other.

It should be understood that, in coupling the CBM to an ACM and optional^ a TMB and/or TBM, the binding characteristics of the CBM and TBM the anticoagulant properties of the ACM and the thrombolytic propertⁱes of the TMB should be changed as little as possible. It may be advantageous to provide a spacer moiety between the CBM and ACM and optionally TMB and TBM to reduce steric hindrance. The CBM may be coupled directly to an ACM

SUBSTITUTE SHEET or indirectl_y via coupling to an TMB and/or TBM coupled to the CBM. Similarl_y, the CBM may be coupled directly to an TMB and/or TBM or indirectly via coupling to an ACM coupled to the CBM. Further, in coupling the CBM to an ACBM and optionally a TMB and/or TBM, the binding characteristics of the CBM, ACBM and TBM and the thrombolytic properties of the TMB should be changed as little as possible. It may be advantageous to provide a spacer moiety between the CBM and ACBM and optionally TMB and TBM to reduce steric hindrance. The CBM may be coupled to directly to an ACBM or indirectly via an TMB and/or TBM. Similarly, the CBM may be coupled directly to an TMB and/or TBM or indirectly via coupling to an ACBM coupled to the CBM.

A CBM/ACM conjugate may be an antibody coupled to an anticoagulant. One method of constructing such a conjugate is the following: (a) preparing F(ab')'₂ fragments of a selected antibody to a clot component by pepsin digestion; (b) reducing and treating the fragments with Ellman's reagent to produce Fab' fragments of the selected antibody; and (c) coupling the Ellman's reagent-treated Fab' fragment to a selected anticoagulant and optionally a TMB and/or TBM, to produce a clot-targeting, anticoagulant molecule. The CBM/ACBM conjugate may be a hybrid antibody. One method of constructing such a conjugate is the following: (a) preparing F(ab')'₂ fragments of a selected antibody to a clot component by pepsin digestion; (b) reducing and treating the fragments with Ellman's reagent to produce Fab' fragments of the selected antibody; (c) thiolysing a selected anticoagulant-specific antibody or a selected anti-coagulant antibody; and (d) coupling the thiolated Fab' fragment to the Ellman's reagent-treated Fab' fragment to produce a hybrid anti-clot antibody anticoagulant specific antibody conjugate.

The CBM/ACBM/TBM conjugate may also be a hybrid antibody. One method of constructing such a conjugate is the following: (a) preparing F(ab')' fragments of a selected antibody to a clot component by pepsin digestion; (b) reducing and treating the fragments with Ellman's reagent to produce Fab' fragments of the selected antibody; (c) thiolysing a selected anticoagulant- specific antibody or a selected anti-coagulant antibody; (d) thiolysing a selected thrombolytic-specific antibody or a selected thrombolytic antibody; and (e) coupling the thioylated Fab' fragments of (c) and (d) to the Ellman's reagent- treated Fab' fragment to produce a hybrid anti-clot antibody anticoagulant specific antibody and thrombolytic-specific antibody conjugate.

SUBSTITUTE SHEET j Another method for constructing a CBM/ ACBM conjugate, comprises: (a) treating an anti-clot component MAb-producing hybridoma and an anticoagulant-specific MAb-producing hybridoma with a distinct site-specific irreversible inhibitor of macromolecular biosynthesis; preferably the inhibitor is selected from the group consisting of emetine, actinomycin D, hydroxyurea, ouabain, cycloheximide, edine and sparsomycin; (b) fusing the two different MAb-producing hybridomas with polyethylene glycol to produce a heterohybridoma; (c) cloning the fused cells by any of a number of cloning methods such as isolation in soft agarose or by limiting dilution; (d) selecting cloned heterohybridomas secreting chimeric anti-clot component antibody- antigen specific antibody with a screening assay appropriate to the antibodies; (e) purifying the antibody product by affinity purification to free it from non- chimeric antibodies. The hybrid or chimeric antibody of the present invention thus comprises two "half molecules," one with specificity for clot component(s) (the CBM) and the other with specificity for an anticoagulant (the ACBM) . In this case the antibody's own disulfide bonds couple the ACBM to the CBM to form an appropriate conjugate.

Such a hybrid antibody, or F(ab')₂ fragment of such a molecule, has advantages which include ease of preparation, the preservation of the correct stoichiometry and stereochemistry of both antibodies and the retention of the binding affinity of each fragment.

When the anticoagulant-binding molecule is also a peptide or protein, the use of a peptide to bind to the clot has the further advantage that the entire conjugate may be prepared without any need for a bifunctional coupling agent. Instead, a DNA sequence encoding the clot-binding peptide and the anticoagulant-binding peptide as a single transcriptional unit is provided, and the desired conjugate is expressed as a fusion protein, with the two moieties joined by a simple peptide bond, or with a peptide spacer of desired length. One particularly preferred example is a DNA sequence encoding a clot-binding antibody fragment (which could be just the binding site, not a complete Fab) and anticoagulant antibody (which could be just the binding site, not a complete

Fab) or antigen as a single transcriptional unit is provided, and the desired conjugate is expressed as a fusion protein, with the two moieties joined by a simple peptide bond, or with a peptide spacer of desired length. Alternatively, the divalent peptide may be prepared by direct chemical synthesis.

The anticoagulant and thrombolytic may be molecules which may be activated in vivo on attachment to the clot by light irradiation (particularly far

SUBSTITUTE SHEET IR light activation) or chemical activation.

In summary, in the present invention, generally, anticoagulants including thrombin inhibitors are targeted to a clot by combining them with clot binding molecules to form a hybrid molecule one part of which can bind to a clot component such as fibrin or platelets with high affinity and the other part being able to bind and inactivate thrombin or other procoagulant molecules located at or within the clot. For example hybrid molecules can be constructed which comprise one half monoclonal antibody with high affinity for a clot component and one half a specific thrombin inhibitor such as a hirudin. The anticoagulant inactivates thrombin and/or other procoagulant molecules located either bound to the clot surface or activated in the vicinity of the clot. This approach has two major advantages over the current antithrombotic agents. Firstly, it targets the clot and thus brings anticoagulant to the clot where thrombolysis causes a local high concentration of procoagulant thrombin. Thus, it enhances the specificity of anticoagulants for ongoing thrombotic processes and increases the local concentration of anticoagulants. Second, the half-life of the desired anticoagulant effect is increased permitting shorter administration of the anticoagulant. These effects result in a decrease in the amount of drug that must be administered, an increase in efficacy and an increase in safety. BEST MODE AND OTHER MODES OF PERFORMING THE

INVENTION

A clot targeted anticoagulant molecule is made by coupling the fibrin specific monoclonal antibody DD-3B6/22 with an anticoagulant peptide (for example PPACK, hirulog or hirudin) or with an anticoagulant (for example heparin, or coumarin) and a thrombolytic agent (example tPA or urokinase). A pharmaceutical composition for treatment selected from the group consisting of a treatment to treat a clot therein, a treatment to reduce a clot therein and a treatment to prevent reformation of a clot therein, is then made by mixing the clot targeted anticoagulant molecule or the clot targeted anticoagulant thrombolytic molecule together with a pharmaceutically acceptable carrier, diluent or excipient. A human may be treated to reduce the clot therein by administering to the human requiring treatment such as treatment to treat a clot therein, treatment to reduce a clot therein and treatment to prevent reformation of a clot therein, an effective clot treating amount of the pharmaceutical composition. Use of monoclonal antibody DD-3B6/22 is especially advantageous to target a clot as there is no cross reaction of the monoclonal antibody with fibrinogen, fibrinogen fragment D, fibrinogen fragment E or non

SUBSTITUTE SHEET 14

cross-linked fibrin.

Examples of dosage forms in accordance with the invention are as follows:

1. Capsule Clot-targeting, anticoagulant molecule 0.1 to 10 mg/kg, generally

0.5 to 8mg/kg

Glycerol 100 to 200 mg

Distilled water 100 to 200 mg

Saccharin 0 to 2 mg Methyl Paraben 1 to 2 mg

Polyvinylpyrrolidone 0 to 2 mg

Liposomes 0.001 to 20 mg

2. Injectable solution

Clot-targeting, anticoagulant molecule 0.1 to 10 mg/kg, generally 0.5 to 9mg/kg

Sodium chloride 8.5 mg

Potassium chloride 3 mg

Calcium chloride 4.8 mg

Water for injection, q.s. to 10 ml EXAMPLES

Example 1-Synthesis of DD-3B6/22 (Fab) - PPACK derivative

(a) Preparation of Antibody fragments

The Fab-SH fragment of DD-3B6/22 was prepared by the method described by John eLfi! Throm. Res. 58 273-281 1990. Briefly purified antibody was digested with pepsin and F(ab')2 fragments purified. Fab-SH is prepared from

F(ab')₂ by mild reduction with beta mercaptoethylamine and gel filtration chromatography at pH 4.5.

(b) Preparation of SPDP labelled PPACK.

The anti-thrombin peptide derivative PPACK was reacted with SPDP (Pharmacia) under the conditions recommended by the manufacturer. Purification of the N-terminally modified derivative was carried out by reverse phase chromatography on a C-18 column using a gradient of 0-60% acetonitrile containing 0.1% trifluoracetic acid. The SPDP-labelled peptide was recovered after freeze drying. (c) Formation of Fab-PPACK derivative.

The pH of the Fab-SH solution was adjusted to pH 7.0 by the addition of 0.2M Sodium phosphate buffer pH 8.0. 10ml of 2mg ml fragment was reacted with

20mg of the freeze dried peptide. After 60 sec the reaction was terminated by the addition of 30mM sodium iodoacetamide and purified by gel filtration , chromatography on a 5X100 cm column of Ultragel Ac44. (d) Activity measurements The activity of the anti-coagulant portion of the Fab-PPACK derivative and the antigen binding capacity for D-Dimer were measured in a number of assays to confirm that both the anticoagulant activity of the reagent and clot binding activities were maintained. 1. Thrombin capture EIA This sandwich assay measures both of the activities of the reagent. The reagent is first bound via the thrombin binding portion and then the ability of the antibody portion to bind D-dimer is then assessed. A positive result in the assay can only be achieved if both segments of the reagent are active.

50μl of human thrombin (3800U/mg) 5μg/ml in phosphate buffered saline (PBS) pH 7.4 was coated onto the wells of a microplate for lh at room temperature and the plate washed 3 times with PBS containing 0.05% tween (PBS/T). 50μl of 0-40μg/ml of conjugate in PBS/T was then added and allowed to incubate for lh at room temperature. After washing a further 3 times with PBS/T, 50μl of lOμg/ml of human D-dimer (prepared as described by John et al Throm. Res. 58 273-281 1990) was added to each well and allowed to incubate for lh. Finally, the presence of bound D-dimer was detected by the addition of 50μl of 1/1000 dilution of HRPO -labelled DD-4D2/182 and subsequent addition of substrate as described by Rylatt et al Throm. Res. 31 767-778 1993. Results:

Conclusion:

The results show that conjugate, but not PPACK or antibody fragment alone can be detected in the thrombin capture EIA. This result confirms that the conjugate contains both the D-dimer antigen binding activity and anticoagulant activity on the same molecule. 2. Chromogenic assay

This assay measures the ability of the reagent to inhibit the action of thrombin on the thrombin specific chromogenic substrate S-2238. Inhibitors of this

reaction are thought to bind directly to the active site.

Briefly 20μl of thrombin (4nM) was incubated for lOmin at room temperature with either 20μl phosphate buffered saline or increasing concentrations of conjugate. 160μl of 500μM S-2238 (Chromagenix) was added to each well and allowed to incubate at 37°C. After 20 min the reaction was stopped by the addition of 67μl of 20% acetic acid and the optical density at 405nm was measured. Results:

Conclusion:

Targeted anticoagulant reagent inhibits the activity of thrombin on the chromogenic substrate in a dose dependant manner.

The Fab fragment of the antibody or the antibody fragment alone has no such inhibitory activity (not shown). 3. Thrombin clotting time inhibition assay

This assay measures the ability of the reagent to inhibit the activity of thrombin to form clots in plasma. Thrombin clotting time was measured as described in "Practical Haematology", Dacie JV and Lewis SM eds, Churchill Livingstone Publishers Edinburgh UK 1 84 pp 218-219.

Briefly 0.2ml of normal plasma was incubated with 0.1ml of 10 NIH U/ml thrombin in the presence or absence of reagent. The time for clot formation was noted.

Conclusion:

Both PPACK alone and the Fab-PPACK conjugate were able to prolong the thrombin dependant clot formation in normal plasma. On a molar basis the conjugate appears to be a more effective thrombin inhibitor. 4. Procoagulant assay

SUBSTITUTE SHEET This assay measures the ability of clot bound targeted anticoagulant reagent to inhibit clot extension. Preparation of plasma and whole blood clots and measurement of thrombin-dependant clot associated procoagulant activity was carried out essentially as described by Eisenberg et al J. Clin. Invest. 91 1877- 1883 1993 (incorporated herein by cross reference). Briefly plasma clots were developed in barium-adsorbed plasma repeated with CaCl₂ to a final concentration of 25mM. The clot formed was washed extensively and then incubated in a solution of citrated plasma containing variable concentrations of inhibitor or buffer as a control. At selected time intervals; the clot was removed, washed with buffer and transferred to a fresh recalcified pool of citrated plasma at 37°C. Plasma concentration of fibrinopeptide A (FPA) as a measure of thrombin activity was monitored over time. Results:

Conclusion:

Clots incubated with targeted anticoagulant reagent are able to inhibit the clot associated procoagulant activity.

Example 2 Synthesis of DD-3B6/22 (Fab) -CYS-7 and DD-3B6/22 (Fab) - CYS-20 hirulog derivatives

(a) Preparation of Antibody fragments

The Fab-SH fragment of DD-3B6/22 was prepared by the method described by John eL_a] Throm. Res. 58 273-281 1990 except that the Fab-TNB derivative was prepared instead of the Fab-SH derivative. Briefly purified antibody was digested with pepsin and F(ab')₂ fragments purified. Fab-SH is prepared from F(ab')₂ by mild reduction with beta mercaptoethylamine and reaction with lO M Ellman's reagent (5,5' Dithio-bis[-2-nitrobenzoic acid]). The derivative was purified by gel filtration chromatography in a 0.2M phosphate buffer at pH 7.4.

(b) Synthesis of synthetic peptides

Peptides were prepared by the Merrifield procedure (Hodges and Merrifield Anal.Biochem. 65 7241 195) essentially as described by Kemp et al Science 241 1352- 1354 1988. The peptides prepared were based on the hirulog structure (Manganore et al Biochemistry 29 7095-7101 1990), but with addition of a cysteine residue to enable conjugation of the peptide to the hinge region of the

antibody by the method outlined below.

Two hirulog variants were synthesised. Native hirulog

D-PhePRPGGGGGAVERYLKQQLLGIWG Cys 7 derivative

D-PhePRPGGCYSGGAVERYLKQQLLGIWG Cys 20 derivative D-PhePRPGGGGGAVERYLKQQLLGIWGCYS

(c) Conjugation

Fab-TNB (-2mg/ml) was a incubated with the cysteine containing hirulog derivatives at a 1:20 molar ratio. The reaction mixture turned yellow in colour after 2min. The reaction was then stopped with the addition of 50μl of 300mM iodoacetamide. This was wrapped in foil and incubated for 30min at room temperature. Unreacted hirulog derivative was removed from conjugate by gel filtration chromatography on a column of Ultragel Ac44 in PBS.

(d) Activity measurements

The thrombin clotting assay was used to establish that the conjugate contained anticoa ulant activit .

Conclusions:

The Fab-hirulog derivatives possessed potent thrombin inhibitor activity. They were more effective than the native cysteine containing hirulog peptides. Example 3 Isolation and Characterisation of Genes Encoding Antibody Fragments

A strategy utilising the polymerase chain reaction (PCR) to identify segments of the genes encoding the antibody and to add linkers and peptide epitopes to those segments to form single chain, antibody-based reagents may adopted, (a) Messenger RNA (mRNA) may prepared from a monoclonal cell line

SUBSTITUTE SHEET 19

((3B6/22), which binds to the cross-linked fibrin product, D-dimer.

From this mRNA template, single and double stranded complementary DNA (ss- and ds-cDNA respectively) may be synthesised. The ds-cDNA may be cloned into lambda-gtlO arms and packaged into a phage library. The heavy chain clone gamma-M/l.T (Tyler et al., Proc. Natl. Acad. of Sci., 1982; 79: 2008-2012) and the light chain clone pH76-kapρa-10 (Adams et al., Biochem., 1980; 19: 2711-2719) were used to source ds-DNA inserts for the screening of the gtlO library. Positive clones were amplified, and the positive insert cDNA sub-cloned into pUC18. To determine the N-terminal sequence of the 3B6/22 light chain, N- terminal residues from the intact 3B6/22 Ig may be removed sequentially by Ed an degradation in an Applied Biosystems sequencer. The N-terminal sequence of the light chain can be thus deduced from the mixed sequence by comparison with the sequence of the cloned heavy chain. The variable region of the kappa light chain not present in gtlO library clones may be amplified by PCR from ss-cDNA. The redundant, forward (sense) primer N960 may be designed from the deduced amino terminal kappa sequence.

Common usage triplet codes found in IgG genes may be used in this procedure - the reverse (antisense) primer N852 was based on the kappa constant region beginning at nucleotide 337, as described by Chiang et al, Biotechniques, 1989 7, 360-366. The amplification reaction yields a single product which when cloned and sequenced shows a coding sequence consistent with a kappa light chain and identical at the 3' end with the overlapping kappa clone K4 AC 1.

(b) A single chain antibody fragment (scFv) was constructed from the 3B6/22 molecule as follows:

₍i₎ Amplification and cloning of the heavy-chain variable domain.

1. The amplification of genes and synthesis of DNA sequences in these genes for cloning were performed by application of the polymerase chain reaction (PCR) as follows. A typical reaction (100 μl volume) contained 1-10 ng of template DNA, 1-2 U of thermostable DNA polymerase, 5 μl of a mixed A,C,G and T deoxynucleotide (dNTP solution) with each base at a concentration of 2 mM, 5 μl of each primer (10 pMolar each) and, where used, 1 μl of internal primers (0.05-0.1 pM), Mg⁺ + to a final concentration of 1-5 mM, a reaction buffer appropriate for the particular polymerase chosen (supplied by manufacturer), and water to 100 μl. The reactants were mixed and

SUBSTITUTE SHEET 20

overlaid with paraffin oil (Sigma biochemical) and subjected to 25-30 cycles in a thermal cycler (Corbett Research, Australia). The general strategy for each of the examples consisted of a denaturation step at 93°C (usually 1 minute), an annealing step between 50 and 65°C for 1 minute and an extension step at 72°C for 2 minutes. Annealing temperatures were adjusted as required to give final product.

2. Olignucleotide primers were synthesised to amplify the variable domain (Vb) from the heavy chain cDNA clone gam a-l.l.la, and add a Thai restriction site at the 5' end and a Bst E2 - peptide epitope -Eco Rl sequence at the 3' end. The product was digested with Thai and Eco Rl, and cloned into the Msc 1/Eco Rl-digested expression vectors pPOW (Power et al, Gene, 1992; 113: 95-99) and transformed into E coli strains TG-1 (Gibson TJ, 1984, "Studies on the Epstein-Barr virus genome", PhD thesis, Cambridge University, England) and 14ρl respectively. 3. Transformed E coli were screened for the presence of plasmids carrying the Vh gene fragment and selected clones (hereafter referred to as pP3B6Vh pr [G3B6Vh) sequenced to check the integrity of the cloning procedure. ₍ii₎ Amplification and cloning of the light chain variable domain and construction of a composite single-chain antibody domain (scFv⁾. 1. Oligonucleotide primers were synthesised to simultaneously amplify (as in i(l) above) and add to the cloned light chain gene, in a PCR amplifⁱcation reaction, a Bst E2 site and a sequence coding for a linker (amino acid sequence - (GGGGS)3-) at the 5' end and a peptide epitope-Eco Rl sequence at the 3' end.

2. The product was digested with Bst E2 and Eco Rl and cloned into the Bst E2,/Eco Rl digested plasmids described as pP3B6Vh pr pG3B6Vh above and transformed into the E coli strains described in i above.

3. Transformed E coli were screened for the presence of the V_h and Vi sequences and selected clones (hereinafter referred to as _PP3B6scFv or _PG3B6scFv) were partly sequenced to check the integrity of the cloning product.

4. Oligonucleotide NSfil5 was used to add a Sfi 1 restriction site to the 5' end of the scFv gene construct in _PP3B6scFv, and (NVKFORNOT) was used to add a Not 1 site to the 3' end of the scFv domain. The product was digested with these restriction enzymes and cloned into the likewise restricted vector pHFA, a derivative of the vector pHEN, and the construct transferred into the E coli strain HB2151, a non-supE phenotypic strain. Clones, referred to as pHFA3B6, were identified by hybridisation and were tested for expression of a

SUBSTITUTE SHEET scFv-peptide fusion the peptide being that recognised by an antibody to the

PPACK peptide.

(c) Expression of recombinant scFv

Recombinant E coli were grown in lOmls of 2X-YT medium (10 gm yeast extract, 19 gm tryptone, 5 gm NaCl per litre overnight. Overnight cultures were diluted to an ODgrjO 0.5 into 100 ml of fresh medium and grown to mid- log phase (OD600 0.5-0.9). Cultures of pG3B6scFv and pHFA 3B6 were induced upon the addition of isopropyl-B-D-thiogalactopyranoside; (IPTG; Sigma 15502) to a concentration of ImM, and growth continued at 30-37°C as required for a further 4 hours. pHFA 3B6/22 cultures were maintained at 30°C for up to 24 hours post-induction.

Cultures of pP3B6scFv were induced by raising the temperature of the medium to 42°C for 15 minutes, and the incubation was continued at 37°C for 2-4 hours. Levels of recombinant proteins in the E coli periplasmic space and the culture supernatant in each case were assayed by ELISA, Western blots of SDS- PAGE gels, and by the agglutination assay.

SUBSTITUTE SHEET

Claims

1. A clot-targeting, anticoagulant molecule comprising a clot-targeting binding molecule coupled to an anticoagulant.

2. The clot-targeting, anticoagulant molecule of claim 1 further comprising a thrombolytic coupled to the clot-targeting binding molecule.

3. The clot-targeting, anticoagulant molecule of claim 1 further comprising a thrombolytic coupled to the anticoagulant.

4. The clot-targeting, anticoagulant molecule of claim 1 wherein the clot- targeting binding molecule is selected from the group consisting of an antibody, a F(ab')₂ fragment of an antibody and a Fab' fragment of an antibody.

5. The clot-targeting, anticoagulant molecule of claim 2 wherein the clot- targeting binding molecule is an antibody, a F(ab')₂ fragment of an antibody and a Fab' fragment of an antibody.

6. The clot-targeting, anticoagulant molecule of claim 3 wherein the clot- targeting binding molecule is an antibody, a F(ab')₂ fragment of an antibody and a Fab' fragment of an antibody.

7. The clot- targeting, anticoagulant molecule of claim 1 wherein the clot- targeting binding molecule is selected from the group consisting of antibodies to cross-linked fibrin, antibodies to fibrin monomer, antibodies to neo-epitopes of fibrin, D-dimer monoclonal antibody DD-3B6/22, monoclonal antibody DD- 1C3/108, monoclonal antibody DD-1D2/48, fibrin specific antibodies, fibrin antifibrin antibody MA 59D8, monoclonal antibody P256, F(ab')₂ fragments of MAb P256, Fab' fragments of MAb P256, bifunctional antibodies which bind to fibrin, bifunctional antibodies which bind to activated platelets, activated platelet binding antibodies, activated platelet binding molecules and an antigen- binding fragment of a clot-targeting antibody.

8. The clot-targeting, anticoagulant molecule of claim 2 wherein the clot- targeting binding molecule is selected from the group consisting of antibodies to cross-linked fibrin, antibodies to fibrin monomer, antibodies to neo-epitopes of fibrin, D-dimer monoclonal antibody DD-3B6/22, monoclonal antibody DD- 1C3/108, monoclonal antibody DD-1D2/48, fibrin specific antibodies, fibrin antifibrin antibody MA 59D8, monoclonal antibody P256, F(ab')2 fragments of MAb P256, Fab' fragments of MAb P256, bifunctional antibodies which bind to fibrin, bifunctional antibodies which bind to activated platelets, activated platelet binding antibodies, activated platelet binding molecules and an antigen- binding fragment of a clot-targeting antibody.

9. The clot-targeting, anticoagulant molecule of claim 3 wherein the clot-

SUBSTITUTE SHEET targeting binding molecule is selected from the group consisting of antibodies to cross-linked fibrin, antibodies to fibrin monomer, antibodies to neo-epitopes of fibrin, D-dimer monoclonal antibody DD-3B6/22, monoclonal antibody DD- 1C3/108, monoclonaf antibody D- lD2/48, fibrin specific antibodies, fibrin antifibrin antibody MA^~ 59D8, monoclonal antibody P256, F(ab')₂ fragments of MAb P256, Fab' fragn.^nts of MAb P256, bifunctional antibodies which bind to fibrin, bifunctional antibodies v.mch bind to activated platelets, activated platelet binding antibodies, activated platelet binding molecules and an antigen- binding fragment of a clot-targeting antibody.

10. The clot-targeting, anticoagulant molecule of claim 1 wherein the clot- targeting binding molecule is selected from the group consisting of MAb DD- 3B6/22, MAb DD-1D2/48 and MAb DD1C3/108.

11. The clot-targeting, anticoagulant molecule of claim 2 wherein the clot- targeting binding molecule is selected from the group consisting of MAb DD- 3B6/22, MAb DD-1D2/48 and MAb DD1C3/108.

12. The clot-targeting, anticoagulant molecule of claim 3 wherein the clot- targeting binding molecule is selected from the group consisting of MAb DD- 3B6/22, MAb DD-1D2/48 and MAb DD1C3/108.

13. The clot-targeting, anticoagulant molecule of claim 1 wherein the anticoagulant is selected from the group consisting of hirudin, C-terminal hirudin peptide, heparin, coumarin, PPACK (d-phe-pro-arg- chloromethylketone), DuP 714 (ac-d-phe-pro-boroarginine), D-Phe P RP G G G GGAVERYLKQQLLGIWG,D-PhePRPGGCYSGGAVERYL KQQLLGIWG,D-PhePRPGGGGGAVERYLKQQLLGIWG CYS, agratropin (2R, 4R)-4- methyl -1- [N2 -(3-methyl-l,2,3,4,-tetrahydro -8- quinolinesulphonyl)-L- arginyl]-2- piperidinecarboxylic acid monohydrate, tick anticoagulant peptide, extrinsic pathway inhibitor, tissue pathway inhibitor, fibrinogen receptor antagonists, cyclo(S,S)-N -acetyl-Cys-(N^α-methyl)Arg- Gly-Asp-Pen NH₂, cyclo(S,S)-(2-mercapto) benzoyl-(N^α-methyl)Arg-Gly-Asp- (2-mercapto),

Ac-Cys-(NMe)Arg-Gly-Asp-Pen-NH2 Ac-Cys-Asn-Dtc-Amf-Gly-Asp-Cys-OH I I ,or I I

Dtc=4,4'Dimethylthiazolidine-5-carboxylicacid Amf=para - aminomethylphenylalanine

factor Xa inhibitor, the prothrombinase complex inhibitor, vitamin-K-dependent

SUBSTITUTE SHEET gamma carboxylation of prothrombin inhibitor, factor X inhibitor, factor VII inhibitor, factor V inhibitor, factor VIII inhibitor, and factor IX inhibitor.

14. The clot-targeting, anticoagulant molecule of claim 10 wherein the anticoagulant is selected from the group consisting of hirudin, C-terminal hirudin peptide, heparin, coumarin, PPACK (d-phe-pro-arg- chloromethylketone), DuP 714 (ac-d-phe-pro-boroarginine), agratropin (2R, 4R)-4- methyl -1- [N2 -(3-methyl-l,2,3,4,-tetrahydro -8-quinolinesulphonyl)-L- arginyl]-2- piperidinecarboxylic acid monohydrate, tick anticoagulant peptide, extrinsic pathway inhibitor, tissue pathway inhibitor, fibrinogen receptor antagonists, D-PhePRP GGGGGA VER YLKQQLLGI WG, D-PheP RP GGCYS GG A VER YLKQ QLLGI WG,D-PhePRP GGGGGA V E R Y L K Q Q L L G I W G CYS, cyclo(S,S)-N^α-acetyl-Cys-(N^α- methyl)Arg-Gly-Asp-Pen NH₂, cyclo(S,S)-(2-mercapto) benzoyl-(N - methyl)Arg-Gly-Asp-(2-mercapto),

Ac-Cys-(NMe)Arg-Gly-Asp-Pen-NH2 Ac-Cys-Asn-Dtc-Amf-Gly-Asp-Cys-OH

I I ,or I I

Dtc=4,4'Dimethylhiazolidine-5-carboxylicacid Amf=para - aminomethylphenylalanine

factor Xa inhibitor, the prothrombinase complex inhibitor, vitamin-K-dependent gamma carboxylation of prothrombin inhibitor, factor X inhibitor, factor VII inhibitor, factor V inhibitor, factor VIII inhibitor, and factor IX inhibitor.

15. The clot-targeting, anticoagulant molecule of claim 11 wherein the anticoagulant is selected from the group consisting of hirudin, C-terminal hirudin peptide, heparin, coumarin, PPACK (d-phe-pro-arg- chloromethylketone), DuP 714 (ac-d-phe-pro-boroarginine), agratropin (2R, 4R)-4- methyl -1- [N2 -(3-methyl-l,2,3,4,-tetrahydro -8-quinolinesulphonyl)-L- arginyl]-2- piperidinecarboxylic acid monohydrate, tick anticoagulant peptide, extrinsic pathway inhibitor, tissue pathway inhibitor, fibrinogen receptor antagonists, D-PhePRP GGGGGA VER YLKQ QLLGI WG,D-PheP RP GGCYS GG A VER YLKQQLLGI WG,D-PhePRP GGGGGA V E R Y L K Q Q L L G I W G CYS, cyclo(S,S)-N^α-acetyl-Cys-(N^α- methyl)Arg-Gly-Asp-Pen NH₂, cyclo(S,S)-(2-mercapto) benzoyl-(N^α- methyl)Arg-Gly-Asp-(2-mercapto),

Ac-Cys-(N e)Arg-Gly-Asp-Pen-NH2 Ac-Cys-Asn-Dtc-Amf-Gly-Asp-Cys-OH I I ,or I I

16. The clot-targeting, anticoagulant molecule of claim 12 wherein the anticoagulant is selected from the group consisting of hirudin, C-terminal hirudin peptide, heparin, coumarin, PPACK (d-phe-pro-arg- chloromethylketone), DuP 714 (ac-d-phe-pro-boroarginine), agratropin (2R, 4R)-4- methyl -1- [N2 -(3-methyl-l,2,3,4,-tetrahydro -8-quinolinesulphonyl)-L- arginyl]-2- piperidinecarboxylic acid monohydrate, tick anticoagulant peptide, extrinsic pathway inhibitor, tissue pathway inhibitor, fibrinogen receptor antagonists, D-Phe PRPGGGGGAVERYLKQQLLGIWG, D-Phe P RP GGCYS GG A VER YLK Q QLL GI W G, D-PhePRP GGGGGA V E R Y L K Q Q L L G I W G CYS, cyclo(S,S)-N^α-acetyl-Cys-(N^α- methyl)Arg-Gly-Asp-Pen NH₂, cyclo(S,S)-(2-mercapto) benzoyl-(N^α- methyl)Arg-Gly-Asp-(2-mercapto),

Ac-Cys-(N e)Arg-Gly-Asp-Pen-NH2 Ac-Cys-Asn-Dtc-Amf-Giy-Asp-Cys-OH

I I ,or 1 I

Dtc=4,4'Dimethytthiazolidine-5-carboxylicacid Amf=para - aminomethylphenylalanine

factor Xa inhibitor, the prothrombinase complex inhibitor, vitamin-K-dependent gamma carboxylation of prothrombin inhibitor, factor X inhibitor, factor VII inhibitor, factor V inhibitor, factor VTII inhibitor, and factor IX inhibitor.

17. The clot-targeting, anticoagulant molecule of claim 2 wherein the thrombolytic is selected from the group consisting of urokinase, scuPA streptokinase and tPA.

18. The clot-targeting, anticoagulant molecule of claim 3 wherein the thrombolytic is selected from the group consisting of urokinase, scuPA streptokinase and tPA.

19. The clot-targeting, anticoagulant molecule of claim 10 wherein the thrombolytic is selected from the group consisting of urokinase, scuPA streptokinase and tPA.

SUBSTITUTE SHEET

20. The clot-targeting, anticoagulant molecule of claim 11 wherein the thrombolytic is selected from the group consisting of urokinase, scuPA streptokinase and tPA.

21. The clot-targeting, anticoagulant molecule of claim 12 wherein the thrombolytic is selected from the group consisting of urokinase, scuPA streptokinase and tPA.

22. The clot-targeting, anticoagulant molecule of any one of claims 1 to 3 further comprising an anti growth factor coupled to a molecule selected from the group consisting of the clot-targeting binding molecule, the anticoagulant and the thrombolytic.

23. A pharmaceutical composition for treatment of a clot in a mammal, comprising a clot-targeting, anticoagulant molecule of claim 1 together with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

24. A pharmaceutical composition for treatment of a clot in a mammal, comprising a clot-targeting, anticoagulant molecule of any one of claims 2 to 21 together with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

25. A method of treating a clot in a mammal, comprising administering to a mammal requiring such treatment an effective amount of a clot-targeting, anticoagulant molecule of any one claims 1 to 21 or a composition of claim 23.

26. A method of treating a clot in a mammal, comprising administering to a mammal requiring such treatment an effective clot treating amount of a clot- targeting binding molecule and an anticoagulant molecule capable of being bound by the clot-targeting, binding molecule in vivo.

27. A kit for treating a clot in a mammal, comprising (a) a clot-targeting, binding molecule; and (b) an anticoagulant capable of being bound by the clot- targeting, binding molecule in vitro and/or in vivo.

28. The kit of claim 27 further comprising a pharmaceutically acceptable carrier, diluent and/or excipient.

29. The clot-targeting, anticoagulant molecule of any of claims 1 to 3 wherein the clot-targeting binding molecule is selected from the group consisting of a fibrin specific antibody, an activated platelet specific antibody, a F(ab')₂ fragment of a fibrin specific antibody, a Fab' fragment of a fibrin specific antibody, a F(ab')₂ fragment of an activated platelet specific antibody and a Fab' fragment of an activated platelet specific antibody.

SUBSTITUTE SHEET