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WO2006061650A2 - Procedes, produits et utilisations relatifs a des plaquettes et/ou la vasculature - Google Patents

Procedes, produits et utilisations relatifs a des plaquettes et/ou la vasculature Download PDF

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Publication number
WO2006061650A2
WO2006061650A2 PCT/GB2005/004764 GB2005004764W WO2006061650A2 WO 2006061650 A2 WO2006061650 A2 WO 2006061650A2 GB 2005004764 W GB2005004764 W GB 2005004764W WO 2006061650 A2 WO2006061650 A2 WO 2006061650A2
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WIPO (PCT)
Prior art keywords
agent
gpvi
binding
acid sequence
vitronectin
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PCT/GB2005/004764
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WO2006061650A3 (fr
Inventor
Götz Münch
Andreas Bültmann
Oliver Vimpany Arnold Boucher
Suresh Babubhai Chahwala
Meinrad Gawaz
Martin Ungerer
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Trigen GmbH
Trigen Ltd
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Trigen GmbH
Trigen Ltd
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Priority claimed from US11/009,106 external-priority patent/US7531178B2/en
Application filed by Trigen GmbH, Trigen Ltd filed Critical Trigen GmbH
Priority to US11/792,857 priority Critical patent/US20090130021A1/en
Priority to EP05849048A priority patent/EP1824979A2/fr
Publication of WO2006061650A2 publication Critical patent/WO2006061650A2/fr
Publication of WO2006061650A3 publication Critical patent/WO2006061650A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present disclosure relates in some aspects to agents which can interfere with the binding of GPVI to various components.
  • Methods of treating disorders or diseases which involve pathological, dysfunctional or non-pathological interactions of GPVI and/or the vasculature are included in the present disclosure.
  • the disclosure also relates to uses of agents for the prevention or therapy of disorders which directly or indirectly involve platelets, as well as other subject matter.
  • Platelet adhesion to the endothelium plays a crucial role in the pathophysiology of reperfusion, sepsis, and cardiovascular diseases (1,2,3,4). Endothelial cell dysfunction allows platelet adhesion even without exposure of extracellular matrices (1,5). Similar to the recruitment of leukocytes 6 , the adhesion of platelets to the endothelial surface is a multistep process, in which platelets are tethered to the vascular wall, followed by subsequent firm adhesion of the platelets. Whereas the adhesion receptors involved in platelet attachment to the subendothelial matrix have been well defined 1 ' 5 , the molecular basis of the regulation of the interaction between platelets and the intact vascular endothelium are incompletely understood.
  • GPVI-collagen interaction is a major trigger of platelet activation and primary Hemostasis; GPVI binding to collagen results in platelet signaling and activation of ⁇ i- and ⁇ 3 -integrins, a critical step for stable platelet adhesion to extracellular matrices 10 .
  • Glycoprotein VI is a 60-65 kDa type I transmembrane glycoprotein, which belongs to the immunoglobulin superfamily (Clemetson, J. M., et al (1999) J Biol.Chem. 274, 29019-29024; Jandrot- Perrus, M., Busfield, et a/ (2000). Blood '96, 1798-1807; Gibbins, J. M., et a/ (1997). FEBS Lett. 413, 255-259; Zheng, Y. M., et a/ (2001). J Biol.Chem.
  • Platelets deficient in GPVI show loss of collagen-induced adhesion and aggregation in vitro (Sugiyama, T., et a/ (1987) Blood 69, 1712-1720).
  • function blocking anti-GPVI monoclonal antibodies attenuate ex vivo platelet aggregation in response to collagen and collagen-related peptide (CRP), which mimics collagen triple helix (Sugiyama, T., Ishibashi, T., Okuma, M. (1993) IntJ.Hematol. 58, 99-104; Schulte, V., ef a/(2001) J Biol.Chem. TJS 1 364-368).
  • GPVI may in fact strictly be required in the process of platelet recruitment under physiological shear stress following vascular injury.
  • both the inhibition or absence of GPVI virtually abolished platelet-vessel wall interactions and platelet aggregation, thereby indicating GPVI as the major determinant of arterial thrombus formation (Massberg, S., eta/. (2003) J.Exp.Med. 197, 41-49).
  • Activated endothelial cells express a variety of adhesion receptors (e.g. ⁇ 3 -integrin, P-selectin, ICAM- 1) (13-21) and molecules (e.g. von Willebrand factor (vWF), vitronectin (Vn), fibronectin (Fn)) (22- 24) that have been shown to play a role in platelet/endothelium adhesion. Most of these adhesion molecules are presented in substantial amounts on the luminal aspect of endothelial cells.
  • adhesion receptors e.g. ⁇ 3 -integrin, P-selectin, ICAM- 13-21
  • molecules e.g. von Willebrand factor (vWF), vitronectin (Vn), fibronectin (Fn)
  • Fibronectin is a large multidomain glycoprotein found in connective tissue, on cell surfaces, and in plasma and other body fluids. It interacts with a variety of macromolecules including components of the cytoskeleton and the extracellular matrix, fibrinolytic, acute phase and complement systems, and with cell-surface receptors on a variety of cells including fibroblasts, neurons, phagocytes and bacteria. It is composed of two peptide chains of approximately 275,000 molecular weight which are linked through two interchain disulfide bonds at the COOH-terminal end of the molecule.
  • Vitronectin is a mixture of two monomeric glycoproteins (65 and 75 kD) present in blood and the extracellular matrix (ECM) of many tissues. Vitronectin and fibronectin are the two major adhesive proteins in plasma and serum. Like many other adhesion molecules, vitronectin binds to cells through an interaction of the Arg-Gly-Asp (RGD) sequence in its cell binding domain with vitronectin-specific cell surface receptors, such as integrins ⁇ v ⁇ 3 and ⁇ v ⁇ .
  • RGD Arg-Gly-Asp
  • Platelets have been shown to be involved in the development of thromboembolic complications of advanced atherosclerotic lesions. However, there is also evidence to suggest that platelets are also involved in the initiation and progression of atherosclerotic plaques. However, the molecules involved and roles thereof have not yet been elucidated in great detail.
  • the present application discloses amongst other things products, for example molecules, which bind to or interact with plural vascular domains or binding sites.
  • Each domain may comprise a different ligand, for example adhesion ormatrix protein, from each other domain.
  • at least one of the domains binds to or interacts with GPVI.
  • all the binding sites bind to or interact with GPVI.
  • All the domains or binding sites may simultaneously be exposed on a dysfunctional or diseased area of blood vessel wall, for example an atherosclerotic or inflamed area.
  • Particular embodiments relate to products which are capable of binding to a dysfunctional or inflamed area of blood vessel wall which does not include an atherosclerotic plaque or lesion.
  • an agent which is capable of binding to a plurality of GPVI-binding sites, optionally a plurality of types of GPVI-binding sites, for example of a dysfunctional, inflamed or atherosclerotic blood vessel area, for example a human blood vessel area.
  • the agent optionally has one or more of the following three capabilities:
  • thrombus formation caused by induction of arterial lesions in the mouse by at least 70% as compared with a control administered with an Fc polypeptide, the relative inhibitions of thrombus formation being determined by measurement of the en face thrombus size as a percentage of an artery wall area (e.g. a human artery wall area) placed on a surface for the purpose of said measurement;
  • an artery wall area e.g. a human artery wall area
  • the agent may be capable of inhibiting the binding of GPVI to collagen and a protein selected from fibronectin, vitronectin and combinations thereof.
  • GPVI GPVI-fibronectin
  • GPVI vitronectin GPVI vitronectin
  • GPVI-fibronectin- vitronectin GPVI-fibronectin- vitronectin.
  • GPVI binding to fibronectin in platelet adhesion a role of GPVI binding to vitronectin in platelet adhesion.
  • the present invention discloses uses of agents which prevent or inhibit the interaction of GPVI with the novel ligands. Methods of treatment utilising the agents are disclosed. Assays utilising the novel ligands of GPVI are included in the disclosure.
  • the present specification discloses the role of GPVI interaction with vitronectin and/or fibronectin in the mediation of platelet adhesion to endothelium in vitro and in vivo.
  • the present specification discloses the role of GPVI interaction with exposed sub-endothelial matrix and/or adhesion proteins other than collagen. This specification contains data demonstrating that inhibition of GPVI binding to fibronectin and/or vitronectin reduces platelet/endothelium adhesion and, as a consequence, vascular remodelling during atherogenesis in vivo.
  • inhibitors of the interaction between GPVI, on the one hand, and fibronectin and/or vitronectin on the other will have use in the prevention of atherogenesis and the progression of atheroscleroma.
  • Such inhibitors will have use in preventing initiation of atheroscleroma or its progression, inter alia, in patients free of active atherosclerotic lesions and/or in chronic therapy.
  • the agent has one or more of the three capabilities enumerated above.
  • the agent may additionally be capable of inhibiting GPVI interaction with collagen.
  • the disclosure includes, but is not necessarily limited to, embodiments in which at least one of collagen, fibronectin and vitronectin are human.
  • the agents of the disclosure are not a fusion protein described in WO 03/104282. Accordingly, the disclosure includes embodiments in which the agent is not a fusion protein comprising: a) an extracellular domain of GPVI or a variant thereof that is functional for binding to collagen; and b) an Fc domain of an immunoglobulin or a functional conservative part thereof, the extracellular domain and the Fc domain being fused via a linker characterised by the amino acid sequence Gly-Gly-Arg.
  • agents of the disclosure comprise an amino acid sequence of or comprised in an extracellular domain of GPVI, or a variant thereof that is functional for binding to at least one protein selected from fibronectin and vitronectin and typically is functional for binding to collagen.
  • agents for example antibody products, which comprise domains which recognize collagen, particularly collagen I and/or collagen III, and one or both of fibronectin and vitronectin.
  • the agents typically bind to such domains.
  • agents which comprise: a first portion capable of binding to an intravascular GPVI-binding domain; and a second portion which is capable of binding to a site other than a GPVI-binding domain and/or comprises a therapeutic or diagnostic moiety.
  • the second portion is capable of binding to a binding site for a platelet-bound ligand other than GPVI, for example GPIIb/IIIa, GPIa/IIa, GPIV, GPIc/IIa, GPIb/IX.
  • the second portion confers inhibitor activity against platelet adhesion, platelet aggregation or coagulation.
  • the second portion may comprise an imaging agent, for example a radio-imaging agent, e.g. Tc ⁇ 9m .
  • the agent may comprise a plurality of said second portions.
  • the first portion may capable of binding to one, two or three proteins selected from collagen, e.g. collagen I and collagen III, vitronectin and fibronectin.
  • the first portion may capable of binding to a plurality of intravascular GPVI-binding domains, each domain optionally being of a different type, for example it may be capable of binding to collagen and to one or both of vitronectin and fibronectin.
  • the first portion may be a distributed portion having spaced apart regions having different binding functions; to look at this concept another way, the agent may contain a plurality of first portions.
  • agents which are functional to bind to collagen and to one or both of vitronectin and fibronectin, and which further have another therapeutic or diagnostic function.
  • agents capable of interfering with a plurality of platelet-blood vessel interactions, of which at least one interaction involves GPVI; the interactions may include interaction with one or both of vitronectin and fibronectin.
  • compositions comprising an agent of the disclosure.
  • implantable, optionally intravascular, devices which in use expose or release a direct or indirect GPVI inhibitor or any agent disclosed herein, for example one which inhibits interaction between GPVI and a protein selected from collagen, vitronectin and fibronectin, and combinations thereof.
  • the devices have a coating or impregnant comprising such an agent.
  • Exemplary devices are selected from a stent, a vascular catheter, a vascular shunt, a balloon catheter, an autologous venous/arterial graft, a prosthetic venous/arterial graft and a guidewire.
  • a method of inhibiting or preventing restenosis, thrombosis, atherogenesis, atheroprogression, atherosclerosis, and/or vascular inflammation in a patient comprising implanting in said patient an intravascular device comprising a direct or indirect GPVI inhibitor adapted to be exposed and/or released when the device is implanted.
  • a particular subject matter of the disclosure is methods for the primary prophylaxis of cardiovascular disease, comprising administering an effective amount of an agent capable of interfering with interaction between GPVI and one or both of vitronectin and/or fibronectin, or of an agent comprising an amino acid sequence derived from an extracellular domain of GPVI.
  • methods for treating a patient suffering from, or at risk of suffering from, a disorder characterised by a pathological interaction between GPVI and fibronectin and/or vitronectin comprising administering an effective amount of an agent which inhibits interaction between GPVI and a protein selected from the group consisting of fibronectin, vitronectin and combinations thereof.
  • the disorder may be initiation of atherosclerosis.
  • a method for secondary prophylaxis of a patient who has suffered, or is at risk or suspicion of having suffered, an atherosclerotic event comprising administering to the patient an effective amount of an agent which inhibits interaction between GPVI and a protein, e.g. two or three proteins, selected from the group consisting of collagen, fibronectin, vitronectin and combinations thereof.
  • a protein e.g. two or three proteins, selected from the group consisting of collagen, fibronectin, vitronectin and combinations thereof.
  • the patient suffered, or is at risk or suspicion of having suffered a disorder selected from the group consisting of thrombosis, myocardial infarction, stroke, transient ischemic attack, occlusive peripheral vascular disease, occlusion of a peripheral artery and complications thereof.
  • Another method disclosed herein is one of treating a patient who has suffered a cardiovascular event comprising administration to the patient of an effective amount of a direct or indirect GPVI inhibitor for at least three months after said cardiovascular event.
  • the GPVI inhibitor may inhibit the interaction between GPVI and a protein selected from the group consisting of fibronectin, vitronectin, collagen and combinations thereof.
  • the GPVI inhibitor may be administered for a period of up to six months after said cardiovascular event.
  • the cardiovascular event may comprise rupture of atherosclerotic plaque. It may be thrombosis or myocardial infarction.
  • the specification discloses also the local administration to an actual or suspected site of an atherosclerotic disorder of an inhibitor of GPVI binding.
  • Such administration is useful in the treatment of a patient suffering from,- or suspected to be suffering from, an atherosclerotic disorder, e.g. an atherosclerotic plaque which may be ruptured.
  • the administration may be via a catheter.
  • a method for treating a patient having, or identified as having, at least one or a combination of risk factors for the formation of atherosclerotic lesions comprising administering an effective amount of an agent which inhibits interaction between GPVI and a protein selected from the group consisting of fibronectin, vitronectin and combinations thereof; • a method of treating a patient who has, or has been identified as having, one or more risk factors associated with developing atherosclerosis, said patient having not yet developed advanced atherosclerotic plaques, wherein said patient is considered to have a risk score of 45 or greater according to the PROCAM study, said method comprising administering an agent which inhibits binding of GPVI to a molecule selected from vitronectin, fibronectin and combinations thereof;
  • a method for preventing or retarding atherosclerotic cardiovascular disease in a patient who is not displaying clinical symptoms of atherosclerosis comprising administering to the patient an agent which inhibits binding of GPVI to a molecule selected from vitronectin, fibronectin and combinations , for example wherein the patient has a 10-year risk of fatal cardiovascular disease according to the SCORE project of at least 3%;
  • a method for the primary prevention of atherosclerotic cardiovascular disease in a patient comprising administering to the patient an agent which inhibits binding of GPVI to a molecule selected from vitronectin, fibronectin and combinations thereof;
  • GPVI to a molecule selected from vitronectin, fibronectin and combinations thereof.
  • a method for treating atherosclerosis in a patient comprising administering to the patient an agent capable of binding to a plurality of GPVI-binding sites at a frequency of once a day or less, e.g. of at most once every 48 hours.
  • an agent which inhibits GPVI interaction with fibronectin for example the agent is capable of inhibiting GPVI binding to, or association with fibronectin.
  • an agent which inhibits GPVI interaction with vitronectin for example the agent is capable of inhibiting GPVI binding to, or association with vitronectin.
  • agents which are capable of inhibiting GPVI binding to, or association with, both vitronectin and fibronectin include agents which are capable of inhibiting GPVI binding to, or association with, both vitronectin and fibronectin.
  • Agents of the disclosure may be capable of inhibiting GPVI binding to, or association with, one or more, or a combination of, collagen, vitronectin and fibronectin.
  • the disclosure also includes methods of treatment which use not a single entity having plural functions but plural entities, each entity having a different function or combination of functions from the other(s).
  • the functions of the entities may include, or be, inhibition of interactions of blood components and/or the vasculature, for example interactions involving any one or more of platelets, monocytes, matrix proteins e.g. collagen, and adhesion proteins e.g. fibronectin and/or vitronectin; such interactions are described in more detail hereinafter in various contexts and all described interactions are applicable to entity combinations.
  • entities may be mentioned antibodies and low molecular weight molecules.
  • the disclosure also includes more generally combination products and therapies involving a product of the disclosure, for example therapy in combination with another antithrombotic drug, particularly one have a mode of action not involving GPVI.
  • Figure 1 Platelet adhesion and aggregation following vascular injury of the common carotid artery in C57BL6/J mice in vivo, (a) Scanning electron micrographs of carotid arteries prior to (left panels) and 2 hrs after (right panels) vascular injury. Endothelial denudation induces platelet adhesion and aggregation, resulting in the formation of a platelet-rich (lower left) thrombus, (b) Platelet- endothelial cell interactions 5 min after vascular injury were investigated by in vivo fluorescence microscopy of the common carotid artery in situ (black columns). Animals without vascular injury served as controls (open columns).
  • the left and right panels summarize transient and firm platelet adhesion, respectively, of eight experiments per group. Platelets were classified according to their interaction with the endothelial cell lining as described 24 and are given per mm 2 of vessel surface. Mean ⁇ s.e.m., asterisk indicates significant difference compared to control, P ⁇ 0.05.
  • microphotographs show representative in vivo fluorescence microscopy images in control animals (upper panel) or following vascular injury (lower panel). White arrows indicate adherent platelets.
  • Figure 2 Inhibition of GPVI abrogates platelet adhesion and aggregation after vascular injury, (a) Platelet adhesion following vascular injury was determined by intravital videofluorescence microscopy. Fluorescent platelets were preincubated with 50 ⁇ g/ml anti-GPVI (JAQl) Fab fragments or control rat IgG. Platelets without mAb preincubation served as control. The left and right panels summarize transient and firm platelet adhesion, respectively.
  • FIG. 3 Platelet adhesion following endothelial denudation in GPVI-deficient mice, (a) JAQl- treated mice lack GPVI. Upper panels: Platelets from mice pretreated with irrelevant control IgG (left) or anti-GPVI (JAQl) (right) were incubated with FITC-labeled JAQl and PE-labeled anti-mouse CD41 for lOmin at room temperature and directly analyzed on a FACScanTM. A representative dot blot of 3 mice per group is presented.
  • FIG. 4 Platelet adhesion to the surface of collagen coated glass coverslips under physiological flow conditions was assessed ex vivo.
  • Left panel Platelets from mice pretreated with irrelevant control IgG immunoadhesin (control) (left) or anti-GPVI immunoadhesin (Fc-GP VI-nt) (right) were investigated for adhesion under physiological flow conditions. The number of platelets was assessed by FACS counting of the washed coverslips at the end of each experiment. Platelet tethering as the first step of platelet adhesion was assessed after 30 seconds and firm platelet adhesion after 5 min under flow conditions. The panels summarize transient and firm platelet adhesion in eight experiments per group. Mean ⁇ s.e.m., asterisk indicates significant difference compared to control IgG, P ⁇ 0.05.
  • FIG. 5 Interaction of Fc-GP VI-nt with collagen was monitored in an ELISA based assay.
  • Adhesion of the immunoadhesin - a GPVI-Fc fusion protein comprising a Gly-Gly-Arg linker and the FC part of an IgG to collagen coated plates with increasing concentrations of Fc-GP VI-nt (0.5 ⁇ g to 10 ⁇ g) was investigated.
  • the binding is visualised with a secondary antibody labelled with peroxidase directed to the Fc part of Fc-GP VI-nt. Peroxidase is finally detected by ELISA.
  • binding of Fc-GP VI-nt to collagen was monitored with sufficient affinity, which reached saturation at ⁇ g concentrations.
  • Figure 7 Amino acid sequence of - a GPVI-Fc fusion protein comprising a Gly-Gly-Arg linker: SEQ ID No: 1.
  • Figure 8 DNA-Sequence of - a GPVI-Fc fusion protein comprising a Gly-Gly-Arg linker: SEQ ID No. 2.
  • Bases 1 to 807 encode the extracellular domain of GP VI.
  • Bases 817 to 1515 encode the Fc part of the IgG.
  • Figure 9 Characterization of - a GPVI-Fc fusion protein comprising a Gly-Gly-Arg linker
  • Middle panel Immunoblotting of Fc, Fc-GPVI-nt, or human platelets using the anti-GPVI monoclonal antibody 5C4.
  • Bound Fc-GPVI-nt was detected by anti-Fc mAb antibody (dilution 1:10.000) and is given relative to the binding observed at 10_g/ml Fc-GPVI-nt.
  • (c f left panel) shows binding of Fc-GPVI-nt (20 ⁇ g/ml) to various substrates.
  • Binding of Fc-GPVI-nt to BSA (10_g/ml) or vWF (10_g/ml) is given as percentage of GPVI-dimer-binding to immobilized collagen. Binding of Fc- GPVI-nt did not occur to BSA or vWF, supporting the specificity of Fc-GPVI-nt binding. Mean ⁇ s.e.m., asterisk indicates significant difference compared to collagen, P ⁇ 0.05. (c, right panel) illustrates binding of Fc-GPVI-nt (20 ⁇ g/ml) or Fc (20 ⁇ g/ml) to immobilized collagen (10 ⁇ g/ml).
  • Fc-GPVI-nt (20 ⁇ g/ml) was preincubated for 10 min with different concentrations of soluble collagen.
  • the binding of the monomer and dimer was assessed on collagen type 1 coated ELISA plates. Increasing concentrations of the GPVI fusion proteins bond to collagen in a saturable manner. Here a Lineweaver Burke plot is demonstrated for affinity assessment (e). The affinity of the monomeric GPVI fusion protein was about 10 times lower compared to equimolar concentrations of the dimeric form Fc-GPVI-nt.
  • Fc-GPVI-nt inhibits CD 62 P activation on human platelets as a parameter of release of intracellular transmitter substances from alpha granules by increasing doses of collagen.
  • Human platelets were isolated from whole blood and incubated with anti-CD 62 antibodies labelled with PE. Fluorescence was determined in a Becton Dickenson FACS device. Representative histograms are shown. Increasing concentrations of collagen from 0 to 10 ⁇ g/ml induced a shift of fluorescence in the presence of the control Fc protein (100 ⁇ g/ml; blue line). In the presence of Fc-GPVI-nt (100 ⁇ g/ml; red line), the shift of fluorescence and hence CD 62 P activation was markedly inhibited.
  • FIG 11 Specific inhibition of collagen-mediated platelet aggregation and release of endogenous transmitters from dense and alpha granules by Fc-GPVI-nt.
  • ATP release was measured simultaneously in the same probes after incubation with control Fc (80 ⁇ g/ml) or Fc-GPVI-nt (80 ⁇ g/ml). The amount of ATP release is given in % of controls without fusion protein,
  • PDGF release was determined in human platelets with an ELISA system specific for human PDGF under basal conditions and after collagen (20 ⁇ g/ml) stimulation. Preincubation with control Fc had no significant effect on PDGF release from collagen-stimulated platelets, whereas Fc- GPVI-nt (100 ⁇ g/ml) reduced the PDGF release significantly. Inhibition of PDGF release did not occur in unstimulated platelets.
  • Fc-GPVI-nt inhibits platelet adhesion to immobilized collagen under flow conditions.
  • Human platelets (2xl0 8 cells/ml) were isolated from whole blood. Plates were coated with immobilized collagen (10_g/ml) or vWF (10_g/ml). Platelet adhesion to the coated plates was determined in a parallel plate flow chamber in the presence of Fc-GPVI-nt or Fc lacking the extracellular GPVI domain (200 ⁇ g/ml). Inhibition of platelet adhesion by Fc-GPVI-nt is given in % of control (Fc control).
  • Fc-GPVI-nt has favourable pharmacokinetics with a prolonged plasma half life after intraperitoneal injection in mice in vivo. Blood concentrations of Fc-GPVI-nt were determined with specific anti-Fc antibodies and ELISA.
  • FIG. 15 Effects of Fc-GPVI-nt on platelet adhesion and aggregation in vivo
  • FIG. 16 Fc-GPVI-nt significantly attenuates atheroprogression in apo e -/- knockout mice in vivo.
  • Apo e -/- mice were treated with Fc-GPVI-nt (4 ⁇ g/g) or control Fc (4 ⁇ g/g) intraperitoneally for 4 weeks twice weekly.
  • Atheroprogression was investigated post mortem after sudan red staining of the large vessels to visualise atheroma and plaque formation.
  • extensive plaque formation of carotid artery preparations was indicated by the red colour in particular in the branching region.
  • In Fc-GPVI-nt treated animals atherosclerosis was almost completely abolished in carotid arteries of apo e -/- mice.
  • FIG. 17 Freshly isolated platelets from patients suffering from diabetes mellitus show reduced expression of the fibrinogen receptor (CD61, top) and increased expression of the Fc receptor (CD32, middle) and therefore increased expression of GPVI. The correlation between CD32 expression and GPVI expression (detected by the specific monoclonal antibody 4C9) is shown on human platelets (bottom).
  • Figure 18 Amino acid sequence of a monomeric fusion protein based on Fc-GPVI-nt.
  • Binding assays using soluble GPVI-Fc (10 ⁇ g/ml) or control Fc (10 ⁇ g/ml) to immobilized bovine collagen type 1 (l ⁇ g/ml), human Vn (l ⁇ g/ml), human Fn (l ⁇ g/ml), and human vWF (l ⁇ g/ml) was performed as described 12 .
  • Bound GPVI-Fc was detected by anti-Fc mAb and is given relative to the binding over non-specific Fc control.
  • FIG. 21 In vivo analysis of GPVI inhibition on vascular remodelling, a, b, ApoE ' ' " mice were treated with irrelevant rat IgG or anti-GPVI JAQl mAb for 4 weeks. Atherosclerotic lesion formation was assessed in the aortic arch and the right coronary artery bifurcation. The extension of fatty streaks ( ⁇ m) was quantified by Sudan III staining. Data represent mean values with SEM (4-6 experiments per group. Depicted are representative photomicrographs of oil red staining of a carotid artery and an aortic arch from control- and JAQl-treated animals.
  • FIG. 23 To assess the efficacy of local balloon-based delivery of GPVt, immunostaining of serial cross sections of carotid arteries were performed. Carotid cross sections were stained with a specific monoclonal antibody (5C4) directed against GPVI. Substantial positive staining for GPVI of injured carotid segments, but not of intact carotid arteries was found.
  • 5C4 monoclonal antibody
  • FIG. 24 Using a modified double-balloon drug-delivery catheter sGPVI (10 ⁇ g/ml) or control protein (Fc (lO ⁇ g/ml) was delivered to site of injury, and the respective carotid segments were incubated for 5 min with SsGPVI or the control. Thereafter, thrombus formation was assessed by direct microscopy. sGPVI treatment of carotid lesions resulted in an approximately 80% reduction of thrombus formation (lO.xx vs. 2.yy, p ⁇ 0.0x).
  • Figure 25 Comparison of the effect on platelet aggregation of GPVI-Fc fusion protein comprising a triple alanine linker and a GPVI-Fc fusion protein comprising a Gly-Gly-Arg linker.
  • Figure 26 Comparison of the effect on ATP release of GPVI-Fc fusion protein comprising a triple alanine linker and a GPVI-Fc fusion protein comprising a Gly-Gly-Arg linker.
  • the extent of protection includes counterfeit or fraudulent products which contain or purport to contain a compound of the invention irrespective of whether they do in fact contain such a compound and irrespective of whether any such compound is contained in a therapeutically effective amount. Included in the scope of protection therefore are packages which include a description or instructions which indicate that the package contains a species or pharmaceutical formulation of the invention and a product which is or comprises, or purports to be or comprise, such a formulation or species.
  • Antibody fragment fragment with specific antigen binding: It has been shown that fragments of a whole antibody can perform the function of binding antigens. Various fragments of antibodies have been defined, including Fab, (Fab')2, Fv, single domain antibodies and single- chain Fv (scFv). These antibody fragments are defined as follows: (1) Fab, the fragment that contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain or equivalent ⁇ by genetic engineering.
  • the Fab fragment therefore contains VL, VH, CL and CHl domains; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab 1 ) 2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction or equivalent ⁇ by genetic engineering; (4) F(Ab') 2, a dimer of two FAb' fragments held together by disulfide bonds(5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (6) single domain antibodies; antibodies whose complementary determining regions are part of a single domain polypeptide; and (7) single chain antibody (“scFV”), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain wherein the VH domain and the VL domain are linked by a peptide linker which allows the
  • Fv,scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains (Y. Reiter etal, Nature Biotech, 14, 1239-1245,1996).
  • Minibodies comprising ascFv joined to a CH3 domain may also be made (S. Hu etal, Cancer Res., 56, 3055-3061,1996). . Methods of making these fragments are routine in the art. Other antibody fragments are also considered.
  • dAB domain antibodies
  • domain antibodies are the smallest functional binding units of antibodies, corresponding to the variable regions of either the heavy (VH) or light (VL) chains of human antibodies.
  • Domain Antibodies have a molecular weight of approximately 13 kDa, or less than one-tenth the size of a full antibody.
  • Domain antibodies may include dAbs which bind to two therapeutic targets. These include: IgG-like molecules; PEGylated fusion proteins; and anti-serum albumin fusion proteins. In the IgG-like antibody, two variable domains bind to two therapeutic targets on each arm of the IgG.
  • bispecific antibodies may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4,446-449 (1993) ), e. g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.
  • Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti- idiotypic reaction.
  • Bispecific diabodies, as opposed to bispecific whole antibodies may also be particularly useful because they can be readily constructed and expressed in E. coli.
  • Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (W094/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against GPVI, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by knobs-into-holes engineering (J. B. B. Ridgeway et al, Protein Eng., 9,616-621,1996). or substance having an antibody antigen-binding domain with the required specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.
  • Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. According to one embodiment of the invention, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678 for example.
  • variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins.
  • VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the disclosure.
  • VHHs are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains such as those derived from Camelidae as described in W09404678 (and referred to hereinafter as VHH domains or Nanobodies ® ). VHH molecules are about 1Ox smaller than IgG molecules.
  • Complementarity-determining region The CDRs are three hypervariable regions within each of the variable light (VL) and variable heavy (VH) regions of an antibody molecule that form the antigen-binding surface that is complementary to the three-dimensional structure of the bound antigen. Proceeding from the N-terminus of a heavy or light chain, these complementarity- determining regions are denoted as "CDRl”, “CDR2”, and “CDR3", respectively. CDRs are involved in antigen-antibody binding, and the CDR3 comprises a unique region specific for antigen-antibody binding.
  • An antigen-binding site therefore, may include six CDRs, comprising the CDR regions from each of a heavy and a light chain V region.
  • Alteration of a single amino acid within a CDR region can destroy the affinity of an antibody for a specific antigen (see Abbas et al., Cellular and Molecular Immunology, 4th ed. 143-5,2000).
  • the locations of the CDRs have been precisely defined, e. g. , by Kabat et al., Sequences of Proteins of Immunologic Interest, U. S. Department of Health and Human Services, 1983.
  • Alignment may be done by hand, or by computer using commonly accepted computer programs; an example of such a program is the Align 2 program discussed in this description. Alignment may be facilitated by using some amino acid residues which are common to most Fab sequences.
  • the light and heavy chains each typically have two cysteines which have the same residue numbers; in VL domain the two cysteines are typically at residue numbers 23 and 88, and in the VH domain the two cysteine residues are typically numbered 22 and 92.
  • Framework residues generally, but not always, have approximately the same number of residues, however the CDRs will vary in size.
  • residues lOOabcde For example, in the case of a CDR from a candidate sequence which is longer than the CDR in the sequence in Kabat to which it is aligned, typically suffixes are added to the residue number to indicate the insertion of additional residues (see, e.g. residues lOOabcde in fig. 5).
  • residues lOOabcde For candidate sequences which, for example, align with a Kabat sequence for residues 34 and 36 but have no residue between them to align with residue 35, the number 35 is simply not assigned to a residue.
  • CDR and FR residues are also determined according to a structural definition (as in Chothia and Lesk, J. MoI. Biol. 196:901-917 (1987). Where these two methods result in slightly different identifications of a CDR, the structural definition is preferred, but the residues identified by the sequence definition method are considered important FR residues for determination of which framework residues to import into a consensus sequence.
  • Aptamer The agent of the present disclosure may be an aptamer.
  • Aptamers have been defined as artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery and re-amplification.
  • RNA aptamers are nucleic acid molecules with affinities for specific target molecules. They have been likened to antibodies because of their ligand binding properties. They may be considered as useful agents for a variety of reasons. Specifically, they are soluble in a wide variety of solution conditions and concentrations, and their binding specificities are largely undisturbed by reagents such as detergents and other mild denaturants. Moreover, they are relatively cheap to isolate and produce. They may also readily be modified to generate species with improved properties. Extensive studies show that nucleic acids are largely non-toxic and non-immunogenic and aptamers have already found clinical application. Furthermore, it is known how to modulate the activities of aptamers in biological samples by the production of inactive dsRNA molecules in the presence of complementary RNA single strands (Rusconi eta/., 2002).
  • RNAs By selecting natural RNAs, based on D-ribose sugar units, against the non-natural enantiomer of the eventual target .
  • molecule for example a peptide made of D-amino acids
  • a spiegelmer directed against the natural L-amino acid target can be created.
  • the Laws of Molecular Symmetry mean that RNAs synthesised chemically based on L-ribose sugars will bind the natural target, that is to say the mirror image of the selection target. This process is conveniently referred to as reflection-selection or mirror selection and the L-ribose species produced are significantly more stable in biological environments because they are less susceptible to normal enzymatic cleavage, i.e.they are nuclease resistant.
  • Cell line/ Cell culture A "cell line” or “cell culture” denotes higher eukaryotic cells gown or maintained in vitro. It is understood that the progeny of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell. "Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide.
  • a promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter.
  • An "isolated" polynucleotide or polypeptide is one that is substantially free of the materials with which it is associated in nature. By substantially free is meant at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of the materials with which it is associated in nature.
  • Epitope The site on an antigen recognized by an antibody as determined by the specificity of the amino acid sequence. Two antibodies are said to bind to the same epitope if each competitively inhibits (blocks) binding of the other to the antigen as measured in a competitive binding assay (see, e. g.
  • two antibodies have the same epitope if most amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • Two antibodies are said to have overlapping epitopes if each partially inhibits binding of the other to the antigen, and/or if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • Fibronectin is a large multidomain glycoprotein found in connective tissue, on cell surfaces, and in plasma and other body fluids. It interacts with a variety of macromolecules including components of the cytoskeleton and the extracellular matrix, circulating components of the blood clotting, fibrinolytic, acute phase and complement systems. It is involved in many cellular processes, including tissue repair, embryogenesis, blood clotting and cell migration/adhesion. It is expressed on the surface of activated endothelial cells.
  • Framework region Relatively conserved sequences flanking the three highly divergent complementarity-determining regions (CDRs) within the variable regions of the heavy and light chains of an antibody.
  • CDRs complementarity-determining regions
  • the variable region of an antibody heavy or light chain consists of a FR and three CDRs.
  • Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the variable region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs.
  • the framework region of an antibody serves to position and align the CDRs.
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • a "human" framework region is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin.
  • Immunoglobulin A protein including one or more polypeptides substantially encoded by immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha
  • Full-length immunoglobulin light chains are generally about 25 Kd or 214 amino acids in length.
  • Full-length immunoglobulin heavy chains are generally about 50 Kd or 446 amino acid in length.
  • Light chains are encoded by a variable region gene at the NH2-terminus (about 110 amino acids in length) and a kappa or lambda constant region gene at the COOH-terminus.
  • Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.
  • the basic structural unit of an antibody is generally a tetramer that consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to an antigen, and the constant regions mediate effector functions.
  • Immunoglobulins also exist in a variety of other forms including, for example, Fv, Fab, and (Fab 1 ) 2, as well as bifunctional hybrid antibodies and single chains (e. g. , Lanzavecchia et al., Eur. J. Immunol. 17: 105,1987 ; Huston et al., Proc. Natl. Acad. Sci. U. S.
  • An immunoglobulin light or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDR's) (e.g. residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop" (e.g.
  • CDR's complementarity determining regions
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • Framework or FR residues are those variable domain residues other than the hypervariable region residues as herein defined.
  • Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species.
  • the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3.
  • a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody although other mammalian species can be used, or the variable region can be produced by molecular techniques. Methods of making chimeric antibodies are well known in the art, e. g. see U. S. Patent No.
  • a "humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin.
  • the non- human immunoglobulin providing the CDRs is termed a "donor” and the human immunoglobulin providing the framework is termed an "acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin.
  • Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i. e. , at least about 85-90%, such as about 95% or more identical.
  • humanized immunoglobulin all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences.
  • a "humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.
  • Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
  • exemplary conservative substitutions are those such as GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, Gin ; Ser, Thr; Lys, Arg; and Phe, Tyr (see U. S. Patent No. 5,585, 089, which is incorporated herein by reference).
  • Humanized immunoglobulins can be constructed by means of genetic engineering, e. g. see U. S. Patent No. 5,225, 539 and U. S. Patent No. 5,585, 089, which are herein incorporated by reference.
  • a human antibody is an antibody wherein the light and heavy chain genes are of human origin.
  • Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing a human B cell secreting the antibody of interest. Immortalization can be accomplished, for example, by EBV infection or by fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell. Human antibodies can also be produced by phage display methods (see, e. g. Dower et al. , PCT Publication No. W091/17271; McCafferty et al., PCT Publication No. W092/001047; and Winter, PCT Publication No.
  • Human antibodies can also be prepared by using transgenic animals carrying a human immunoglobulin gene (e. g., see Lonberg et al. PCT Publication No. W093/12227 ; and Kucherlapati, PCT Publication No. WO91/10741, which are herein incorporated by reference.)
  • Monoclonal antibody An antibody produced by a single done of B- lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells.
  • Immunoreactivity A measure of the ability of an agent, sometimes an antibody, to recognize and bind to a specific antigen.
  • Specifically binds refers to the ability of individual agents or antibodies to specifically immunoreact with an antigen. This binding is a non-random binding reaction between an agent, for example but not limited to a antibody molecule, and the antigen.
  • the antigen is glycoprotein VI (GPVI), fibronectin and/or vitronectin. Binding specificity is typically determined from the reference point of the ability of the agent to differentially bind the antigen of interest and an unrelated antigen, and therefore distinguish between two different antigens, particularly where the two antigens have unique epitopes.
  • specificity may be determined by means of a binding assay such as ELISA employing a panel of antigens.
  • An agent according to the present invention may recognise GPVI on cells of the platelet/megakaryocyte lineage, and not other human blood cells, in particular granulocytes, lymphocytes and erythrocytes.
  • Inhibit A species which retards, blocks or prevents an interaction, for example binding between GPVI and a ligand or substrate, is considered to inhibit the interaction. Typically, inhibition does not result in 100% blockage but rather reduces the amount and/or speed of interaction. Thus inhibition of interaction of platelets with the surface of a blood vessel may not completely prevent binding and/or activation of platelets but will desirably have a clinically useful effect in slowing and/or reducing the severity of atherosclerosis.
  • Low molecular weight compounds the term will be understood by those skilled in the art. The term may also be understood to include any chemical compound which possesses a molecular weight of below 2,000, e.g. below 1,000.
  • Low molecular weight thrombin inhibitor the term will be understood by those skilled in the art.
  • the term may also be understood to include any composition of matter (e.g. chemical compound) which inhibits thrombin to an experimentally determinable degree in in vivo and/or in in i/y ⁇ r ⁇ tests, and which possesses a molecular weight of below 2,000, e.g. below 1,000.
  • Particular low molecular weight thrombin inhibitors include low molecular weight peptide-based, amino acid-based, and/or peptide analogue-based, thrombin inhibitors.
  • low molecular weight peptide-based, amino acid-based, and/or peptide analogue-based, thrombin inhibitors will be well understood by one skilled in the art to include low molecular weight thrombin inhibitors with one to four peptide linkages, and includes those described in the review paper by Claesson in Blood Coagul. Fibrin. (1994) 5, 411, as well as those disclosed in U.S. Pat. No.
  • Low molecular weight peptide-based thrombin inhibitors include HOOC— CH2— (R)Cha— Pic— Nag— H (wherein Cha represents cyclohexylalanine, Pic represents (S)-pipecolinic acid and Nag represents noragmatine; known as inogatran; see International Patent Application WO 93/11152) and, especially, HOOC— CH2— (R)CgI- Aze— Pab— H (known as melagatran; see above and International
  • prodrug of a low molecular weight thrombin inhibitor includes any compound that releases a low molecular weight thrombin inhibitor, for example one that, following oral or parenteral administration, is. metabolised in vivo to form a low molecular weight thrombin inhibitor (as defined herein), in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)), following oral or parenteral administration.
  • Prodrugs of the thrombin inhibitor melagatran that may be mentioned include those disclosed in international patent application WO 97/23499.
  • Preferred prodrugs of melagatran are those of the formula R ⁇ C- CH2— (R)CgI- Aze— Pab— OH (see the list of abbreviations above or in
  • R 1 represents C ⁇ -IQ alkyl or benzyl, such as linear or branched Cj-s alkyl (e.g. Ci_4 alkyl, especially methyl, propyl and, particularly, ethyl) and the OH group replaces one of the amidino hydrogens in Pab.
  • Cj-s alkyl e.g. Ci_4 alkyl, especially methyl, propyl and, particularly, ethyl
  • Low molecular weight thrombin inhibitors also include Cbz-(R)-Phe-(S)-Pro-(R)-boroMpg (also known as TRI 50c) and its salts and prodrugs, where Cbz represent benzyloxycarbonyl, Phe and Pro have their normal meanings, and boroMpg represents methoxypropyl glycine in which the carboxy group has been replaced by a boronyl group -B(OH)2- Prodrugs of boronic acids include species in which the boronyl group is reversibly derivatised.
  • nucleic acid is a polymeric form of nucleotides of any length, which contain
  • nucleic acids may have any three-dimensional structure, and may perform any function, known or unknown.
  • the term "nucleic acid” includes double-, single-stranded, and triple-helical molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a nucleic acid encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form.
  • Pathological a pathological or dysfunctional interaction of GPVI with fibronectin and/or vitronectin and/or collagen represents an interaction which is abnormal compared to the interaction in healthy subjects during the normal function of the body. A pathological or dysfunctional interaction may result in disease in a subject.
  • Polypeptide A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L- isomers being preferred.
  • polypeptide or protein as used herein is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
  • polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
  • fragment refers to a portion of a polypeptide that is at least 8, 10, 15, 20 or 25 amino acids in length.
  • functional fragments of a polypeptide refers to all fragments of a polypeptide that retain an activity of the polypeptide (e. g., the binding of an antigen).
  • Biologically functional fragments can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell.
  • soluble refers to a form of a polypeptide that is not inserted into a cell membrane.
  • Pharmaceutical agent or drug A chemical or biological compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.
  • compositions and formulations suitable for pharmaceutical delivery of the IL-2 receptor antagonists herein disclosed are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co, Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the IL-2 receptor antagonists herein disclosed.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions e. g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, salts, amino acids, and pH buffering agents and the like, for example sodium or potassium chloride or phosphate, Tween, sodium acetate or sorbitan monolaurate.
  • auxiliary substances such as wetting or emulsifying agents, preservatives, salts, amino acids, and pH buffering agents and the like, for example sodium or potassium chloride or phosphate, Tween, sodium acetate or sorbitan monolaurate.
  • PR-15 a dimer of a protein having the sequence of SEQ ID NO l(see Figure 7).
  • purified does not require absolute purity or isolation; rather, it is intended as a relative term.
  • a purified or isolated protein preparation is one in which protein is more enriched than the protein is in its generative environment, for instance within a cell or in a biochemical reaction chamber.
  • a preparation of protein is purified such that the protein represents at least 50% of the total protein content of the preparation.
  • substantially purity of 90%, 95%, 98% or even 99% or higher of the active agent can be utilized.
  • Sequence identity The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or orthologs of the IL-2R antibodies or antigen binding fragments, and the corresponding cDNA sequence, will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or cDNAs are derived from species that are more closely related, compared to species more distantly related (e. g. , human and murine sequences).
  • a sequence described herein with reference to a wild type sequence may unless otherwise specified have a nature-identical sequence or it may be a non-natural mutant; it may comprise a fragment of a natural or non-natural protein.
  • the sequence may comprise or consist of a sequence having at least 90% homology with a wild type sequence, e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • percent homology of two amino acid sequences or of two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and may be performed either for prophylaxis or during the course of clinical pathology. Desirable effects include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, lowering the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. Unless otherwise specified or required by the context, treatment may be therapeutic, for example, restoring vessel patency, or prophylactic, for example preventing thrombosis or initiation or development of atherosclerosis.
  • Variable region also variable domain or V domain: The regions of both the light chain and the heavy chain of an Ig that contain antigen-binding sites.
  • the regions are composed of polypeptide chains containing four relatively invariant "framework regions” (FRs) and three highly variant “hypervariable regions” (HVs). Because the HVs constitute the binding site for antigen(s) and determine specificity by forming a surface complementarity to the antigen, they are more commonly termed the “complementarity-determining regions,” or CDRs, and are denoted CDRl, CDR2, and CDR3. Because both of the CDRs from the heavy and light chain domains contribute to the antigen- binding site, it is the three-dimensional configuration of the heavy and the light chains that determines the final antigen specificity.
  • the framework regions surround the CDRs. Proceeding from the N- terminus of a heavy or light chain, the order of regions is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the term "variable region” is intended to encompass a complete set of four framework regions and three complementarity-determining regions. Thus, a sequence encoding a "variable region” would provide the sequence of a complete set of four framework regions and three complementarity-determining regions.
  • Vectors may be any of a number of nucleic acids into which a desired sequence may be inserted.
  • Vectors include, but are not limited to, plasmids, phagemids and virus genomes.
  • a cloning vector is one which is able to replicate in a host cell, and which typically is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis.
  • replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • Vectors may further contain one or more selectable marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e. g.,p-galactosidase,luciferase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e. g., various fluorescent proteins such as green fluorescent protein, GFP).
  • Preferred vectors are those capable of autonomous replication, also referred to as episomal vectors.
  • vectors may be adapted to insert into a chromosome, so called integrating vectors.
  • the vector of the invention is typically provided with transcription control sequences (promoter sequences) which mediate cell/tissue specific expression. These promoter sequences may be cell/tissue specific, inducible or constitutive.
  • Promoter is a term recognised in the art and, for the sake of clarity, includes the following features which are provided by example only, and not by way of limitation.
  • Enhancer elements are cis acting nucleic acid sequences often found 5' to the transcription initiation site of a gene (enhancers can also be found 3 1 to a gene sequence or even located in intronic sequences and is therefore position independent). Enhancers function to increase the rate of transcription of the gene to which the enhancer is linked. Enhancer activity is responsive to trans acting transcription factors (polypeptides) which have been shown to bind specifically to enhancer elements.
  • Thebinding/activity of transcription factors is responsive to a number of environmental cues which include, by example and not by way of limitation, intermediary metabolites, environmental effectors.
  • Promoter elements also include so called TATA box, RNA polymerase initiation selection (RIS) sequences and CAAT box sequence elements which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.
  • RIS RNA polymerase initiation selection
  • Adaptations also include the-provision of autonomous replication sequences which both facilitate the maintenance of said vector in either the eukaryotic cell or prokaryotic host, so called”shuttle vectors".
  • Vectors which are maintained autonomously are referred to as episomal vectors.
  • Episomal vectors are desirable since these molecules can incorporate large DNA fragments (30-SOkb DNA).
  • Vitronectin is a mixture of two monomeric glycoproteins (65 and 75 kD) present in blood and the extracellular matrix of many tissues. Vitronectin is expressed on the surface of activated endothelial cells. Vitronectin is one of the major adhesive proteins in plasma and serum. Vitronectin has been considered to be involved in promoting cell attachment, spreading, proliferation, and differentiation of many normal and neoplastic cells, and has been used to study cell migration.
  • the disclosure includes diverse products which are capable of inhibiting binding of one or more components of the blood to one or more potential binding sites for them in the cardiovascular system.
  • the agents may inhibit GPVI binding to the ligand molecule(s) (i.e. fibronectin, vitronectin, or both).
  • ligand molecule(s) i.e. fibronectin, vitronectin, or both.
  • One class of agents binds to GPVI.
  • Another class of agents binds to one or both of the ligand molecules.
  • Some agents bind to GPVI at a binding site of GPVI for the ligand(s) concerned.
  • agents which bind to the ligand(s) concerned optionally at a GPVI binding site of the ligand(s).
  • agents which bind to vitronectin agents which bind to fibronectin and agents which bind to both of fibronectin and vitronectin.
  • the disclosure thus includes agents which inhibit GPVI interaction with fibronectin.
  • the agents may inhibit GPVI binding to fibronectin.
  • the agents bind to fibronectin, optionally, for example, at a GPVI binding site of fibronectin. It is proposed that inhibiting the interaction between GPVI and fibronectin inhibits platelet adhesion; it is proposed that such activity inhibits platelet aggregation.
  • the disclosure includes agents which inhibit GPVI interaction with vitronectin.
  • the agents may inhibit GPVI binding to vitronectin.
  • the agents bind to vitronectin, optionally, for example, at a GPVI binding site of vitronectin. It is proposed that inhibiting the interaction between GPVI and vitronectin inhibits platelet adhesion; it is proposed that such activity inhibits platelet aggregation.
  • Embodiments of the present disclosure reside in a protein which binds to collagen at a site at which membrane bound GPVI binds to collagen.
  • Embodiments of the disclosure reside in a protein which binds to fibronectin at a site at which membrane-bound GPVI binds to fibronectin.
  • Embodiments of the disclosure reside in a protein which binds to vitronectin at a site at which membrane bound GPVI binds to vitronectin.
  • species comprising an amino acid sequence of, or derived from, an extracellular domain of GPVI.
  • Other examples include antibody products (e.g. antibodies, antibody fragments, engineered antibodies and antibody fragments), aptamers and molecules derived from snake venom.
  • products for example molecules, which bind to or interact with plural vascular domains or binding sites.
  • the domains and more normally all the domains (or binding sites) are potential sites of interaction with platelets; in embodiments, therefore, the products can hinder or block tethering or binding of platelets to blood vessel walls having such domains or binding sites
  • Each domain may comprise a different ligand, for example adhesion protein, from each other domain.
  • the products are exemplified below by products of which at least one of the domains binds to or interacts with GPVI. In one class of products, all the binding sites bind to or interact with GPVI.
  • the products mentioned in this paragraph can provide enhanced binding properties as compared to single domain-binding products, for example enabling adhesion to a blood vessel wall or atherosclerotic plaque for a period of at least 12 hours, particularly at least 24 hours, e.g. 48 hours or more as explained next.
  • PR-15 binds to injured blood vessels for at least 48 hours after intravenous administration.
  • PR-15 is a representative of agents interacting with plural vascular domains (in this case selected from collagen, vitronectin and fibronectin) and the data indicate that such agents may advantageously be administered at a frequency of twice daily or less, e.g. at periods of once a day or longer, e.g. once every two, three or four days or once a week. Methods involving administration at such periods or more are included in the disclosure.
  • the domains or binding sites may simultaneously be exposed on a dysfunctional or diseased area of blood vessel wall, for example an atherosclerotic or inflamed area.
  • the products may, as already indicated, function when bound to a vascular surface to retard or prevent tethering and/or adhesion of platelets; particular embodiments relate to products which are capable of binding to a dysfunctional or inflamed area of blood vessel wall which does not include an atherosclerotic plaque or lesion; such products can have value in inhibiting, for example preventing or retarding, initiation of atherosclerosis.
  • the disclosure includes agents which are capable of binding to a plurality of GPVI-binding sites of a dysfunctional, inflamed or atherosclerotic blood vessel area, for example a human blood vessel area.
  • the binding sites may all be of different types; alternatively stated, they may all comprise different ligands, e.g. adhesion proteins.
  • Exemplary are agents, or products, which are capable of binding to two or three ligands selected from collagen, vitronectin and fibronectin. Such products are exemplified below by products (agents) which can bind to all of collagen, fibronectin and vitronectin.
  • an aspect of the invention is products which are capable of binding to all of collagen, fibronectin and vitronectin.
  • certain agents which bind to as few as a single one of these proteins are also included in the disclosure, as described next.
  • GPVI bound to the surface of platelets, binds to vitronectin and fibronectin.
  • GPVI binds to vitronectin and fibronectin.
  • Some bind small molecules such as epinephrine or thromboxane A2 (TxA2), which help activate the platelet.
  • TxA2 thromboxane A2
  • Other receptors on the surface of platelets help the platelet tether and/or adhere to sites of vascular injury but also help tethering and/or adherence to atherosclerotic blood vessel areas.
  • Fibronectin and vitronectin are proteins which are expressed on the surface of activated endothelial cells.
  • the endothelium may be activated in response to or association with many stimuli, including inflammation.
  • Fibronectin and vitronectin are present on dysfunctional endothelium.
  • the present disclosure proposes inter alia use of an agent capable of binding at least to one or both of fibronectin and vitronectin to inhibit interaction between GPVI on membrane bound platelets and an inflamed or dysfunctional endothelium.
  • Platelets briefly adhere to an activated but intact endothelium but tend not to adhere strongly but to subsequently 'bounce away' from the endothelium.
  • the brief adherence of platelets to the activated endothelium may activate platelets which subsequently release activatory molecules.
  • vitronectin and fibronectin play a role in platelet binding to the endothelium, in particular via GPVL
  • vitronectin and fibronectin are considered not to activate platelets by themselves, or to have a minor role in platelet activation.
  • the adhesion of platelets to an activated, e.g. dysfunctional, but intact endothelium may be a first step in the formation of an atherosclerotic plaque. Inhibition of this interaction may prevent the subsequent activation of platelets which have adhered, albeit perhaps briefly, to the activated endothelium.
  • agents of the present disclosure as well as methods and uses thereof may be useful in preventing initiation of atherosclerotic lesions or plaques.
  • the agents may act to inhibit the adhesion of platelets to an activated endothelium by inhibition of interaction between GPVI and fibronectin and/or vitronectin.
  • the present disclosure has therefore new therapeutic applications in patients who do not yet show symptoms of atherosclerotic plaque but who are potentially at risk of developing atherosclerotic plaques in the future.
  • Such patients may have been identified as having a risk factor for cardiovascular disease and/or who are at risk of suffering from cardiovascular disease, e.g. coronary heart disease.
  • Risk assessment calculations may include the PROCAM coronary heart disease risk function and the Framingham coronary heart disease risk function.
  • the PROCAM risk function estimates the probability of developing coronary death or first myocardial infarction within ten years and employs age, systolic blood pressure, LDL and HDL cholesterol, triglycerides, cigarette use, diabetes and family history of myocardial infarction as risk factors.
  • the FRAMINGHAM risk function estimates the probability of developing coronary death, myocardial infarction (recognised and unrecognised), angina pectoris or coronary insufficiency (total CHD end points) within ten years taking age, blood pressure, LDL and HDL cholesterol, cigarette use and diabetes as risk factors. (Anderson K M et al, Circulation 1991; 83:356-362).
  • agents of the present disclosure may be used in the treatment, particularly chronic or long-term treatment, of patients who have been identified as having a risk factor of 45 or more.
  • agents of the present invention may be administered to patients who have been identified as having a PROCAM score of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60.
  • Such treatment is prophylactic, to prevent, delay or reduce the risk of future cardiovascular disease.
  • Methods, uses and agents of the present disclosure can be utilised for or in the treatment of patients who are at risk of development of atherosclerotic plaques. Such patients may have been identified as having some or all of the risk factors associated with atherosclerosis.
  • one class of patients which could be treated using agents, methods and uses of the present invention are patients who have been identified using the Framingham risk factor as having a defined level of risk of developing atherosclerotic lesions and complications thereof.
  • the defined level of risk may be determined according to the points obtained by the patient using the Framingham method.
  • the male patient may have a Framingham points score of 11, 12, 13, 14, 15, 16, 17 or greater.
  • the patient is a male subject.
  • the patient may have been identified as having a risk factor, using the Framingham study, wherein the patient has been awarded 20 or more points according to the Framingham point scores.
  • the female patient may have 20, 21, 22, 23, 24, 25 or more according to the Framingham points score.
  • SCORE Systematic COronary Risk Evaluation
  • a patient may be considered at risk justifying prophylactic medication if he/she has a 10-year risk of fatal cardiovascular disease according to the SCORE project of at least 3%, for example at least 5 as in the case of patients having a risk of at least 10%.
  • the disclosure includes primary prophylaxis of cardiovascular disease using an agent which inhibits interaction of GPVI with one or both of fibronectin and vitronectin.
  • a method of treating a patient who has previously suffered a cardiovascular event associated with, or suspected of being associated with, atherosclerosis e.g. plaque rupture, disruption or fissure
  • the method comprises administering an effective amount of an agent of the present disclosure.
  • GPVI As well as binding to the fibronectin and vitronectin, GPVI also binds to collagen. In particular, GPVI binds to collagen Types I and III. Immunohistochemical studies are reported to have shown enrichment of collagen types I, III, V, and VI, vitronectin, fibronectin, fibrinogen/fibrin, and thrombospondin in the atherosclerotic plaque. The GPVI ligands collagen (specifically collagen I and collagen III), vitronectin and fibronectin are therefore exposed when an atherosclerotic lesion is disrupted and/or ruptures.
  • Collagen and adhesion proteins are also thought to be exposed for some time after rupture or fissure of the plaque, when the lesion is healing.
  • platelets bind to GPVI-binding sites (to be mentioned are collagen, vitronectin and fibronectin) when an atherosclerotic plaque ruptures or fissures, become activated and cause aggregation of platelets at the site of atherosclerotic rupture or disruption.
  • GPVI-binding sites are collagen, vitronectin and fibronectin
  • inhibition of GPVI interaction with one or more of its ligands, e.g. at least collagen can inhibit thrombus formation at the site of atherosclerotic lesion rupture.
  • agents of the present disclosure or direct and indirect inhibitors of GPVI may be useful in secondary prophylaxis after a cardiovascular event, as well as in primary prophylaxis against atherosclerosis and/or thrombosis.
  • the agent used in this context is capable of binding to plural GPVI-binding sites (e.g. to all of collagen, vitronectin and fibronectin) to provide relatively strong binding to a ruptured or fissured surface.
  • a method of treating a patient who has suffered a cardiovascular event, particularly one which is or is suspected of being a result of rupture or disruption of an atherosclerotic lesion comprising administering an effective amount of agent according to the present disclosure, or a GPVI inhibitor, to said patient over an extended period of time.
  • an extended period of time may be of at least one month, e.g. from three to six months or more following the initial cardiovascular event.
  • the treatment of the patient may be considered a chronic treatment.
  • the administration may take place periodically throughout the term of the treatment, e.g. at periods of twice a day, once a day or longer. Substantially continuous administration by, for example, infusion is not excluded.
  • the mode of administration of the agent of the invention may be intravenous, inter-arterial or subcutaneous injection or infusion, or by oral administration.
  • the method comprising administering the agent to the patient over a time scale of at least three months, following the initial cardiovascular event.
  • the method may comprise administering an effective amount of an agent of the disclosure for three, four, five, six or more months following a cardiovascular event, for example thrombosis or myocardial infarction.
  • Circulating monocytes are among the earliest cells recruited into experimentally induced vascular lesions in animals and spontaneous atherosclerosis in human arteries. As a result, monocytes have been hypothesized to serve as markers, initiators, and promoters of arterial occlusive diseases. It has been reported that monocytes adhere to the luminal surface of stent-injured arteries and then penetrate the healing arterial wall, with resultant neointimal hyperplasia. Mechanisms of monocyte adhesion that have been defined in vitro include vitronectin-dependent binding of urokinase-type plasminogen activator receptors on the cell surface. Monocytes have been shown to adhere to fibronectin.
  • agents of the invention will inhibit monocyte recruitment.
  • the agents of the disclosure may be used to inhibit interaction between monocytes and the endothelium thus inhibiting or retarding monocyte recruitment to a site of inflammation on the endothelial. It is contemplated that such activity will help prevent or retard initiation and development of atherosclerosis. It is further contemplated that inhibition of monocyte adhesion and migration into mechanically injured arteries will alter vascular repair, limit the extent of neointimal hyperplasia, and potentially control a (tenopathies that follow vascular intervention or injury.
  • the disclosure includes a method for inhibiting (for example blocking or retarding) monocyte recruitment to an injured, dysfunctional or diseased blood vessel area, comprising contacting the blood vessel area with a product described herein which binds to one or both of vitronectin and fibronectin.
  • the method may be performed in vitro for screening or research purposes, or in vivo for medical purposes (prophylaxis or therapy) by administering an effective amount of such product to a patient.
  • One class of patients has had a cardiovascular intervention, particularly implantation of a cardiovascular device (see later in this specification for further description of such devices).
  • the disclosure also teaches the unpredicted benefits of co-administering, whether or not by fixed combination, (i) an agent of the disclosure, e.g. an inhibitor of interaction between GPVI and a protein selected from vitronectin, fibronectin and combinations thereof, and, in particular, (ii) at least one other antiplatelet agent (e.g. acetyl salicylic acid or clopidogrel), a direct or indirect matrix metalloprotease inhibitor, and/or an anticoagulant.
  • an agent of the disclosure may be co-administered with acetyl salicylic acid and clopidogrel.
  • Another hitherto unpredicted benefit taught by this disclosure arises from combining or co-administering an anticoagulant, particularly a low molecular weight thrombin inhibitor, with an agent described herein.
  • an agent described herein may be used in various methods of treating vascular disorders.
  • the agents of the disclosure bind effectively to an atherosclerotic disorder, particularly a ruptured or fissured plaque, when delivered locally, thus avoiding the necessity of systemic administration at clinical levels.
  • local administration e.g. by a catheter, of an agent of the disclosure to a site, or suspected site, of an atherosclerotic disorder.
  • such an agent may be administered to a patient via a catheter to treat, for example, plaque disruption (e.g. rupture, fission), stenosis or restenosis or other cardiovascular disorders.
  • the agent is a fusion protein comprising an extracellular domain of GPVI fused to an immunoglobulin Fc portion via a linker.
  • the fusion protein is PR-15, a fusion protein comprising the sequence disclosed in Figure 7.
  • the administration of an agent of the present invention via a catheter may provide an alternative method of treating spontaneous or other types of arterial lesions to systemic antiplatelet therapy.
  • the agents of the disclosure bind to the target molecule with a high degree of affinity.
  • Binding affinity of an agent for example an antibody or fusion protein may be measured using for example BIACORE systems.
  • the strength of the binding of an agent to a peptide sequence of GPVI may be analysed using chemiluminescence and quantified by measuring signal intensity.
  • the agents may be, for example, antibodies, antibody fragments, proteins, polypeptides, fusion proteins, aptamers or compounds (particularly low molecular weight compounds) or combinations thereof.
  • the agent is an antibody or fragment thereof
  • the antibody or fragment thereof may be, although is not limited to, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a human antibody or a humanized antibody and the fragment is a fragment of such an antibody.
  • Other agents which bind the ligands for example, GPVI, fibronectin and/or vitronectin, as defined herein are encompassed within the present invention.
  • agents which comprise natural amino acid sequences e.g. a sequence found in the extracellular domain of GPVI, or variants thereof.
  • variants are those that vary from a reference (natural) amino acid sequence by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics.
  • amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and asparatic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan.
  • conservative modifications of a reference amino acid sequence do not affect the biological function of the sequence.
  • any reference to a GPVI protein or fragment thereof encompasses all GPVI polymorphisms and expressed GPVI proteins thereof.
  • the proteins encoded by the GPVIa and GPVIb alleles differ by five amino acids. Three of these are in the stem (S219P, K237E, T249A), and two are in the cytoplasmic domain (Q317L and H322N). Both GPVIa and GPVIb are considered "GPVI".
  • variants which show enhanced biological activity relative to an original amino acid sequence, for example, a sequence found in the extracellular domain of GPVI. Further definition of an antibody and fragments thereof are included herein and form part of the invention.
  • the agent is an antibody fragment
  • the antibody fragment is a Fab fragment, a F ⁇ b 7 ⁇ fragment, a scFv, a Fv fragment or a single domain antibody.
  • the agent may be a humanized antibody or fragment thereof.
  • a "humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin.
  • the non-human immunoglobulin providing the CDRs is termed a "donor” and the human immunoglobulin providing the framework is termed an "acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin.
  • Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical, e.g. at least 96%, 97%, 98% or 99%.
  • all parts of a humanized immunoglobulin, except possibly the CDRs are substantially identical to corresponding parts of natural human immunoglobulin sequences.
  • a "humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • the method of isolation may depend on the immunoglobulin isotype.
  • Purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and antiimmunoglobulin.
  • the agent of the invention is purified by using Protein G-Sepharose columns.
  • the agent may also be a single domain antibody such as, for example, a VHH directed to a target mentioned herein, for example, GPVI, collagen, fibronectin and/or vitronectin.
  • the VHH may belong to a class having human-like sequences.
  • the class is characterised in that the VHHs carry an amino acid from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine at position 45, such as, for example, L45 according to the Kabat numbering.
  • peptides belonging to this class show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation.
  • the agent may comprise a polypeptide construct comprising two or more single domain antibodies which have been joined.
  • the single domain antibodies may be identical in sequence and directed against the same target or antigen, for example, GPVI, collagen, fibronectin and/or vitronectin.
  • a multivalent VHH may be bivalent (2VHHs), trivalent (3VHHs), tetravalent (4 VHHs) or have a higher valency molecules.
  • a single domain antibody directed towards fibronectin there is provided.
  • a single domain antibody which is capable of inhibiting fibronectin binding with GPVL
  • the single domain antibody binds to fibronectin.
  • the single domain antibody binds to GPVI and prevents GPVI binding to fibronectin.
  • a single domain antibody directed towards vitronectin that is to say, there is provided a single domain antibody which is capable of inhibiting vitronectin binding with GPVI.
  • the single domain antibody binds to vitronectin.
  • the single domain antibody binds to GPVI and prevents GPVI binding to vitronectin.
  • a protein or polypeptide derived from a snake venom protein which is capable of inhibiting the interaction between GPVI and fibronectin and/or vitronectin.
  • the snake venom derived protein or polypeptide may bind to GPVI and prevent binding of GPVI to fibronectin.
  • the snake venom derived protein may bind to fibronectin and prevent binding of GPVI to fibronectin.
  • the snake venom derived protein or polypeptide may bind to GPVI and prevent binding of GPVI to vitronectin.
  • the snake venom derived protein may bind to vitronectin and prevent binding of GPVI to vitronectin.
  • Snake venoms are mixtures of biologically active proteins and peptides. Many snake venoms affect hemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting the endothelium. Snake venom components can be classified into various families, such as C-type lectins, metalloproteinases, serine proteases, disintegrins and phospholipases. The various members of a particular family may act selectively on different blood coagulation factors, blood cells or tissues (Lu et al; Toxicon, 2005 Jun 15;45(8): 1089-98 )
  • proteins derived from Crovidisiri a collagen-binding protein isolated from snake venom of Crotalus viridis.
  • such proteins may be recombinant proteins which have homology to Crovidisin and additionally include one or both of a vitronectin- binding domain and a fibronectin-binding domain.
  • a particular class of agents comprise an amino acid sequence of or comprised in an extracellular domain of GPVl, or a variant thereof that is functional for binding to one, two or three proteins selected from the group consisting of collagen, vitronectin and fibronectin.
  • agents which are functional to bind to collagen and to at least one protein selected from fibronectin and vitronectin.
  • Such agents may be soluble in plasma; for example, it is known that glycosylation may be modified, e.g. increased, to improve solubility.
  • polypeptides comprising a sequence of or contained in an extracellular domain of GPVI or a function conservative variant of fragment thereof.
  • polypeptides may include a sequence having a homology of at least 90%, e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, with an extracellular domain of GPVl.
  • Products of the disclosure include formulations which contain a plurality of agents, each targeting a different binding site (e.g. respectively targeting two or three of vitronectin, fibronectin and collagen), e.g. a first agent which interferes with GPVI-vitronectin interaction, a second agent which interferes with GPVI-fibronectin interaction and an optional third agent which intereferes witih GPVI- collagen interaction.
  • agents each targeting a different binding site (e.g. respectively targeting two or three of vitronectin, fibronectin and collagen)
  • a first agent which interferes with GPVI-vitronectin interaction
  • a second agent which interferes with GPVI-fibronectin interaction
  • an optional third agent which intereferes witih GPVI- collagen interaction.
  • Methods of the disclosure include co-administration, whether or not in fixed combination, of a plurality of agents (often two or three agents), each targeting a different binding mode, e.g. different vascular binding sites, for platelets, monocytes or other components of blood.
  • a plurality of agents forten two or three agents
  • Products of the disclosure include formulations, packages or kits comprising a plurality of active agents, each agent being capable of interrupting a different mode of binding, for example different receptor-ligand interaction, of a blood component, particularly a platelet or monocyte.
  • Such a plurality of agents may be used in a method of treatment,
  • the plurality of agents are all antibody products, for example antibody fragments.
  • co-administration and/or combinations e.g. combination formulations, combination packages, combination kits
  • plural single domain antibodies e.g. two or three
  • methods or products involve plural (e.g. two or three) domain antibodies (dAb's) as described above.
  • agents particularly polypeptides, comprise an amino acid sequence encoded by:
  • nucleic acid sequence which encodes a wild type GPVI extracellular domain
  • nucleic acid sequence which hybridises to said nucleic acid sequence (i) and which encodes a polypeptide that is capable of binding to a plurality of types of GPVI-binding sites of a dysfunctional, inflamed or atherosclerotic blood vessel area; or
  • agents particularly polypeptides, which comprise an amino acid sequence encoded by:
  • nucleic acid sequence which encodes a wild type GPVI extracellular domain
  • nucleic acid sequence which hybridises to said nucleic acid sequence (i) and which encodes a polypeptide that is which is capable of inhibiting the binding of GPVI to collagen and to one or both of fibronectin and vitronectin; or
  • nucleic acid sequence which differs from said nucleic acid sequence (i) by virtue of the degeneracy of the genetic code.
  • agents particularly polypeptides, which comprise an amino acid sequence encoded by:
  • agents particularly polypeptides, comprising an amino acid sequence encoded by:
  • nucleic acid sequence of bases 1 to 807 of SEQ ID No. 2 (i) a nucleic acid sequence of bases 1 to 807 of SEQ ID No. 2 (figure 8); (ii) a nucleic acid sequence which hybridises to bases 1 to 807 of SEQ ID No. 2 (figure 8) and which encodes a polypeptide that is which is capable of inhibiting the binding of GPVI to collagen and to one or both of fibronectin and vitronectin; or
  • the disclosure includes amongst other things fusion proteins which directly or indirectly inhibit collagen-induced platelet activation and have a longer plasma half-life than the extracellular domain of GPVI as an isolated protein.
  • derivatisation of proteins e.g. by sugars or by polymers such as, for example, PEG, can increase plasma half-life.
  • fusion proteins containing an antibody-derived sequence as in the case of proteins containing at least part of a heavy chain constant region (e.g. at least the hinge) linked to an active sequence (particularly one derived from an extracellular domain of GPVI as mentioned herein) through a linker.
  • Other GPVI fusion proteins, apart from antibody derived fusion proteins, are also encompassed in the present invention.
  • the linker comprises the amino acid sequence GIy-X-Z, where Z is a hydrophilic amino acid and X is any amino acid.
  • X is selected from GIy and Ala.
  • X may be GIy.
  • Z may be selected from Arg, His, Lys, Ser, Thr, Asp, GIu, Tyr, Asn and GIn. In some embodiments, Z may be selected from Arg, Ser, Lys and His: particularly Z is Arg.
  • the 20 common amino acids may be classified as hydrophobic, polar, positively charged and negatively charged as follows :
  • Polar and charged amino acids may be considered hydrophilic, e.g. serine, arginine and lysine.
  • One class of disclosed proteins comprises a linker having a hydrophilic amino acid selected from positively charged amino acids, e.g. arginine or lysine.
  • a further class comprises a linker having a hydrophilic amino acid selected from negatively charged amino acids.
  • a third class comprises a linker having a hydrophilic amino acid selected from polar uncharged charged amino acids.
  • the linker may contain additional amino acids to the N-terminal or C-terminal sides of GIy-X-Z, or both sides.
  • GIy is one possible such amino acid.
  • the linker may include the amino acid sequence GIy- Gly-Arg-Gly.
  • linkers comprising a polyglycine amino acid sequence e.g. containing 2, 3, 4, 5 or more contiguous glycine residues.
  • the linker comprises the amino acid sequence Gly-Arg-Gly.
  • the linker may comprise the amino acid sequence XfGIy-Z, where
  • Xi is not cysteine.
  • Xi may be GIy or Ala or another amino acid with a hydrophobic side chain, e.g. VaI, He, Leu, Met, Phe, Pro or Trp.
  • X 1 is Asp, GIn, Ser, Thr or Tyr.
  • the linker may contain additional amino acids to the N-terminal or C-terminal sides of Xi-GIy-Z, or both sides.
  • GIy is one possible such amino acid.
  • the linker comprises the sequence Gly-Gly-Arg.
  • Gly-Gly-Lys is another linker sequence.
  • the active sequence is at the N-terminal side of the linker and the antibody-derived sequence is at the C-terminal side of the linker.
  • the linker comprises at least one hydrophilic amino acid.
  • the hydrophilic amino acid may be a residue Z as described above.
  • Some linkers are of the amino acid sequence GIy-X-Z as previously described.
  • linker may be flexible.
  • the flexible peptide linker is of about 20 or fewer amino acids in length. More preferably, the peptide linker should have at least two amino acids in length. Furthermore, it is even more preferable to use apeptide linker comprising two or more of the following amino acids: glycine, serine, alanine and threonine.
  • linker could be at any residue within the extracellular domain of GPVI which would allow the extracellular domain of GPVI to flexibly bind with fibronectin, vitronectin and/or collagen. It will be apparent to one skilled in the art that alternative linkers can be used to link first and second parts.
  • suitable linkers might be a nucleic acid (egoligonucleotide); a peptide nucleic acid; a chemical crosslinker (e.g. polyoxyethlene).
  • fusion proteins which comprise an antibody-derived polypeptide.
  • the antibody-derived polypeptide includes an Ig heavy chain constant region; in a sub-class of protein, the antibody-derived polypeptide includes a hinge region of an immunoglobulin and is functional to prolong the plasma half-life of the protein beyond that of a fusion protein which does not contain the antibody-derived polypeptide.
  • the antibody-derived polypeptide includes a hinge region and a CH2 region of an immunoglobulin.
  • the antibody-derived polypeptide includes a hinge region, a CH2 and CH3 region of an immunoglobulin.
  • the antibody-derived polypeptide may be an Fc domain of an immunoglobulin, for example.
  • Particular proteins comprise an antibody-derived polypeptide which is an Fc domain of an IgG molecule.
  • the IgG may be an IgGl.
  • Fc species referred to herein may be IgG derived, particularly IgGl derived, e.g. derived from such a mouse IgG.
  • the antibody-derived polypeptide is a polypeptide which has the properties of an Fc domain of an IgG molecule or a polypeptide which can confer such properties to a fusion protein.
  • properties may include, for example, a prolonged serum half-life and thus the incorporation of such a sequence into a fusion protein confers a prolonged serum half-life on the fusion protein as compared to a protein which does not include the antibody-derived polypeptide.
  • the protein may be conjugated with polyethylene glycol (“PEG”), such conjugation being termed "pegylation” (See Abuchowski et al.,JBiol Chem., 252: 3582-3586 (1977)).
  • PEG polyethylene glycol
  • pegylation See Abuchowski et al.,JBiol Chem., 252: 3582-3586 (1977)
  • PEG is typically characterised as a non-immunogenic uncharged polymer with three water molecules per ethylene oxide monomer. PEG is believed to slow renal clearance by providing increased hydrodynamic volume in pegylated proteins (Maxfield et al, Polymer, 16: 505-509 (1975)).
  • US 5,849,535 also describes humanGH (hGH) variants which are conjugated to one or more polyols, such as poly (ethylene glycol) (PEG).
  • the protein may be a dinner, for example a dimer containing disulfide bonds between Cys residues of two polypeptides.
  • the dimer is a homodimer but heterodimers are not excluded.
  • the dimer may be of a polypeptide containing at least part of an Fc hinge region, for example of a polypetide as described above containing at least a CH2 region and a hinge region, such as CH2, CH3 and hinge regions, for example.
  • polypeptides which comprise an antibody derived amino acid sequence linked to an amino acid sequence derived from an extracellular domain of GPVI and which include conservative substitutions, insertions, and/or deletions, but which still retain the biological activity of the fusion protein of Figure 7 (or SEQ ID No. 1).
  • the number of amino acid residues which are modified, whether by substitution, insert or deletion may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • an extracellular domain of GPVI includes fragments or analogues thereof which have the biological activity of the extracellular domain of GPVI as herein defined.
  • the biological activity of the extracellular domain of GPVI is considered to include, amongst others, collagen binding activity and/or fibronectin binding activity and/or vitronectin binding activity. It will be appreciated that fragments or analogues of GPVI may have greater or less binding affinity to collagen, fibronectin and/or vitronectin than the extracellular domain of GPVI, but nonetheless, will in practice have sufficient binding activity for the protein to be therapeutically useful.
  • an Fc domain of an immunoglobulin includes fragments or analogues thereof which have the properties of an Fc domain of an immunoglobulin as herein defined.
  • the IgG Fc may contain amino acid mutations as compared to naturally occurring IgG Fc, for example, to make it non-lytic.
  • an Fc is a human Fc; this applies for example to Fc molecules used as a control in certain experiments described herein to assess the activity of PR-15, where an Fc of a human IgGl may be used.
  • a particularly advantageous GPVI fusion protein is the protein known as PR-15, which has the amino acid sequence of SEQ ID NO 1 (see Fig 7).
  • PR-15 is characterised by the linker Gly-Gly-Arg and is therefore a member of a class of a fusion proteins comprising: a) an extracellular domain of GPVI or a variant thereof that is functional for binding to collagen; and b) an Fc domain of an immunoglobulin or a functional conservative part thereof, the extracellular domain and the Fc domain being fused via a linker characterised by the amino acid sequence Gly-Gly-Arg.
  • the active, or most active, fusion proteins described in the previous paragraph are dimers thereof. These are capable of binding to a plurality of GPVI-binding sites, optionally a plurality of types of GPVI-binding sites, of a dysfunctional, inflamed or atherosclerotic blood vessel area.
  • PR-15 is a representative of classes of agents (whether fusion proteins, antibody products, aptamers or otherwise) having one (or more) of the following capabilities shown in Example 13 below:
  • PR-15 is a representative of classes of agents (whether fusion proteins, antibody products, aptamers or otherwise) having one (or more) of the following characteristics:
  • the agent optionally being capable of inhibiting collagen-induced ATP release from, human platelets by at least 65%, e.g. least 70% as in the case of inhibition by at least 80%, e.g. at least 90%;
  • the agent capable of binding to a plurality of GPVI-binding sites, optionally a plurality of types of GPVI-binding sites, of a dysfunctional, inflamed or atherosclerotic blood vessel area, the agent being capable of inhibiting platelet aggregation in response to collagen by at least 50%, optionally by at least 60%, for example at least 70%, e.g. 75% or more;
  • PR-15 and members of the above mentioned classes of agent to which PR-15 belongs are used to interfere with or inhibit an interaction with one of both of fibronectin and vitronectin.
  • examples of such uses are prophylaxis against adhesion of platelets and/or monocytes to blood vessels in patients asymptomatic for atherosclerosis and/or thrombosis, and in patients considered at risk of suffering atherosclerosis and/or cardiovascular disease.
  • an agent is used to interfere with or inhibit an interaction with collagen; in. sub-embodiments, the agent is not PR-15 or another member of the class of a fusion proteins comprising: a) an extracellular domain of GPVI or a variant thereof that is functional for binding to collagen; and b) an Fc domain of an immunoglobulin or a functional conservative part thereof, the extracellular domain and the Fc domain being fused via a linker characterised by the amino acid sequence Gly-G
  • agents which are fusion proteins as described in the preceding paragraph may be applied to (i) agents which are fusion proteins as described in the preceding paragraph; (ii) agents which are not fusion proteins as described in the preceding paragraph; (iii) agents which are GPVI fusion proteins; (iv) agents which are not GPVI fusion proteins; (v) agents which do not comprise a GPVI-derived sequence; (vi) agents which do comprise a GPVI-derived sequence; (vii) agents which are antibody products (e.g.
  • agents which are aptamers agents which comprise a sequence derived from a snake venom; (x) agents which do fall within any one or more of paragraphs Al to A105 below; (xi) agents which do not fall within any one or more of paragraphs Al to A105 below.
  • nucleic acid comprising a nucleic acid sequence, which sequence encodes an agent as described herein when the agent is a polypeptide product, for example an antibody, a fragment thereof, a fusion protein, or a protein.
  • the present disclosure includes a nucleic acid sequence which encodes an agent comprising a wild type amino acid sequence but whose coding sequence for said wild type amino acid sequence differs from the wild-type nucleic acid sequence as a result of the degeneracy of the genetic code.
  • the present disclosure also includes nucleic acids that share at least 90% homology with a nucleic acid sequence which encodes an agent of the present disclosure.
  • the nucleic acid may have 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98% or 99% homology to such a nucleic acid. Included are such nucleic acids wherein the agent has a wild type sequence, and polypeptides encoded by such nucleic acids.
  • nucleic acid molecule which hybridises under stringent conditions to a nucleic acid molecule which encodes an agent of the disclosure.
  • Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other.
  • the stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993).
  • the T m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following have been found as exemplary for hybridization conditions but without limitation:
  • Hybridization 5x SSC at 65 0 C for 16 hours Wash twice: 2x SSC at room temperature (RT) for 15 minutes each
  • Hybridization 6x SSC at RT to 55 0 C for 16-20 hours
  • a common formula for calculating the stringency conditions required to achieve hybridisation between nucleic acid molecules of a specified homology is:
  • nucleic acid sequences which differ from those described above by virtue of the degeneracy of the genetic code.
  • isolated nucleic acid molecules e.g., cDNAs
  • isolated nucleic acid molecule comprises one of the nucleotide sequences set forth in Sequence SEQ ID NO: 2 (figure 8) or the coding region or a complement thereof.
  • the isolated nucleic acid molecule comprises a nucleotide sequence which hybridizes to or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence as in SEQ ID NO: 2 (figure 8), or a portion thereof.
  • the isolated nucleic acid molecule encodes one of the amino acid sequences set forth in SEQ ID NO: 1 (figure 7).
  • an expression vector which comprises a nucleic acid as described herein and associated regulatory sequences necessary for expression of a protein or polypeptide in a host cell.
  • a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted.
  • Vectors include, but are not limited to, plasmids, phagemids and virus genomes.
  • said viral based vector is based on viruses selected from the group consisting of adenovirus; retrovirus; adeno associated virus; herpesvirus; lentivirus; baculovirus.
  • host cells comprising a nucleic acid as described herein or a vector as described herein.
  • Such host cells are transfected or transformed so that they contain the nucleic acid or vector in such a way that they are effective in expressing the desired polypeptide/protein when cultured in appropriate media under the necessary growth conditions.
  • the host cell is selected from a HeLa cell and a CHO (Chinese Hamster Ovary) cell.
  • the host cells to be used are not particularly circumscribed so as long as they can be transfected by a vector to be used and can express the DNA of the present disclosure.
  • bacteria such as Escherichia coli, yeast such as Sacchammyces cerevisiae, and an animal cell such as a COS cell, a CHO cell, etc. can be used.
  • prokaryotic host cells examples include E coli.
  • eukaryotic host cells include avian, insect, plant, and animal cells such as C0S7, HeLa, and CHO cells.
  • a polypeptide agent for example a fusion protein, antibody or antibody fragment
  • a polypeptidic agent By cultivating a transformant or transfected cell, a polypeptide agent, for example a fusion protein, antibody or antibody fragment, can be produced in a cell or a culture medium. Then, by collecting the produced polypeptide, e.g. an antibody (or antibody fragment), a polypeptidic agent can be obtained.
  • the obtained polypeptide e.g. antibody or protein can be isolated and purified by appropriately combining methods, for example, centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion-exchange chromatography, or gel-filtration chromatography.
  • the cells can be cultured in a suitable medium, and spent medium can be used as an polypeptide source.
  • suitable medium can be used as an polypeptide source.
  • matrix-coated channels or beads and cell cocultures may be included to enhance growth of transformed cells e.g. antibody-producing cells.
  • the method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse.
  • the mammal is optionally primed for ascites production by prior administration of a suitable composition, for example, Pristane.
  • Antibodies may also be obtained by employing routine recombinant methods such as described in Sambrook et a/. (1989) supra.
  • nucleic acid sequences of the disclosure can be cloned into a suitable expression vector (which contains control sequences for transcription, such as a promoter).
  • the expression vector is in turn introduced into a host cell.
  • the host cell is grown under suitable conditions such that the polynucleotide is transcribed and translated into a protein. Heavy and light chains of antibodies may be produced separately, and then combined by disulfide bond rearrangement. Alternatively, vectors with separate polynucleotides encoding each chain of an antibody of the disclosure, or a vector with a single polynucleotide encoding both chains as separate transcripts, may be transfected into a single host cell which may then produce and assemble the entire molecule.
  • the host cell is a higher eukaryotic cell that can provide the normal carbohydrate complement of the molecule. The antibody is thus produced in the host cell can be purified using standard techniques in the art.
  • the agents of this disclosure can be made by any suitable procedure, including by recombinant methods or by chemical synthesis, as appropriate. Peptides which are produced may then be separated from each other by techniques known in the art, including but not limited to, gel filtration chromatography, gel electrophoresis, and reverse-phase HPLC.
  • polypeptidic agents of the disclosure can be chemically synthesized using information provided in this disclosure, in conjunction with standard methods of protein synthesis.
  • a suitable method is the solid-phase Merrifield technique.
  • Automated peptide synthesizers are commercially available, such as those manufactured by Applied Biosystems, Inc. (Foster City, Calif.).
  • a fusion protein comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to collagen; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z or Z-P-Q, wherein X is an amino acid, P and Q are each independently amino acids provided that at least one of P and Q is GIy, and Z is a hydrophilic amino acid.
  • A5 The fusion protein of any preceding Paragraph, wherein the first polypeptide is functional for binding to collagen at the platelet-bound GPVI binding site of collagen.
  • A6 The fusion protein of any preceding Paragraph, wherein the second polypeptide comprises an amino acid sequence of an Ig heavy chain constant part.
  • A7 The fusion protein of any preceding Paragraph, wherein the second polypeptide comprises a hinge region of an immunoglobulin and is functional to prolong the plasma half-life beyond that of a protein consisting of the first polypeptide and the linker.
  • A8 The fusion protein of any preceding Paragraph, wherein the second polypeptide comprises a hinge region and a CH2 region of an immunoglobulin.
  • A9 The fusion protein of any preceding Paragraph, wherein the second polypeptide comprises a hinge region, a CH2 region and a CH3 region of an immunoglobulin.
  • the fusion protein of any preceding Paragraph wherein the first polypeptide comprises an extracellular domain of GPVI or a variant thereof that is functional for binding to collagen; and the second polypeptide comprises an Fc domain of an immunoglobulin or a functional conservative part thereof.
  • A14 The fusion protein of any preceding Paragraph, wherein the linker comprises the amino acid sequence GIy-X-Z and X is GIy.
  • A15 The fusion protein of any preceding Paragraph, wherein P is GIy.
  • A16 The fusion protein of any preceding Paragraph, wherein the linker comprises a diglycine sequence.
  • A17 The fusion protein of any preceding Paragraph, which is expressed in a mammalian cell.
  • a fusion protein comprising: a) an amino acid sequence which, when the protein is administered, results in inhibition of adhesion of platelets to collagen; and b) an antibody-derived amino acid sequence, wherein sequence (a) is linked at its C- terminus to the N-terminus of sequence (b) through a linker comprising a hydrophilic amino acid.
  • sequence (a) is linked at its C- terminus to the N-terminus of sequence (b) through a linker comprising a hydrophilic amino acid.
  • A21 The fusion protein of any of Paragraphs A18 to A20, wherein the hydrophilic amino acid is selected from Arg, Ser, Thr, Lys, His, GIu, Asp and Asn.
  • a fusion protein comprising: a) an amino acid sequence comprising an extracellular domain of GPVI or a variant thereof that is functional for binding to collagen; b) a linker comprising an amino acid sequence GIy-GIy-Z, wherein Z is a hydrophilic amino acid; and c) an amino acid sequence comprising an Fc domain of an immunoglobulin or a functional conservative part thereof.
  • A24 The fusion protein of Paragraph A23, wherein the amino acid sequence (a) is encoded by: (i) a nucleic acid sequence of bases 1 to 807 of SEQ ID No. 2 (figure 8); (ii) a nucleic acid sequence which hybridises to bases 1 to 807 of SEQ ID No. 2 (figure 8); or (iii) a nucleic acid sequence which differs from bases 1 to 807 of SEQ ID No. 2 (figure 8) by virtue of the degeneracy of the genetic code.
  • A26 The fusion protein of any of Paragraphs A23 to A25, wherein the hydrophilic amino acid is selected from Arg, His and Lys.
  • A27 The fusion protein of any of Paragraphs A23 to A26, wherein amino acid sequence (c) is encoded by:
  • a dimer of a polypeptide comprising a) an extracellular domain of GPVI or a variant thereof that is functional for binding to collagen; and b) a linker comprising an amino acid sequence GIy-GIy-Z, wherein Z is a hydrophilic amino acid; and c) an Fc domain of an immunoglobulin or a functional conservative part thereof.
  • a method of treating or preventing thrombosis in a subject comprising administering to the subject a therapeutically effective amount of a fusion protein comprising: a) an amino acid sequence which, when the protein is administered, results in inhibition of collagen-induced platelet activation; and b) a antibody-derived amino acid sequence, wherein sequence (a) is linked at its C- terminus to the N-terminus of sequence (b) through a linker comprising a hydrophilic amino acid.
  • amino acid sequence (a) comprises an extracellular domain of GPVI or a variant thereof that is functional for binding to collagen; and the antibody- derived amino acid sequence comprises an Fc domain of an immunoglobulin or a functional conservative part thereof.
  • the fusion protein comprises sequentially in an N-terminus to C-terminus direction, a first amino acid sequence, a second amino acid sequence and a third amino acid sequence wherein said first amino acid sequence comprises:
  • a dimer of a polypeptide comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to collagen; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z or Z-P-Q, wherein X is an amino acid, P and Q are each independently amino acids provided that at least one of P and Q is GIy, and Z is a hydrophilic amino acid.
  • a dimer of a polypeptide comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to collagen; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z or Z-P-Q, wherein X is an amino acid, P and Q are each independently amino acids provided that at least one of P and Q is GIy, and Z is a hydrophilic amino acid, wherein the first polypeptide binds to collagen competitively with platelet-bound GPVI, the second polypeptide comprises a hinge region of an immunoglobulin and is functional to prolong the plasma half-life beyond that of a protein consisting of the first polypeptide and the linker and wherein the linker comprises the amino acid sequence GIy-X-Z, wherein Z is selected from the group consisting of Arg, Ser, Thr, Asp, GIu , Tyr, Asn and GIn.
  • a dimer of a polypeptide comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to collagen; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z, wherein X is GIy and Z is selected from the group consisting of Lys and Arg, wherein the first polypeptide binds to collagen competitively with platelet-bound GPVI and wherein the second polypeptide comprises a hinge region of an immunoglobulin and is functional to prolong the plasma half-life beyond that of a protein consisting of the first polypeptide and the linker.
  • a fusion protein comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to fibronectin; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z or Z-P-Q, wherein X is an amino acid, P and Q are each independently amino acids provided that at least one of P and Q is GIy, and Z is a hydrophilic amino acid.
  • A40 The fusion protein of any of Paragraphs A36 to A39, wherein the first polypeptide is functional for binding to fibronectin at the platelet-bound GPVI binding site of fibronectin.
  • A41 The fusion protein of any of Paragraphs A36 to A40, wherein the second polypeptide comprises an amino acid sequence of an Ig heavy chain constant region.
  • A42 The fusion protein of any of Paragraphs A36 to A41, wherein the second polypeptide comprises a hinge region of an immunoglobulin and is functional to prolong the plasma half-life beyond that of a protein consisting of the first polypeptide and the linker.
  • A44 The fusion protein of any of Paragraphs A36 to A43, wherein the second polypeptide comprises a hinge region, a CH2 region and a CH3 region of an immunoglobulin.
  • A45 The fusion protein of any of Paragraphs A36 to A44, wherein the first polypeptide comprises an extracellular domain of GPVI or a variant thereof that is functional for binding to fibronectin; and the second polypeptide comprises an Fc domain of an immunoglobulin or a functional conservative part thereof.
  • A46 The fusion protein of any of Paragraphs A36 to A45 wherein Z is selected from the group consisting of Arg, Ser, Thr, Asp, GIu , Tyr, Asn and GIn.
  • A51 The fusion protein of any of Paragraphs 36 to 50, wherein the linker comprises a diglycine sequence.
  • A52 The fusion protein of any of Paragraphs A36 to A51, which is expressed in a mammalian cell.
  • a fusion protein comprising: a) an amino acid sequence which, when the protein is administered, results in inhibition of adhesion of platelets to fibronectin; and b) an antibody-derived amino acid sequence, wherein sequence (a) is linked at its C-terminus to the N-terminus of sequence (b) through a linker comprising a hydrophilic amino acid.
  • the fusion protein of Paragraph A53 or Paragraph A54, wherein the amino acid sequence which results in the inhibition of adhesion of platelets to fibronectin is an extracellular domain of GPVl.
  • A56 The fusion protein of any of Paragraphs A53 to A55, wherein the hydrophilic amino acid is selected from Arg, Ser, Thr, Lys, His, GIu, Asp and Asn.
  • a fusion protein comprising: a) an amino acid sequence comprising an extracellular domain of GPVI or a variant thereof that is functional for binding to fibronectin; b) a linker comprising an amino acid sequence GIy-GIy-Z, wherein Z is a hydrophilic amino acid; and c) an amino acid sequence comprising an Fc domain of an immunoglobulin or a functional conservative part thereof.
  • A59 The fusion protein of Paragraph A58, wherein the amino acid sequence (a) is encoded by: (i) a nucleic acid sequence of bases 1 to 807 of SEQ ID No. 2 (figure 8); (ii) a nucleic acid sequence which hybridises to bases 1 to 807 of SEQ ID No. 2 (figure 8); or
  • nucleic acid sequence which differs from bases 808 to 816 of SEQ ID No. 2 (figure 8) by virtue of the degeneracy of the genetic code A61.
  • A63 A protein having the characteristics of a protein obtained by expressing in a mammalian cell under non-reducing conditions a DNA sequence comprising in a 5' to 3' direction :
  • a method of treating or preventing thrombosis in a subject comprising administering to the subject a therapeutically effective amount of a fusion protein comprising: a) an amino acid sequence which, when the protein is administered, results in inhibition of fibronectin-induced platelet activation; and b) a antibody-derived amino acid sequence, wherein sequence (a) is linked at its C- terminus to the N-terminus of sequence (b) through a linker comprising a hydrophilic amino acid.
  • A66 The method of Paragraph A65, wherein amino acid sequence (a) comprises an extracellular domain of GPvT or a variant thereof that is functional for binding to fibronectin; and the antibody- derived amino acid sequence comprises an Fc domain of an immunoglobulin or a functional conservative part thereof..
  • A67 The method of Paragraph A65 or Paragraph A66, wherein the fusion protein comprises sequentially in an N-terminus to C-terminus direction, a first amino acid sequence, a second amino acid sequence and a third amino acid sequence wherein said first amino acid sequence comprises: A) i) an amino acid sequence encoded by a nucleic acid sequence of bases 1 to
  • a dimer of a polypeptide comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to fibronectin; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z or Z-P-Q, wherein X is an amino acid, P and Q are each independently amino acids provided that at least one of P and Q is GIy, and Z is a hydrophilic amino acid.
  • a dimer of a polypeptide comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to fibronectin; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z or Z-P-Q, wherein X is an amino acid, P and Q are each independently amino acids provided that at least one of P and Q is GIy, and Z is a hydrophilic amino acid, wherein the first polypeptide binds to fibronectin competitively with platelet-bound GPVI, the second polypeptide comprises a hinge region of an immunoglobulin and is functional to prolong the plasma half-life beyond that of a protein consisting of the first polypeptide and the linker and wherein the linker comprises the amino acid sequence GIy-X-Z, wherein Z is selected from the group consisting of Arg, Ser, Thr, Asp, GIu , Tyr, Asn and GIn.
  • a dimer of a polypeptide comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to fibronectin; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z, wherein X is GIy and Z is selected from the group consisting of Lys and Arg, wherein the first polypeptide binds to fibronectin competitively with platelet-bound GPVI and wherein the second polypeptide comprises a hinge region of an immunoglobulin and is functional to prolong the plasma half-life beyond that of a protein consisting of the first polypeptide and the linker.
  • a fusion protein comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to vitronectin; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z or Z-P-Q, wherein X is an amino acid, P and Q are each independently amino acids provided that at least one of P and Q is GIy, and Z is a hydrophilic amino acid.
  • A74 The fusion protein of any of Paragraphs A71 to A73, wherein the first polypeptide binds to vitronectin competitively with platelet-bound GPVI.
  • A75 The fusion protein of any of Paragraphs A71 to A73, wherein the first polypeptide is functional for binding to vitronectin at the platelet-bound GPVI binding site of vitronectin.
  • A76 The fusion protein of any of Paragraphs A71 to A74, wherein the second polypeptide comprises an amino acid sequence of an Ig heavy chain constant region.
  • A77 The fusion protein of any of Paragraphs A71 to A75, wherein the second polypeptide comprises a hinge region of an immunoglobulin and is functional to prolong the plasma half-life beyond that of a protein consisting of the first polypeptide and the linker.
  • A78 The fusion protein of any of Paragraphs A71 to A76, wherein the second polypeptide comprises a hinge region and a CH2 region of an immunoglobulin.
  • A79 The fusion protein of any of Paragraphs A71 to A78, wherein the second polypeptide comprises a hinge region, a CH2 region and a CH3 region of an immunoglobulin.
  • A80 The fusion protein of any of Paragraphs A71 to A79, wherein the first polypeptide comprises an extracellular domain of GPVI or a variant thereof that is functional for binding to vitronectin; and the second polypeptide comprises an Fc domain of an immunoglobulin or a functional conservative part thereof.
  • A81 The fusion protein of any of Paragraphs A71 to A80 wherein Z is selected from the group consisting of Arg, Ser, Thr, Asp, GIu , Tyr, Asn and GIn.
  • A87 The fusion protein of any of Paragraphs A71 to A86, which is expressed in a mammalian cell.
  • a fusion protein comprising: a) an amino acid sequence which, when the protein is administered, results in inhibition of adhesion of platelets to vitronectin; and b) an antibody-derived amino acid sequence, wherein sequence (a) is linked at its C- terminus to the N-terminus of sequence (b) through a linker comprising a hydrophilic amino acid.
  • A91 The fusion protein of any of Paragraphs A88 to A90, wherein the hydrophilic amino acid is selected from Arg, Ser, Thr, Lys, His, GIu, Asp and Asn.
  • a fusion protein comprising: a) an amino acid sequence comprising an extracellular domain of GPVI or a variant thereof that is functional for binding to vitronectin; b) a linker comprising an amino acid sequence GIy-GIy-Z, wherein Z is a hydrophilic amino acid; and c) an amino acid sequence comprising an Fc domain of an immunoglobulin or a functional conservative part thereof.
  • A94 The fusion protein of Paragraph A93, wherein the amino acid sequence (a) is encoded by: (i) a nucleic acid sequence of bases 1 to 807 of SEQ ID No. 2 (figure 8); (H) a nucleic acid sequence which hybridises to bases 1 to 807 of SEQ ID No. 2 (figure 8); or
  • nucleic acid sequence which differs from bases 808 to 816 of SEQ ID No. 2 (figure 8) by virtue of the degeneracy of the genetic code A96.
  • A98 A protein having the characteristics of a protein obtained by expressing in a mammalian cell under non-reducing conditions a DNA sequence comprising in a 5' to 3' direction :
  • a dimer of a polypeptide comprising a) an extracellular domain of GPVI or a variant thereof that is functional for binding to vitronectin; and b) a linker comprising an amino acid sequence GIy-GIy-Z, wherein Z is a hydrophilic amino acid; and c) an Fc domain of an immunoglobulin or a functional conservative part thereof.
  • a method of treating or preventing thrombosis in a subject comprising administering to the subject a therapeutically effective amount of a fusion protein comprising: a) an amino acid sequence which, when the protein is administered, results in inhibition of vitronectin -induced platelet activation; and b) a antibody-derived amino acid sequence, wherein sequence (a) is linked at its C- terminus to the N-terminus of sequence (b) through a linker comprising a hydrophilic amino acid.
  • amino acid sequence (a) comprises an extracellular domain of GPVI or a variant thereof that is functional for binding to vitronectin; and the antibody- derived amino acid sequence comprises an Fc domain of an immunoglobulin or a functional conservative part thereof.
  • the fusion protein comprises sequentially in an N-terminus to C-terminus direction, a first amino acid sequence, a second amino acid sequence and a third amino acid sequence wherein said first amino acid sequence comprises: A) i) an amino acid sequence encoded by a nucleic acid sequence of bases 1 to
  • a dimer of a polypeptide comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to vitronectin; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z or Z-P-Q, wherein X is an amino acid, P and Q are each independently amino acids provided that at least one of P and Q is GIy, and Z is a hydrophilic amino acid.
  • a dimer of a polypeptide comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to vitronectin; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z or Z-P-Q, wherein X is an amino acid, P and Q are each independently amino acids provided that at least one of P and Q is GIy, and Z is a hydrophilic amino acid, wherein the first polypeptide binds to vitronectin competitively with platelet-bound GPVI, the second polypeptide comprises a hinge region of an immunoglobulin and is functional to prolong the plasma half-life beyond that of a protein consisting of the first polypeptide and the linker and wherein the linker comprises the amino acid sequence GIy-X-Z, wherein Z is selected from the group consisting of Arg, Ser, Thr, Asp, GIu , Tyr, Asn and GIn.
  • a dimer of a polypeptide comprising: a) a first polypeptide which is capable of inhibiting adhesion of platelets to vitronectin; b) a second, antibody-derived polypeptide; and c) a linker comprising an amino acid sequence GIy-X-Z, wherein X is GIy and Z is selected from the group consisting of Lys and Arg, wherein the first polypeptide binds to vitronectin competitively with platelet-bound GPVI and wherein the second polypeptide comprises a hinge region of an immunoglobulin and is functional to prolong the plasma half-life beyond that of a protein consisting of the first polypeptide and the linker.
  • polypeptides which comprise some or all of a GPVI protein which is capable of binding to fibronectin and/or vitronectin. Such a polypeptide may competitively inhibit or prevent platelet-bound GPVI binding to fibronectin and/or vitronectin.
  • soluble GPVI proteins i.e. soluble proteins comprising a sequence derived from GPVI.
  • a soluble GPVI protein may be a GPVI fusion protein.
  • the fusion protein may comprise an extracellular domain of GPVI fused to a heterologous peptide sequence.
  • the heterologous peptide sequence may be an Fc constant region.
  • a soluble GPVI protein for example a GPVI-Fc fusion protein
  • a soluble GPVI protein for example a GPVI-Fc fusion protein
  • vitronectin activity or function for example a GPVI-Fc fusion protein
  • an agent described herein may be used in various methods of treating vascular disorders.
  • the agents of the disclosure bind effectively to an atherosclerotic disorder, particularly a ruptured or fissured plaque, when delivered locally, thus avoiding the necessity of systemic administration at clinical levels.
  • local administration e.g. by a catheter, of an agent of the disclosure to a site, or suspected site, of an atherosclerotic disorder.
  • such an agent may be administered to a patient via a catheter to treat, for example, plaque disruption (e.g. rupture, fission) stenosis or restenosis or other thrombotic complications.
  • the agent is a fusion protein comprising an extracellular domain of GPVI fused to an immunoglobulin Fc portion via a linker.
  • the fusion protein is PR-15, a fusion protein comprising the sequence disclosed in Figure 7.
  • the agents of the disclosure bind to the target molecule with a high degree of affinity.
  • Binding affinity of an agent for example an antibody or fusion protein may be measured using for example BIACORE systems.
  • the strength of the binding of an agent to a peptide sequence of GPVI may be analysed using chemiluminescence and quantified by measuring signal intensity.
  • amino acid sequence of the ligand for example, GPVI, fibronectin and/or vitronectin, to which the agent binds may be modified by one or more changes in sequence which do not eliminate the underlying biological function and utility of the agents as described herein.
  • the disclosure includes agents which have the function of inhibiting GPVI interaction and/or association with fibronectin and/or vitronectin and/or collagen, including all variant forms which have such functionality, in particular clinically useful functionality, especially ability to inhibit GPVI binding with one, two or particularly all three of fibronectin, vitronectin and collagen.
  • proteins and other poly(amino acids) may be derivatised as by glycosylation, for example, to modify their properties.
  • Other modifications included within the disclosure include without limitation attachment of natural or synthetic polymers (e.g. a polyethylene glycol or dextran) albumin affinity tags (see for example Bioorg Med Chem Lett. 2002 Oct 21;12(20):2883-6).
  • peptides containing one or more d-amino acids are also included.
  • an indwelling device may be any suitable medical substrate that can be placed in a human or veterinary patient.
  • the device may not be limited to permanently implanted devices and can include any device that comes into contact with blood, for example a surgeon's knife.
  • a coating or impregnant can be formed on or in an indwelling device or prosthesis or other medical instruments or articles.
  • the agent will be released, e.g. at a desired rate and for a predetermined duration of time, at the site of implantation.
  • a bioactive agent can be coupled to the surface of a medical device by surface modification, embedding or integration and released from within polymeric materials (matrix-type), or surrounded by and released through a carrier (reservoir-type).
  • polymeric materials in such applications should optimally act as a biologically inert barrier and not induce further inflammation within the body.
  • a coating composition can be used to coat a device or implant surface using any suitable means, e.g., by dipping, spraying and the like.
  • the suitability of the coating composition for use on a particular material, and in turn, the suitability of the coated composition can be evaluated by those skilled in the art, given the present description.
  • the polymeric materials should therefore be non-inflammatory and, in the case of a stent, capable of being stretched without flaking or delaminating from the stent, and be able to deliver the drug at a sustained, controlled and predictable rate.
  • Polymer systems that can meet these requirements are, for example, biodegradable polymers, such as polyglycolic-polylactic acid, polyethyleneoxidepolybutylene terephthalate, and polyorthoester or non-degradable polymers, such as Biogold polyamine-heparin, and methacryloylphosphorylcholine-laurylmethacrylate, for example.
  • Exemplary implantable devices include but are not limited to drug-delivering vascular stents; other vascular devices (e.g., grafts, catheters, valves, artificial hearts, heart assist devices), implantable defibrillators, blood oxygenator devices; surgical devices e.g.
  • tissue-related materials tissue-related materials
  • membranes cell culture devices
  • chromatographic support materials biosensors, shunts for hydrocephalus, wound management devices, endoscopic devices, infection control devices, orthopedic devices, dental devices, urological devices, colostomy bag attachment devices, ophthalmic devices, glaucoma drain shunts, synthetic prostheses, intraocular lenses, respiratory, peripheral cardiovascular, spinal, neurological, dental, and ear/nose/throat devices (e.g., ear drainage tubes), renal devices, and dialysis articles (e.g., tubing, membranes, grafts).
  • contemplated devices include self-expanding stents (e.g., made from nitinol), balloon-expanded stents (e.g., prepared from stainless steel), degradable coronary stents, non-degradable coronary stents, peripheral coronary stents, urinary catheters (e.g., surface-coated with antimicrobial agents), penile implants, sphincter devices, urethral devices, bladder devices, renal devices, vascular implants and grafts, intravenous catheters (e.g., treated with antithrombotic agents), small diameter grafts, artificial lung catheters, electrophysiology catheters, anastomosis devices, vertebral disks, bone pins, suture anchors, hemostatic barriers, clamps, surgical staples/sutures/screws/plates/clips, atrial septal defect closures, electro-stimulation leads for cardiac rhythm management (e.g., pacer leads), glucose sensors (long- term and
  • Implantable devices may be categorised, such as belonging to the groups medical devices, dental devices, orthopedic devices, diagnostic devices, surgical devices, cell culture devices, urological devices, wound management devices, endoscopic devices, infection control devices, urological devices, colostomy bag attachment devices, ophthalmic devices, renal devices and dialysis.
  • the devices as listed above may be microporous.
  • a device may comprise nano bumps in order to closer mimic a smooth muscle vessel.
  • Indwelling devices e.g. implants, suitably further comprise a further bioactive agent or drug material, e.g. one selected from the group consisting of protein kinase inhibitors, antiproliferative agents, antimitotic agents, antibiotics, antimetabolites, anticoagulants, fibrolytic agents, antimigratory agents, antisecretory agents, anti-inflammatory agents, non-steroidal agents, angiogenic agents, anti-angiogenic agents, immunosuppressive agents, pyrimidine analogues, purine analogues, spleen tyrosine kinase (SYK) inhibitors and combinations thereof.
  • a further bioactive agent or drug material e.g. one selected from the group consisting of protein kinase inhibitors, antiproliferative agents, antimitotic agents, antibiotics, antimetabolites, anticoagulants, fibrolytic agents, antimigratory agents, antisecretory agents, anti-inflammatory agents, non-steroidal agents, angiogenic agents, anti-angiogenic
  • An. aspect of the invention is a method of inhibiting or preventing restenosis, thrombosis, atherogenesis, atheroprogression, atherosclerosis, and/or vascular inflammation in a patient, said method comprising implanting in said patient an intravascular device comprising a direct or indirect GPVl inhibitor adapted to be exposed and/or released when the device is implanted.
  • One particular device is a stent.
  • Stents Intravascular stenting of arteries is one of the most frequent operations in cardiovascular surgery.
  • Stents metal woven fabric cylinders
  • Stents are inserted into a vessel with injured inner wall to avoid its destruction, thrombus formation or vasoconstriction.
  • metal stents provoke thrombus formation.
  • the coronary artery response to stent implantation leads to a complex and largely predictable sequence of events.
  • the events are related to inflammation and repair processes, which are known to be natural compensatory mechanisms.
  • the temporal sequence of events to stent implantation can be divided into acute and chronic phases.
  • thrombosis disturbances predominate.
  • tissue remodeling reaches primacy.
  • neointimal hyperplasia and restenosis are protracted in duration, and deeper into the vessel wall during the chronic phase.
  • a stent having a coating or impregnant which contains at least one disclosed agent is useful for a variety of medical procedures, including, by way of example, treatment of obstructions caused by tumors in bile ducts, esophagus, trachea/bronchi and other biological passageways.
  • a stent having the above-described coating is particularly useful for treating occluded regions of blood vessels caused by abnormal or inappropriate migration and proliferation of smooth muscle cells, thrombosis, and restenosis.
  • Stents may be placed in a wide array of blood vessels, both arteries and veins. Representative examples of sites include the iliac, renal, and coronary arteries.
  • DES drug-eluting stents
  • antimicrobial agents e.g., antibiotics such as vancomycin or norfloxacin
  • incorporation of antimicrobial agents may also prove beneficial as additional active agents.
  • the present agent is for the treatment of a thromboembolic disorder which may be selected from unstable angina, an acute coronary syndrome, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from (a) prosthetic valves or other implants, (b) indwelling catheters, (c) stents, (d) cardiopulmonary bypass, (e) hemodialysis, or (f) other procedures in which blood is exposed to an artificial surface that promotes thrombosis
  • a thromboembolic disorder which may be selected from unstable angina, an acute coronary syndrome, first myocardial infarction
  • the product of the present invention has a dual action, for example, it has a direct or indirect diagnostic function or a therapeutic (including prophylactic) function by another mode of action.
  • the disclosure includes an agent which comprises: a first portion capable of binding to an intravascular GPVI-binding domain; and a second portion which is capable of binding to a site other than a GPVI-binding domain and/or comprises a therapeutic or diagnostic moiety, or a combination thereof.
  • the second portion may be capable of binding to a binding site for a platelet-bound ligand other than GPVI, for example GPIa, GPIb, GPIc, GPIIa, GPIIb, GPIIIa, GPIX, GPIIb/IIIa, GPIa/IIa, GPIV, GPIc/IIa, GPIb/IX.
  • the second portion may comprise an amino acid sequence derived from a platelet receptor other than GPVI, or a sequence having homology to at least part of such sequence.
  • agents having a second portion which: is functional to interfere with, modulate or confer the activity of platelet derived growth factor (PDGF), glycoprotein IBa, thrombomodulin, vascular epidermal growth factor, transforming growth factor- ⁇ l, basic fribroblast growth factor, angiotensin II, factor VIII, von Willebrand factor; or comprises a protein kinase inhibitor, an antiproliferative agent, an antimitotic agent, an antibiotic, an antimetabolite, an anticoagulant, a fibrolytic agent, an antimigratory agent, an antisecretory agent, an anti-inflammatory agent, a non-steroidal agent, an angiogenic agent, an anti- angiogenic agent, an immunosuppressive agent, a pyrimidine analogue, a purine analogue, or a spleen tyrosine kinase (SYK) inhibitor.
  • PDGF platelet derived growth factor
  • glycoprotein IBa glycoprotein IBa
  • thrombomodulin vascular
  • the second portion may confer anti-platelet activity, for example inhibitory activity against platelet adhesion and/or platelet aggregation.
  • the second portion may confer anticoagulant activity.
  • the second portion comprises a matrix metalloprotease inhibitor.
  • the second portion is either proliferative (particularly to aid wound healing) or antiproliferative (to help prevent atherosclerosis or (re)stenosis.
  • the disclosure includes embodiments in which the second portion comprises an amino acid sequence derived from platelet derived growth factor (PDGF), a platelet receptor protein (e.g. a platelet-bound glycoprotein) hirudin, thrombomodulin, vascular epidermal growth factor, transforming growth factor- ⁇ l, basic fibroblast growth factor, angiotensin II, factor VIII, von Willebrand factor, tick anticoagulant protein (TAP) or nematode anticoagulant protein (NAP), or a sequence having homology to at least part of such sequence.
  • PDGF platelet derived growth factor
  • a platelet receptor protein e.g. a platelet-bound glycoprotein
  • hirudin hirudin
  • thrombomodulin hirudin
  • vascular epidermal growth factor transforming growth factor- ⁇ l
  • basic fibroblast growth factor
  • the second portion may comprise a domain selected from one or more of the group consisting of protein kinase inhibitors, antiproliferative agents, antimitotic agents, antibiotics, antimetabolites, pyrimidine analogs, purine analogs, anticoagulants, fibrinolytic agents, antiplatelet agents, antimigratory agents, antisecretory agents, anti-inflammatory agents, non-steroidal agents, immunosuppressive agents, angiogenic agents, ACE inhibitors, actin inhibitors, analgesics, anesthetics, anti-hypertensives, anti polymerases, antisecretory agents, anti-AIDS substances, anticancer substances, anti-cholinergics, anti-coagulants, anti-convulsants, anti-depressants, anti- emetics, antifungals, anti-glaucoma solutes, antihistamines, antihypertensive agents, antiinflammatory agents (such as NSAIDs), anti metabolites, antimitotics, antioxidants, anti-parasite and/or anti-
  • the dual action products may include an additional agent which interacts with other factors, including protein factors, that are involved in the clotting cascade which may include coagulation factors I-XIII (for example, fibrinogen, prothrombin, tissue thromboplastin, calcium, proaccelerin (accelerator globulin), proconvertin (serum prothrombin conversion accelerator), antihemophilic factor, plasma thromboplastin component, Stuart factor (autoprothrombin C), plasma thromboplastin antecedent (PTA), Hageman factor, and fibrin-stabilizing factor (FSF, fibrinase, protransglutaminase)).
  • coagulation factors I-XIII for example, fibrinogen, prothrombin, tissue thromboplastin, calcium, proaccelerin (accelerator globulin), proconvertin (serum prothrombin conversion accelerator), antihemophilic factor, plasma thromboplastin component, Stuart factor (autoprothrombin C), plasma thromboplastin
  • the second portion may comprise an immunosuppressive agent, e.g. rapamycin or a rapamycin derivative.
  • Rapamycin may be used for the treatment of AV graft stenosis since it has been shown to significantly reduce in-stent restenosis and prevent chronic organ rejection. Rapamycin is a potent inhibitor of cytokine and growth factor-mediated smooth muscle cell proliferation. Other surface adhesion molecule or cell-cell adhesion molecules may also function to promote coagulation or thrombosis.
  • Exemplary cell adhesion molecules or attachment proteins include laminin, collagen, elastin, tenascin, fibrinogen, thrombospondin, osteopontin, von Willebrand Factor, bone sialoprotein (and active domains thereof), or a hydrophilic polymer such as hyaluronic acid, chitosan or methyl cellulose, and other proteins, carbohydrates, and fatty acids.
  • Exemplary cell-cell adhesion molecules include N-cadherin and P-cadherin and active domains thereof. It is therefore contemplated that certain products of the present invention will interact with at least one of these.
  • a conjugate comprising an agent of the present disclosure linked to one or more heterologous molecules.
  • a conjugate of the present disclosure may be used as, for example but not limited to, a drug delivery agent or a multifunctional therapeutic agent.
  • a pharmaceutical composition comprising a conjugate according to the present disclosure. Methods of treatment and uses of the conjugates to manufacture a medicament to treat a disorder are also included in the present disclosure. Further aspects of the disclosure include kits comprising conjugates of the present disclosure, and pharmaceutical compositions comprising a conjugate of the present disclosure together with another active agent or drug. Such active agents and drugs include, but are not limited to, those described herein.
  • the conjugate comprises (1) an agent, which is a polypeptide, linked to (2) a second polypeptide.
  • the second polypeptide has a different activity from the polypeptide agent.
  • the agent is a soluble GPVI polypeptide, and is optionally a GPVI fusion protein.
  • the conjugate comprises a GPVI-Fc fusion protein linked to a second polypeptide.
  • the agent is linked to a non- polypeptide molecule such as a small molecule drug.
  • the conjugate in other embodiments, comprises other agents of the disclosure as described herein.
  • conjugates of the disclosure may be bi- or multifunctional in their activities.
  • the agent is conjugated to a molecule which is capable of modulating the expression or activity of factors involved in, for example but not limited to, blood coagulation, leukocyte recruitment, immune system activation, tissue fibrosis and tumorigenesis.
  • conjugates of the disclosure may be rendered multifunctional so that they are active against more than platelet interaction with the vasculature, for example another component in the clotting cascade (eg thrombin activity), or the intracellular signaling cascade (eg growth factor).
  • another component in the clotting cascade eg thrombin activity
  • the intracellular signaling cascade eg growth factor
  • the conjugate comprises the agent linked to a cytotoxic drug or toxin.
  • the agent is a monoclonal antibody which is capable of being internalized by a target cell. Upon administration to a patient, the conjugate binds to target cells through their antibody portions and becomes internalized, allowing the drugs or toxins to exert their cytotoxic or cytostatic effects.
  • the components of the conjugate may be covalently bonded to each other either directly or through a linking group.
  • the conjugate may comprise a linker between the agent and the heterologous molecule.
  • the agent and heterologous molecule may be linked directed.
  • the linker is selected from the group consisting of: a flexible linker, an inflexible linker, a linker which is capable of being cleaved further. It will be apparent to one skilled in the art that alternative linkers can be used to link first and second parts of the conjugate.
  • a flexible linker may be about 20 or fewer amino acids in length.
  • the conjugate comprises an agent which is capable of acting as a presenter protein ligand.
  • a conjugate which is a bifunctional molecule is synthesized by covalently linking an endogenous presenter protein ligand to a drug moiety, either directly or through a linking group.
  • the host endogenous presenter protein ligand of the conjugate may then act, when administered, to direct the drug moiety to a target presenter protein in the host.
  • the conjugate comprises an agent which binds to a protein selected from GPVI, fibronectin, vitronectin and/or collagen, linked to a drug moiety.
  • the agent When administered, the agent may then act to direct the drug moiety to presenter protein, namely a protein which interacts with the agent.
  • the agent binds to a protein which is a target for the drug moiety.
  • the agent binds to one or more of the proteins selected from GPVI, fibronectin, vitronectin and collagen.
  • the drug moiety is active against one or more of the proteins to which the agent binds.
  • the conjugate binds to the presenter protein to produce a binary complex.
  • the presenter protein to which the ligand of the conjugate binds may be any protein that is present in the host at the time the conjugate is introduced to the host, i.e. the presenter protein will be endogenous to the host.
  • the conjugate comprises (1) a GPVl fusion protein, linked to (2) a drug moiety or a peptide moiety.
  • the conjugate comprises a GPVI fusion protein which comprises an extracellular domain of GPVI linked to an Fc portion via a linker comprising a GIy- Gly-Arg sequence.
  • the conjugate comprises a GPVI fusion protein as described herein.
  • soluble GPVI is capable of adhering to the location where it is locally released.
  • a soluble GPVI for example in the form of a GPVI fusion protein, in a conjugate with a drug moiety or peptide moiety would act to ensure that the drug or peptide was not swept away in the blood following local delivery.
  • the use of soluble GPVI in a conjugate with a drug moiety or peptide moiety may act to address the problem of locally delivered drugs being swept away by the blood after delivery.
  • soluble GPVI is considered to include any protein or polypeptide which comprises at least an extracellular domain of GPVI or functional portion thereof. Soluble GPVI may include, but is not limited to, GPVI fusion proteins.
  • the drug moiety may include, but is not limited to, any of the compounds or drug described herein.
  • the conjugate comprises a cleavable linker between the agent and the drug moiety or peptide moiety.
  • the agent may act to "slow down” or retard the removal of the drug by the blood stream from the local area of delivery.
  • the drug moiety may be any molecule, as well as a fragment for example a binding portion, thereof, that is capable of modulating a biological process in a living host, either by itself or in the context of the presenter protein/conjugate binary complex.
  • the drug moiety is a small organic molecule that is capable of binding to a target of interest.
  • the drug moiety of the conjugate will include one or more functional groups necessary for structural interaction with the target, e.g. groups necessary for hydrophobic, hydrophilic, electrostatic or even covalent interactions, depending on the particular drug and its intended target.
  • the target is a protein
  • the drug moiety will include functional groups necessary for structural interaction with proteins, such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, etc., and will typically include at least an amine, amide, sulfhydryl, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the drug moiety will also comprise a region that may be modified and/or participate in covalent linkage to the other components of the conjugate, such as the presenter protein ligand or linker, without substantially adversely affecting the moiety's ability to bind to its target.
  • the drug moiety of the conjugate may be derived from a naturally occurring or synthetic compound that may be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the preparation of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc.
  • a diagnostic or visualisation agent comprising an agent of the present disclosure linked to a moiety to enable the location of the agent to be detected e.g. a detectable moiety or a moiety for binding to a dectable moiety or entity.
  • detectable moiety may include molecules that are readily detectable by a medical imaging system.
  • the detectable moiety is selected from the group consisting of electron- opaque molecules, for example gold particles, radioisotopes, for example technetium, and stable isotope-labeled molecules.
  • the detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety is selected from a radiolabel, a fluorophore, a chromophore, an imaging agent and a metal ion.
  • the detectable moiety may be a radioisotope, for example 1-123, 1-124 and 1-131, Tc-99m, Re-186 and Re-188, particularly Tc-99m; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • radioiodine radioiodine (radioactive iodine isotopes) as a component of an agent of the disclosure. Particularly, radioiodine is excluded when the agent is the agent of Paragraph 15 below.
  • Radio-labelling of biomolecules has been used as a means to track and detect the pathway or location of a particular biomolecule when administered to a patient or subject.
  • Such radio-labelled biomolecules are capable of emitting low levels of radiation, which can be detected and pin-pointed to a target organ or other substrate.
  • Radionuclides such as rhenium-186m and, particularly, technetium-99m, are useful for biomolecule labelling since they are known to form relatively stable bonds with a variety of biomolecules and may be used in the imaging of patients.
  • the detetable moiety may be administered as a separate entity which binds to the visualisation imaging or locating agent in vivo through a binding partner e.g. an antibody product.
  • the agent is a protein, antibody or fragment thereof.
  • the protein is a soluble GPVI protein, for example a GPVI fusion protein.
  • Other agents described herein are also encompassed in this aspect of the disclosure.
  • a method for diagnosis is also provided in which the imaging agent is administered to a subject suspected of having an atherosclerotic disorder, for example a dysfunctional endothelium, microscopic atheroscelerotic lesions and/ or macroscopic atherosclerotic lesions, and the distribution of the imaging agent within the body of the subject is measured or monitored.
  • the imaging agents of the disclosure are useful for in vivo imaging, wherein an imaging agent linked to a detectable moiety such as a radio-opaque agent or radioisotope is administered to a subject, preferably into the bloodstream, and the presence and location of the imaging agent in the host is assayed.
  • This imaging technique is useful in the staging and treatment of atherosclerosis or thrombosis or other cardiovascular disorders.
  • the technique may also be useful in the staging and treatment of cancers.
  • the imaging agent may comprise any moiety that is detectable in a host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • the imaging agent may further comprise polymerized vesicles, which are composed of polymerized lipids and lipids that are chelated to gadolinium. These vesicles counteract the instability of pure liposomes while avoiding the clumping and tangling of pure polymers.
  • the resulting vesicle is round and flexible, and it interacts well with surfaces where biomarkers such as collagen, fibronectin and/ or vitronectin may be located.
  • Agents for example proteins and antibodies, specific to the biomarker can be attached to the vesicle and injected into the blood.
  • the amount of imaging agent accumulating at target sites may increase substantially over imaging agents without the vesicles.
  • the present disclosure further includes methods of amplifying the signal produced by the imaging agent. These are important to ensure that a signal sufficient for detection is produced. Thus, accumulation of the imaging agent at the target site may be helpful. In this instance, use in the imaging agent of an agent which binds to plural binding sites as described herein, e.g. selected from fibronectin, vitronectin and collagen, will potentially ensure that the imaging agent accumulates at site which expresses fibronectin, vitronectin and/or collagen in quantities sufficient to produce a detectable signal.
  • the imaging agent may comprise a molecule which causes amplification of the imaging agent, for example an enzyme.
  • the present disclosure also includes methods of detecting an imaging agent of the present disclosure comprising administering the imaging agent to a subject and using a method to detect the signal produced by the imaging agent.
  • Such methods may include CT, MR, optical imagining, ultrasound, and PET.
  • a method of diagnosing one of the following conditions in a subject (a) inflamed endothelium of a blood vessel;
  • a method for preparing a hybridoma cell-line producing monoclonal antibodies comprising the steps of: i) immunising an immunocompetent mammal with an immunogen for example, GPVI, fibronectin, vitronectin or fragments thereof; ii) fusing lymphocytes of the immunised immunocompetent mammal with myeloma cells to form hybridoma cells; iii) screening monoclonal antibodies produced by the hybridoma cells of step (ii) for binding activity to the amino acid sequences of (i); iv) culturing the hybridoma cells to proliferate and/or to secrete said monoclonal antibody; and v) recovering the monoclonal antibody from the culture supernatant.
  • an immunogen for example, GPVI, fibronectin, vitronectin or fragments thereof
  • fusing lymphocytes of the immunised immunocompetent mammal with myeloma cells to form hybridoma cells
  • the said immunocompetent mammal is a mouse.
  • said immunocompetent mammal is a rat.
  • an agent to bind human glycoprotein VI, fibronectin and/or vitronectin or fragments thereof may be tested by immunoassay. Any form of direct binding assay is suitable.
  • the human glycoprotein VI, fibronectin and/or vitronectin or alternatively the agent is labeled. Suitable labels include radioisotopes such as 125 I, enzymes such as peroxidase, fluorescent labels such as fluorescein, and chemiluminescent labels.
  • the other binding partner is insolubilized (for example, by coating onto a microtiter plate) to facilitate washing. After combining the labeled component with the insolubilized component, the solid phase is washed and the amount of bound label is determined.
  • the agent is titered for its ability to decrease the binding of, for example, an anti-GPVI antibody e.g. hGP 5C4 Fab to human glycoprotein VI, or human glycoprotein VI to fibronectin and/or vitronectin. Either of the binding pairs in the reaction to be inhibited is labeled, while the other is typically insolubilized in order to facilitate washing.
  • an anti-GPVI antibody e.g. hGP 5C4 Fab to human glycoprotein VI, or human glycoprotein VI to fibronectin and/or vitronectin.
  • the present invention relates in some respects to treating a subject with a condition in which inhibition cardiovascular disease, particularly atherosclerosis and/or thrombosis, is required or desired.
  • the referenced agents find application in the primary prophylaxis of atherosclerotic disorders. They find application in the secondary prophylaxis of atherosclerotic disorders, after an atherosclerotic event or suspected atherosclerotic event, e.g. atherothrombosis.
  • an atherosclerotic event or suspected atherosclerotic event e.g. atherothrombosis.
  • venous thrombosis e.g, DVT
  • pulmonary embolism e.g. in myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis
  • systemic embolism usually from the atrium during arterial fibrillation or from the left ventricle after transmural myocardial infarction, or caused by congestive heart failure
  • prophylaxis of re- occlusion ie thrombosis
  • PTA percutaneous trans-luminal angioplasty
  • coronary bypass operations the prevention of re-thrombosis after microsurgery and vascular surgery in general.
  • Further indications include the therapeutic and/or prophylactic treatment of disseminated intravascular thrombosis caused by bacteria, multiple trauma, or any other mechanism; anticoagulant treatment when blood is in contact with foreign surfaces in the body such as vascular grafts, vascular stents, vascular catheters, mechanical and biological prosthetic valves or any other medical device; and anticoagulant treatment when blood is in contact with medical devices outside the body such as during cardiovascular surgery using a heart-lung machine or in haemodialysis; the therapeutic and/or prophylactic treatment of idiopathic and adult respiratory distress syndrome, pulmonary fibrosis following treatment with radiation or chemotherapy, septic shock, septicemia, inflammatory responses, which include, but are not limited to, edema, acute or chronic atherosclerosis such as coronary arterial disease, cerebral arterial disease, peripheral arterial disease, reperfusion damage, and restenosis after percutaneous trans-luminal angioplasty (PTA).
  • PTA percutaneous trans-luminal angioplasty
  • Conditions which may be treated include thrombosis, especially DVT, including distal and proximal DVT.
  • the present disclosure finds particular utility in the prophylactic treatment of DVT resulting from surgery, such as gastrointestinal, or orthopaedic, surgery (e.g. hip or knee replacement). This includes DVT resulting from immobilisation after surgery.
  • Suitable doses of the described agents, in the therapeutic and/or prophylactic treatment of mammalian, especially human, patients may be determined routinely by the medical practitioner or other skilled person.
  • the physician, or the skilled person will be able to determine the actual dosage which will be most suitable for an individual patient, which is likely to vary with the condition that is to be treated, as well as the age, weight, sex and response of the particular patient to be treated.
  • the above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this disclosure.
  • the package defined herein may comprise more than one dosage unit, in order to provide for repeat dosing. If more than one dosage unit is present, such units may be the same, or may be different in terms of the dose of active agent composition and/or physical form.
  • a further aspect of the disclosure provides a method of treatment of a condition in which inhibition of thrombin is required or desired, which comprises administration of:
  • treatment includes therapeutic and/or prophylactic treatment.
  • the disclosure provides an agent or a ligand as hereinbefore described for use as a pharmaceutical.
  • polypeptides of the disclosure are at least partially purified from other cellular constituents.
  • the polypeptide is at least about 50% pure, as a weight percent of total protein. More preferably, the protein is at least about 50-75% pure.
  • the polypeptide is preferably at least about 90% pure, typically 98% pure or more.
  • a pharmaceutical formulation comprising an agent, a ligand or a humanised antibody as hereinbefore described.
  • the formulation may contain at least one additional pharmaceutically acceptable component, e.g. an excipient, diluent or carrier.
  • the formulation may be for parenteral administration.
  • the formulation is for oral administration.
  • the agent may be, for example, a single-chain antibody or other antibody fragment which is suitable for oral administration; it may by way of example be a VHH or aAb.
  • the product which comprises an agent of the disclosure may be comprised within a pharmaceutical formulation which is for chronic administration.
  • the pharmaceutical formulation may be for the treatment of atherosclerosis in a subject who has atherosclerotic plaques or who does not have atherosclerotic plaques but is at risk thereof.
  • a pharmaceutical formulation comprising a ligand described herein, for example, GPVI, fibronectin and/or vitronectin or fragments thereof; in embodiments the formulation is a composition comprising the ligand and a pharmaceutically acceptable diluent, carrier or excipient.
  • the described ligands for use as a pharmaceutical may be an intravenous formulation.
  • the agents of the present disclosure may also be used in methods of inhibiting platelet aggregation, wherein the method comprises contacting platelets with an effective amount of the agent as described herein.
  • the method may involve platelets that are in vitro or in vivo.
  • the agents of the present disclosure may also be used in methods of treating a disease or disorder associated with pathological, dysfunctional or non-pathological interaction between platelet-bound GPVI and fibronectin.
  • the method may comprise administering an agent, or a product which comprises an agent, which inhibits the binding of platelet bound GPVI to fibronectin to a subject with the disease or disorder or at risk of developing the disease or disorder.
  • a therapeutically effective amount of the agent or product is administered.
  • the agents of the present disclosure may also be used in methods of treating a disease or disorder associated with pathological, dysfunctional or non-pathological between platelet-bound GPVI and vitronectin comprising administering an agent or a product which comprises an agent which inhibits the binding of platelet bound GPVI to vitronectin to a subject with the disease or disorder or at risk of developing the disease or disorder.
  • an agent or a product which comprises an agent which inhibits the binding of platelet bound GPVI to vitronectin to a subject with the disease or disorder or at risk of developing the disease or disorder.
  • a therapeutically effective amount of the agent or product is administered.
  • the treatment is therapeutic or prophylactic and the disease or disorder is selected from acute coronary syndromes, cardiovascular thrombosis, cerebrovascular thrombosis, unstable angina, stable angina, angina pectoris, embolus formation, deep vein thrombosis, hemolytic uremic syndrome, hemolytic anemia, acute renal failure, thrombolytic complications, thrombotic thrombocytopenic purpura, disseminated intravascular comgelopathy, thrombosis, coronary heart disease, thromboembolic complications, myocardial infarction, restenosis, and atrial thrombosis formation in atrial fibrillation, chronic unstable angina, transient ischemic attacks and strokes, peripheral vascular disease, arterial thrombosis, pre-eclampsia, embolism, restenosis and/or thrombosis following angioplasty, carotid endarterectomy, anastomosis of vascular grafts, and chronic exposure to cardiovascular devices.
  • acute coronary syndromes
  • the treatment is for therapy or prevention of thromboembolism and reocculsion occurring during and after thrombolytic therapy, after angioplasty, and after coronary artery bypass,
  • the agents of the present disclosure may also be used in methods of preventing or retarding initiation and/or progression of atherosclerotic lesions in a subject with the atherosclerotic lesions or at risk of developing atherosclerotic lesions.
  • a therapeutically effective amount of the agent or a product comprising the agent is administered.
  • the agents of the present disclosure may also be used in methods of treating or reducing advanced atherosclerotic lesions in a subject.
  • a therapeutically effective amount of the agent or a product comprising the agent is administered.
  • the agents of the present disclosure may also be used in the manufacture of a medicament for the treatment of a disease or disorder associated with pathological, dysfunctional or non-pathological interaction between platelet-bound GPVI and vitronectin.
  • the agents of the present disclosure may also be used in the manufacture of a medicament for the treatment of a disease or disorder associated with pathological, dysfunctional or non-pathological interaction between platelet-bound GPVI and fibronectin.
  • the treatment is therapeutic or prophylactic and the a disease or disorder is selected from acute coronary syndromes, cardiovascular thrombosis, cerebrovascular thrombosis, unstable angina, stable angina, angina pectoris, embolus formation, deep vein thrombosis, hemolytic uremic syndrome, hemolytic anemia, acute renal failure, thrombolytic complications, thrombotic thrombocytopenic purpura, disseminated intravascular comgelopathy, thrombosis, coronary heart disease, thromboembolic complications, myocardial infarction, restenosis, and atrial thrombosis formation in atrial fibrillation, chronic unstable angina, transient ischemic attacks and strokes, peripheral vascular disease, arterial thrombosis, pre-eclampsia, embolism, restenosis and/or thrombosis following angioplasty, carotid endarterectomy, anastomosis of vascular grafts, and chronic exposure to cardiovascular devices.
  • the medicament is for treating or preventing thromboembolism and reocculsion occurring during and after thrombolytic therapy, after angioplasty, and after coronary artery bypass.
  • agents which bind GPVI with fibronectin and/or vitronectin and/or collagen can be used in the treatment of any disease, state or condition involving the interaction of GPVI with fibronectin and/or vitronectin and/or collagen.
  • agents of the disclosure which interrupt the interaction between GPVI and fibronectin and/or vitronectin may be useful in treating or preventing atherosclerosis.
  • Based on the recent improvements in imaging techniques by intravascular ultrasound or nuclear magnetic resonance imaging it is possible to identify patients with atherosclerosis being at risk of acute clinical complications such as acute coronary or carotid syndrome, whereby the patients have active lesions as possible causes for intravascular thrombosis. It is then possible by the present teachings to prevent or delay the formation of intravascular thrombosis by the administration of a medicament containing the agents of the invention, advantageously without undesired or unacceptable side effects.
  • Atherosclerotic lesions can be investigated e.g. by intravascular ultrasound or thermography (e.g., Fayed and Fuster, Clinical imaging of the high-risk or vulnerable atherosclerotic plaque. Circulation 2001; 89:305-316) or nuclear resonance imaging (Helft et al., Progression and
  • aspects of the disclosure provide methods of treatment comprising, administration of an agent as provided, pharmaceutical compositions comprising such a agent, and use of such an agent in the manufacture of a medicament for administration, for example in a method of making a medicament or pharmaceutical composition comprising formulating the agent with a pharmaceutically acceptable excipient.
  • the agents of the disclosure may be used in the prophylaxis of atherosclerotic disorders, e.g. initiation or development of atherosclerosis, in patients having one or more markers of vascular inflammation. Patients identified as having such markers, particularly at a clinically significant level, may be at risk of an atherosclerotic event, e.g. angina or myocardial infarction.
  • inflammatory markers may be a powerful method for identifying increased inflammatory activity and predicting future cardiovascular events in individuals.
  • markers include liver proteins, such as high-sensitivity-C- reactive protein (hs-CRP), serum amyloid A (SAA), secretory phospholipase A2 and fibrinogen; cytokines, such as interleukin-1 (IL-I), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF- ⁇ r); and vascular markers, such as soluble intercellular adhesion molecule-1 (sICAM-1).
  • these variables have been identified as prospective risk markers of cardiovascular disease, and together provide a consistent message of the involvement of inflammation in cardiovascular risk.
  • markers of vascular disease include brain natriuretic peptide (BNP) and N-terminal pro-BNP (NT-proBNP) and elevated ADMA is now widely recognized as a risk marker for vascular disease.
  • BNP brain natriuretic peptide
  • NT-proBNP N-terminal pro-BNP
  • ADMA ADMA is now widely recognized as a risk marker for vascular disease.
  • Other markers of vascular disease may also be used to predict future vascular disorders in individuals.
  • Clinical indications in which an agent of the present disclosure may be used to provide therapeutic benefit include any condition in which fibronectin and/or vitronectin and/or collagen recognition by GPVI potentially has pathological consequences, for example in cardiovascular conditions such as thrombosis, including for example arterial thrombosis occurring at a blood vessel wall disease (e. g. coronary artery thrombosis, which causes myocardial infarction). Similar thrombotic processes may occur in other serious conditions at diverse anatomical locations, for instance in the cerebral vasculature, leading to stroke, or in the peripheral extremities. In the latter case for instance, patients with intermittent claudication may be treated.
  • cardiovascular conditions such as thrombosis, including for example arterial thrombosis occurring at a blood vessel wall disease (e. g. coronary artery thrombosis, which causes myocardial infarction).
  • thrombotic processes may occur in other serious conditions at diverse anatomical locations, for instance in the cerebral vasculature, leading to stroke,
  • Agent-mediated blockade of GPVI may be used and be beneficial during therapeutic procedures which induce damage to the blood vessel wall, for instance vascular surgery.
  • vascular surgery may include, but are not limited to, coronary artery bypass grafting, balloon angioplasty and stenting.
  • circulating platelets may be exposed to fibronectin or vitronectin where they may contribute to local thrombotic effects and to the inflammatory processes which ensue.
  • An example of the latter occurs in hepatitis where the hepatic circulation is compromised by the disease.
  • diseases of generalised platelet activation such as thrombocytopenic purpura and haemolytic uraemic syndrome and other clinical conditions with disseminated intravascular coagulation may be ameliorated.
  • multi-organ damage because of arterial insufficiency in patients with homozygous sickle disease may be beneficially affected by inhibiting the activation of platelets via GPVI.
  • kidney damage by platelet and fibrin disposition on the glomerular membrane and other conditions such as micro-angiopathic vasculitides may be treated by agent-mediated GPVI and/or fibronectin and/or vitronectin blockade.
  • VTE Venous thromboembolic events
  • venous thromboembolism which includes deep vein thrombosis (DVT) and resulting pulmonary emboli (PE) is the second leading cause of death in people affected by cancer, after death from the cancer itself.
  • thrombosis is a common complication in patients with malignant disease, with up to 15 percent of patients developing a clot with major symptoms and serious outcomes.
  • the occurrence of a blood clot can have a significant impact on a cancer patient's quality of life and in some instances can be life-threatening. Up to 1 in 7 cancer patients will develop thromboembolism at some point.
  • Types of treatment may include;
  • UCAD Unstable coronary artery disease
  • Platelet aggregation and activation in cancer patients may be triggered by the shedding of membrane glycolipids into the host circulation by tumor cells. Platelet activation and aggregation can then lead to thrombus formation in the cancer patient.
  • Thrombotic manifestations in cancer patients may present as one of the following: migratory thrombophlebitis or Trousseau syndrome, deep venous thrombosis (DVT), pulmonary embolism, TTP/HUS, arterial thrombosis, and DIC.
  • migratory thrombophlebitis or Trousseau syndrome deep venous thrombosis (DVT)
  • pulmonary embolism TTP/HUS
  • arterial thrombosis and DIC.
  • the agents of the disclosure have application in primary prophylaxis of thrombosis, and this includes such prophylaxis in cancer patients.
  • Trousseau syndrome is a classically described variant form of venous thrombosis characterized by a recurrent and migratory pattern preferentially involving superficial veins of the arms and chest. Trousseau syndrome is highly associated with adenocarcinomas and laboratory evidence of DIC. Migratory thrombophlebitis has also been associated with the use of somatostatin or octreotide therapy for malignant carcinoid syndrome.
  • VTE Venous thromboembolism
  • DVT pulmonary embolism
  • pulmonary embolism is diagnosed in up to 15% of cancer patients.
  • the overall incidence of cancer-related VTE in post-mortem studies is much higher, however, ranging between 35% and 50%.
  • Cancer patients at greatest risk for VTE include those with mucin-secreting tumors (e.g. pancreatic and gastrointestinal cancer), cancers of the lung, brain, prostate, breast, and ovary, and patients with acute promyelocytic leukaemia and myeloproliferative disorders, specifically polycythemia vera and essential thrombocythemia.
  • VTE often complicates the care of cancer patients undergoing major surgery and of patients receiving chemotherapy and/or hormonal therapy.
  • the risk of developing thrombosis in cancer patients is influenced by the age and hormonal status of the patient.
  • Postmenopausal women with advanced breast cancer receiving tamoxifen in addition to adjuvant chemotherapy have a higher risk for thrombotic events than do premenopausal women with breast cancer.
  • Other antineoplastic agents that are associated with VTE include cisplatin, cyclophosphamide, methotrexate, and 5-fluorouracil.
  • Thromboembolic events have been also reported with thalidomide therapy.
  • Mechanical causes of VTE may result from vascular invasion by tumors of the kidney, stomach, lung, liver, adrenal cortex, and testicle, and form the use of central venous catheters.
  • Anti-platelet therapy for example therapy utlizing the agents of the present disclosure, may be useful in the prevention of strokes, thrombosis, myocardial infarction and vascular death in a patient suffering from cancer.
  • Such therapy may be acute therapy immediately or shortly following a cardiovascular event or, alternatively a chronic or long-term therapy to treat, prevent or reduce the likelihood of vascular disease in patients suffering from cancer and/or being treated for cancer.
  • VTE may be diagnosed using several techniques including determining the present probability of VTE by measuring of plasma D-dimer.
  • Other techniques include diagnostic imaging modalities for DVT such as ascending contrast venography, compression ultrasonography, impedance plethysmography, and magnetic resonance venography.
  • diagnostic techniques may be used to determine whether a patient suffering from cancer would benefit form treatment using an agent of the present disclosure.
  • VTE VTE
  • a soluble GPVI protein may be considered to cause lower amounts of bleeding than some conventional anti-coagulant drugs, and therefore may be particularly useful in treating VTE in cancer patients.
  • Agents of the present disclosure for example but not limited to a soluble GPVI protein or an anti GPVI single domain antibody, may be particularly useful in treating VTE in patients suffering from brain metastases, since agents of the disclosure may be considered not to significantly increase bleeding, unlike other anti-coagulant therapies.
  • cancer chemotherapies can cause significant changes in platelet counts and marked fluctuation in anticoagulant responses, which may further increase the risk for bleeding.
  • An agent of the disclosure for example a soluble GPVI protein, may not be affected by changes in platelet counts and therefore may be useful in treating VTE in patients being treated by chemotherapy.
  • Treatment of VTE using the methods, agents and products of the present disclosure described herein may be considered to include treatment of recurrent VTE in cancer patients. It is also considered that agents of the disclosure can be used to coat catheters, for example although not limited to, long-term central venous catheters which are necessary for some cancer patients, thus preventing or reducing the risk of thrombotic complications as a result of catheter use. It is considered that use of agents of the disclosure in such a method may further comprise any other of the features described herein.
  • Agents of the disclosure may also be used prophylatically to treat cancer patients who require long- term catheter use to reduce or prevent thrombotic and/or cardiovascular complications of catheter use.
  • Anti-GPVI and/or anti-fibronectin and/or anti-vitronectin treatment in accordance with the present disclosure may be used to provide clear benefit for patients with cardiovascular disease, especially those who have undergone corrective vessel surgery or angioplasties with or without stenting.
  • Anti- GPVI, and/or anti-fibronectin and/or vitronectin treatment may be given by injection (e. g. intravenously) or by local delivery methods (e.g. pre- coating of stents or other indwelling devices).
  • Anti-GPVI, and/or anti-fibronectin and/or vitronectin may be delivered by gene-mediated technologies.
  • Alternative formulation strategies may provide preparations suitable for oral or suppository route.
  • the route of administration may be determined by the physicochemical characteristics of the treatment, by special considerations for the disease, to optimise efficacy or to minimise side-effects.
  • the agents of the disclosures may be used to treat and/or protect against a variety of disorders, including, for example, seizures, transient ischemic shock, strokes, focal ischemia originating from thrombus or cerebral hemorrhage,global ischemia originating from cardiac arrest, trauma, neonatal palsy, hypovolemic shock, and hyperglycemia and associated neuropathies.
  • agents of the present disclosure may be useful in treating or preventing metastasis in a subject.
  • agents of the present disclosure may be useful in the treatment of inflammatory disorders, such as rheumatoid arthritis and psoriasis, and cardiovascular diseases, such as atherosclerosis and restenosis.
  • the agents of the present disclosure may be useful for the treatment or prevention of other diseases including, but not limited to, thromboembolic disorders, asthma, allergies, adult respiratory distress syndrome, graft versus host disease, organ transplant rejection, septic shock, eczema, contact dermatitis, inflammatory bowel disease, and other autoimmune diseases.
  • the agents of the present disclosure may also be useful for wound healing.
  • the products of the present disclosure are also useful for the treatment, including prevention, of angiogenic disorders.
  • the term angiogenic disorders as used herein includes conditions involving abnormal neovascularization. Where the growth of new blood vessels is the cause of, or contributes to, the pathology associated with a disease, inhibition of angiogenisis will reduce the deleterious effects of the disease.
  • an example of such a disease target is diabetic retinopathy.
  • inhibition of angiogenisis will reduce the blood supply to the tissue and thereby contribute to reduction in tissue mass based on blood supply requirements.
  • Examples include growth of tumors where neovascularization is a continual requirement in order that the tumor grow and the establishment of solid tumor metastases.
  • the agents of the present disclosure inhibit tumor tissue angiogenesis, thereby preventing tumor metastasis and tumor growth.
  • the inhibition of angiogenesis using the agents of the present disclosure can ameliorate the symptoms of the disease, and, in some cases, can cure the disease.
  • the present inventive method includes the administration to an animal, such as a mammal, particularly a human, in need of the inhibition of platelet activation of an effective amount, e. g., a therapeutical effective amount, of one or more of the aforementioned present inventive agents, alone or in combination with one or more other pharmaceutically active agents.
  • an effective amount e. g., a therapeutical effective amount
  • the agents of the disclosure may be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route, as an oral or nasal spray or via inhalation,
  • the agents may be administered in the form of pharmaceutical preparations in a pharmaceutical acceptable dosage form.
  • the compositions may be administered at varying doses.
  • an agent of the disclosure is to be administered to an individual, it is particularly at least 80% pure, more preferably it is at least 90% pure, even more preferably it is at least 95% pure and free of pyrogens and other contaminants.
  • the percent purity is calculated as a weight percent of the total protein content of the preparation, and does not include constituents which are deliberately added to the composition after the agent is purified.
  • the present disclosure provides a method of treatment of a human or animal body in need of treatment or prevention of acute or chronic vascular diseases associated with intraarterial and/or intravenous thrombosis, which comprises administration to a human or animal of a pharmaceutically effective amount of an inhibitor or agent of the disclosure.
  • the dosage may be in the range of from 0.1 to 100 mg/patient/ day.
  • the agents are used as lyophilised powders solubilised in PBS/succrose/manitol-buffer prior to parenteral administration.
  • the disclosure provides a method for treating a disease or disorder selected from therapeutic or prophylactic cardiovascular conditions, thrombosis, heart attack, stroke, intermittent coagulation, conditions with disseminated intravascular coagulation, thrombocytopenic purpura, haemolytic uraemic syndrome, damage to blood vessel wall resulting from surgery or therapy, collagen-induced inflammation, homozygous sickle disease, kidney damage by platelet and fibrin disposition on the glomerular member and micro-angiopathic vasculitides comprising administering an agent of the disclosure, to a subject with the disease or disorder or at risk of developing the disease or disorder.
  • the treatment may be therapeutic and/or prophylactic.
  • the disclosure provides for the use of an agent of the disclosure for the manufacture of a medicament to treat or prevent of a disease or disorder selected from cardiovascular conditions, thrombosis, heart attack, stroke, intermittent coagulation, conditions with disseminated intravascular coagulation, thrombocytopenic purpura, haemolytic uraemic syndrome, damage to blood vessel wall resulting from surgery or therapy, collagen-induced inflammation, homozygous sickle disease, kidney damage by platelet and fibrin disposition on the glomerular member and micro-angiopathic vasculitides.
  • a disease or disorder selected from cardiovascular conditions, thrombosis, heart attack, stroke, intermittent coagulation, conditions with disseminated intravascular coagulation, thrombocytopenic purpura, haemolytic uraemic syndrome, damage to blood vessel wall resulting from surgery or therapy, collagen-induced inflammation, homozygous sickle disease, kidney damage by platelet and fibrin disposition on the glomerular member and micro-angiopathic vasculitides.
  • Agents of the disclosure are advantageously used as drugs for the treatment and prevention of atherosclerosis.
  • the agents also solve the problem of treatment of atherosclerosis by inhibition of platelet secretion.
  • the products or agents of this disclosure are administered prophylactically.
  • the disclosure provides inhibitors of GPVI-fibronectin interactions, which in embodiments are selective GPVI-fibronectin interaction inhibitors. It will also be appreciated from the above that the disclosure provides inhibitors of GPVI-vitronectin.
  • the products of the invention may also be combined and/or co-administered with any antithrombotic agent with a different mechanism of action, such as the inhibitors of thrombin or other coagulation enzymes (e.g. Factor IXa or X), antiplatelet agents acetylsalicylic acid, tidopidine, clopidogrel, thromboxane receptor and/or synthetase inhibitors, matrix metalloprotease inhibitors, fibrinogen receptor antagonists, prostacyclin mimetics and phosphodiesterase inhibitors and ADP-receptor (P 2 T) antagonists.
  • thrombin or other coagulation enzymes e.g. Factor IXa or X
  • antiplatelet agents acetylsalicylic acid, tidopidine, clopidogrel, thromboxane receptor and/or synthetase inhibitors
  • matrix metalloprotease inhibitors acetylsalicylic acid
  • the agents of the invention may further be combined and/or co-administered with thrombolytics such as tissue plasminogen activator (natural, recombinant or modified), streptokinase, urokinase, prourokinase, anisoylated plasminogen-streptokinase activator complex (APSAC), animal salivary gland plasminogen activators, and the like, in the treatment of thrombotic diseases, in particular myocardial infarction.
  • tissue plasminogen activator naturally, recombinant or modified
  • streptokinase urokinase
  • prourokinase prourokinase
  • anisoylated plasminogen-streptokinase activator complex APSAC
  • animal salivary gland plasminogen activators and the like
  • Such agent can be one or more agents selected from, but not limited to several major categories, namely, a lipid-lowering drug, including an IBAT (ileal Na + /bile acid cotransporter) inhibitor, a fibrate, niacin, a statin, a CEETP (cholesteryl ester transfer protein) inhibitor, and a bile acid sequestrant, an anti-oxidant, including vitamin E and probucol, a Ilb/IIIa antagonist (e.g.
  • abciximab eptifibatide, tirofiban
  • an aldosterone inhibitor e.g. spirolactone and epoxymexrenone
  • an adenosine A2 receptor antagonist e.g. losartan
  • an adenosine A3 receptor agonist e.g. beta- blocker, acetylsalicylic acid, a loop diuretic, an angiotensin receptor blocker and an ACE (angiotensin converting enzyme) inhibitor.
  • the products of the disclosure may be combined and/or co-administered with a cardioprotectant, for example an adenosine Al or A3 receptor agonist.
  • a cardioprotectant for example an adenosine Al or A3 receptor agonist.
  • a method for treating a cardiovascular disease in a patient that comprises treating the patient with a product of the disclosure and an NSAID, e.g., a COX-2 inhibitor. Accordingly, the products of the disclosure may be combined and/or co-administered with an NSAID.
  • an NSAID e.g., a COX-2 inhibitor
  • the agents of the present disclosure may be combined more than one other active agents.
  • the agents may be combined with antiplatelets drugs and/or anti-coagulant drugs.
  • methods of treatment of vascular diseases which comprise administering a combination of anti-platelet drugs and/or anti-coagulant drugs to a patient, wherein the administration may be sequential or simultaneous.
  • any of the agents disclosed herein may be used in combination with other drugs, for example anti-thrombotic or anti-platelet drugs, in the treatment of any of the disorders disclosed herein, for example cardiovascular disorders.
  • the agent and the drug may be for simultaneous, sequential or separate administration to a patient. That is to say that administration of the agent and other drug(s) may be separately, sequentially or simultaneously.
  • a dosage form for administration of the agent and the drug "simultaneously" is not particularly limited provided that the agent and the other drug can be administered almost at the same time.
  • One dosage form is a single unit dosage form containing the agent and the drug.
  • a dosage form for administration of the agents and the drug "separately or sequentially " is not particularly limited provided that the two agents can be separately administered wherein there is a time interval between the first administration, of the agent, and the second administration, of the other drug. For example, after a pre-determined time from the first administration of the agent, the drug is administered, or after pre-determined time from the first administration of the agent, the drug is administered.
  • triple antiplatelet therapy aspirin plus cilostazol and clopidogrel or ticlopidine - reduces the early risk of thrombotic complications after coronary stenting with a bare metal stent compared with dual therapy.
  • the increased efficacy is not associated with an increased risk of major bleeding [JAm Co// ⁇ J ⁇ _// ⁇ /2005;46:1833-1837.)
  • the treatment may comprise triple antiplatelet therapy comprising treating the patient with a composition comprising an agent of the present disclosure together with at least three agents, the agent all having different mods of antiplatelet activity, each being selected from the group consisting of aspirin, cilostazol, clopidogrel and ticlopidine.
  • Triple anti-platelet therapy as disclosed herein may include administering compositions containing the agents of the disclosure with other anti-platelet or, for example, to reduce the early risk of thrombotic complications after coronary surgery, for example stenting.
  • the agents of the present disclosure may be administered with a drug which acts by way of a different mechanism to the agent of the present disclosure.
  • a drug which acts by way of a different mechanism to the agent of the present disclosure.
  • cilostazol which selectively inhibits phosphodiesterase III
  • a patient may be treated with a combination of drugs including the agents of the invention and clopidogrel or ticlopidine, which are adenosine diphosphate receptor antagonists.
  • Actual dosage levels of active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration (referred to herein as a "therapeutically effective amount").
  • the selected dosage level will depend upon the activity of the particular compound, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required for to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • a parenteral formulation including a product or an agent as described herein.
  • the formulation may consist of the product alone or it may contain additional components, in particular the product may be in combination with a pharmaceutically acceptable diluent, excipient or carrier, for example a tonicity agent for the purpose of making the formulation substantially isotonic with the body of the subject to receive the formulation, e.g. with human plasma.
  • a pharmaceutically acceptable diluent, excipient or carrier for example a tonicity agent for the purpose of making the formulation substantially isotonic with the body of the subject to receive the formulation, e.g. with human plasma.
  • the formulation may be in ready-to-use form or in a form requiring reconstitution prior to administration.
  • Parenteral preparations can be administered by one or more routes, such as intravenous, subcutaneous, intradermal and infusion; a particular example is intravenous.
  • routes such as intravenous, subcutaneous, intradermal and infusion; a particular example is intravenous.
  • a formulation disclosed herein may be administered using a syringe, injector, plunger for solid formulations, pump, or any other device recognized in the art for parenteral administration.
  • Liquid dosage forms for parenteral administration may include solutions, suspensions, liposome formulations, or emulsions in oily or aqueous vehicles.
  • the liquid dosage forms may contain other compounds.
  • Tonicity agents for the purpose of making the formulations substantially isotonic with the subject's body, e.g. with human plasma
  • Tonicity agents such as, for instance, sodium chloride, sodium sulfate, dextrose, mannitol and/or glycerol may be optionally added to the parenteral formulation.
  • a pharmaceutically acceptable buffer may be added to control pH.
  • Thickening or viscosity agents for instance well known cellulose derivatives (e.g. methylcellulose, carboxymethylcellulose, hydroxyethylcellulose and hydroxypropylmethylcellulose), gelatin and/or acacia, may optionally be added to the parenteral formulation.
  • Solid dosage forms for parenteral administration may encompass solid and semi-solid forms and may include pellets, powders, granules, patches, and gels.
  • the active compound is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier.
  • the disclosed products may be presented as solids in finely divided solid form, for example they may be milled or micronised.
  • the formulations may also include antioxidants and/or preservatives.
  • antioxidants may be mentioned thiol derivatives (e.g. thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol, glutathione), tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, sulfurous acid salts (e.g. sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) and nordihydroguaiareticacid.
  • Suitable preservatives may for instance be phenol, chlorobutanol, benzylalcohol, methyl paraben, propyl paraben, benzalkonium chloride and cetylpyridinium chloride.
  • the parenteral formulations may be prepared as large volume parenterals (LVPs), e.g. larger than 100 ml, more particularly about 250 ml, of a liquid formulation of the active compound. Examples of LVPs are infusion bags.
  • the parenteral formulations may alternatively be prepared as small volume parenterals (SVPs), e.g. about 100 ml or less of a liquid formulation of the active compound. Examples of SVPs are vials with solution, vials for reconstitution, prefilled syringes for injection and dual chamber syringe devices.
  • One class of formulations disclosed herein is intravenous formulations.
  • the active compound or compounds can be present at varying concentrations, with a carrier acceptable for parenteral preparations making up the remainder.
  • the carrier is water, particularly pyrogen free water, or is aqueous based.
  • the carrier for such parenteral preparations is an aqueous solution comprising a tonicity agent, for example a sodium chloride solution.
  • aqueous based is meant that formulation comprises a solvent which consists of water or of water and water-miscible organic solvent or solvents; as well as containing a product of disclosure in dissolved form, the solvent may have dissolved therein one or more other substances, for example an antioxidant and/or an isotonicity agent.
  • organic cosolvents may be mentioned those water- miscible solvents commonly used in the art, for example propyleneglycol, polyethyleneglycol 300, polyethyleneglycol 400 and ethanol.
  • organic co-solvents are only used in cases where the active agent is not sufficiently soluble in water for a therapeutically effective amount to be provided in a single dosage form.
  • the solubility of the active compound in the present formulations may be such that the turbidity of the formulation is lower than 50 NTU, e.g. lower than 20 NTU such as lower than 10 NTU.
  • parenteral formulations are administered at or near physiological pH. It is believed that administration in a formulation at a high pH (i.e., greater than 8) or at a low pH (i.e., less than 5) is undesirable. In particular, it is contemplated that the formulations would most desirably be administered at a pH of between 6.0 and 7.0 such as a pH of 6.5.
  • the pH values mentioned in this paragraph are not critical, however, and formulations may fall outside them.
  • the parenteral formulation may be purged of air when being packaged.
  • the parenteral formulation may be packaged in a sterile container, e.g. vial, as a solution, suspension, gel, emulsion, solid or a powder.
  • a sterile container e.g. vial
  • Such formulations may be stored either in ready-to-use form or in a form requiring reconstitution prior to administration.
  • Parenteral formulations according to the disclosure may be packaged in containers.
  • Containers may be chosen which are made of material which is non-reactive or substantially non-reactive with the parenteral formulation.
  • Glass containers or plastics containers e.g. plastics infusion bags, may be used.
  • a concern of container systems is the protection they afford a solution against UV degradation. If desired, amber glass employing iron oxide or an opaque cover fitted over the container may afford the appropriate UV protection.
  • Plastics containers such as plastics infusion bags are advantageous in that they are relatively light weight and non-breakable and thus more easily stored. This is particularly the case for Large Volume parenterals.
  • the intravenous preparations may be prepared by combining the active product or products with the carrier. After the formulation is mixed, it may be sterilized, for example using known methods. Once the formulation has been sterilized, it is ready to be administered or packaged, particularly in dark packaging (e.g. bottles or plastics packaging), for storage. It is envisaged, however, that the disclosed products might not be stored in solution but as dry solids, particularly a finely divided form such as, for example, a lyophilisate, in order to prolong shelf life; this would of course apply to other parenteral formulations, not only intravenous ones.
  • the intravenous preparations may take the form of large volume parenterals or of small volume parenterals, as described above.
  • kits for producing a single-dose administration unit.
  • the products may each contain both a first container having the active compound (optionally combined with additives, for example anti-oxidant, preservative and, in some instances, tonicity agent) and a second container having the carrier/diluent (for example water, optionally containing one or more additives, for example tonicity agent).
  • additives for example anti-oxidant, preservative and, in some instances, tonicity agent
  • carrier/diluent for example water, optionally containing one or more additives, for example tonicity agent.
  • Such dual chamber syringes or binary syringes will have in one chamber a dry preparation including or consisting of the active compound and in another chamber a suitable carrier or diluent such as described herein.
  • the two chambers are joined in such a way that the solid and the liquid mix to form the final solution.
  • formulations disclosed herein comprises subcutaneous or intradermal formulations (for example formulations for injection) in which the active product (or active agent combination) is formulated into a parenteral preparation that can be injected subcutaneously or intradermally.
  • the formulation for administration will comprise the active product and a liquid carrier.
  • the carrier utilized in a parenteral preparation that will be injected subcutaneously or intradermally may be an aqueous carrier (for example water, typically containing an additive e.g. an antioxidant and/or an isotonicity agent) or a nonaqueous carrier (again one or more additives may be incorporated).
  • a non-aqueous carrier for such parenteral preparations may be mentioned highly purified olive oil.
  • the active compound and the carrier are typically combined, for example in a mixer. After the formulation is mixed, it is preferably sterilized, such as with U.V. radiation. Once the formulation has been sterilized, it is ready to be injected or packaged for storage. It is envisaged, however, that the disclosed products will not be stored in liquid formulation but as dry solids, in order to prolong shelf life.
  • the active product may suitably be formulated together with one or more polymers that are gradually eroded or degraded when in use, e.g. silicone polymers, ethylene vinylacetate, polyethylene or polypropylene.
  • Transdermal formulations may be prepared in the form of matrices or membranes, or as fluid or viscous formulations in oil or hydrogels or as a compressed powder pellet.
  • an adhesive which is compatible with the skin may be included, such as polyacrylate, a silicone adhesive or polyisobutylene, as well as a foil made of, e.g., polyethylene, polypropylene, ethylene vinylacetate, polyvinylchloride, polyvinylidene chloride or polyester, and a removable protective foil made from, e.g., polyester or paper coated with silicone or a fluoropolymer.
  • water or organic solvents or mixtures thereof may be used.
  • Transdermal gels may furthermore contain one or more suitable gelling agents or thickeners such as silicone, tragacanth, starch or starch derivatives, cellulose or cellulose derivatives or polyacrylic acids or derivatives thereof.
  • Transdermal formulations may also suitably contain one or more substances that enhance absorption though the skin, such as bile salts or derivatives thereof and/or phospholipids.
  • Transdermal formulations may be prepared according to a method disclosed in, e.g., B W Barry, "Dermatological Formulations, Percutaneous Absorption", Marcel Dekker Inc., New York- Basel, 1983, or Y W Chien, "Transdermal Controlled Systemic Medications", Marcel Dekker Inc., New York-Basel, 1987.
  • the pharmaceutical products of the invention may be administered orally or parenterally ("parenterally” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.) to a host to obtain an protease-inhibitory effect.
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the compounds may be administered alone or as compositions in combination with pharmaceutically acceptable diluents, excipients or carriers.
  • a pharmaceutical composition including a described product, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
  • compositions of this invention for parenteral injection or infusion suitably comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • solvents co-solvents or additives for parenteral, e.g. intravenous, or other formulations may be mentioned:
  • Acids e.g. with pH greater than 1.8
  • Cremophor EL e.g. up to 25% in water
  • Tween surfactants e.g. up to 2% • Water.
  • microsphere-based delivery systems composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide-co-glycolide (PLG).
  • PLG poly-DL-lactide-co-glycolide
  • lipophilic carbohydrate excipients termed oligosaccharide ester derivatives.
  • OEDs which have been used to modify pharmacokinetic profiles of drugs
  • SoliDoseTM technology Elan Pharmaceuticals
  • This technology offers the ability to formulate drug molecules with modified-release characteristics and improved bioavailability.
  • Another technology from the same company makes use of select carbohydrate excipients, such as trehalose and sucrose to stabilize molecules in the dry state, thereby preventing their physical and chemical degradation at ambient temperatures and above.
  • Intravenous and other parenteral compositions may be provided as ready-to-use solutions or as lyophilisates or dry powders for reconstitution prior to administration.
  • Parenteral and other compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol or phenol sorbic acid. It may also be desirable to include isotonic agents such as sugars or sodium chloride, for example. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents (for example aluminum monostearate and gelatin) which delay absorption.
  • adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol or phenol sorbic acid. It may also be desirable to include isotonic agents such as sugars or sodium chloride, for example. Prolonged absorption of the injectable pharmaceutical form
  • the absorption of the drug in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are suitably made by forming microencapsule matrices of the drug in biodegradable polymers, for example polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations may also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or one or more: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid, for example; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, for example; c) humectants such as glycerol, for example; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, for example; e) solution retarding agents such as paraffin, for example; f) absorption accelerators such as quaternary ammonium compounds, for example; g) wetting
  • the dosage form may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycol, for example.
  • oral formulations contain a dissolution aid.
  • the dissolution aid is not limited as to its identity so long as it is pharmaceutically acceptable.
  • examples include nonionic surface active agents, such as sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters (e.g., sorbitan trioleate), polyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monofatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylene sorb
  • the solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings such as multiple coatings, for example, well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, and/or in delayed fashion. Examples of embedding compositions which can be used include polymeric substances and waxes.
  • the active compounds may also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • the active compounds may be in finely divided form, for example it may be micronised.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.
  • inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol,
  • the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth and mixtures thereof.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to a compound of the present invention, stabilisers, preservatives, excipients and the like.
  • the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p 33 et seq.
  • Dosage forms for topical administration of an agent invention include powders, sprays, ointments and inhalants.
  • the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants which may be required.
  • Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
  • a liquid dosage form for parenteral administration for example a solution, suspension, liposome formulation, or emulsion in oily or aqueous vehicles.
  • the liquid dosage forms may contain e.g. tonicity agents (for the purpose of making the formulations substantially isotonic with the subject's body, e.g. with human plasma) such as, for instance, sodium chloride, sodium sulfate, dextrose, mannitol and/or glycerol may be optionally added to the parenteral formulation.
  • a pharmaceutically acceptable buffer may be added to control pH.
  • Thickening or viscosity agents for instance well known cellulose derivatives (e.g. methylcellulose, carboxymethylcellulose, hydroxyethylcellulose and hydroxypropylmethylcellulose), gelatin and/or acacia, may optionally be added to the parenteral formulation.
  • LVPs are infusion bags.
  • SVPs are vials with solution, vials for reconstitution, prefilled syringes for injection and dual chamber syringe devices.
  • Solid dosage forms for parenteral administration may encompass solid and semi-solid forms and may include pellets, powders, granules, patches, and gels.
  • Subcutaneous or intradermal formulations for example formulations for injection
  • the active product or active agent combination
  • the formulation for administration will comprise the active product and a liquid carrier.
  • a parenteral preparation having an aqueous carrier for example water, typically containing an additive e.g. an antioxidant and/or an isotonicity agent, for example solutions, dispersions, suspensions or emulsions.
  • aqueous carrier for example water, typically containing an additive e.g. an antioxidant and/or an isotonicity agent
  • solutions, dispersions, suspensions or emulsions for example solutions, dispersions, suspensions or emulsions.
  • a parenteral formulation having a nonaqueous carrier for example solutions, dispersions, suspensions or emulsions.
  • Pharmaceutically acceptable non-aqueous carriers can be fully saturated, or partially or fully unsaturated.
  • examples of non-aqueous carriers include, but are not limited to: (i) Vegetable oils (such as cottonseed oil, corn oil, sesame oil, soybean oil, olive oil, fractionated coconut oils, peanut oil, sunflower oil, safflower oil, almond oil, avocado oil, palm oil, palm kernel oil, babassu oil, beechnut oil, linseed oil, rape oil, and the like), mineral oils, synthetic oils, and combinations thereof.
  • Fully saturated non-aqueous carriers examples of which include, but are not limited to, medium to large chain fatty acids (e.g. capric acid and/or caprylic acid) and particularly esters thereof (such as fatty acid triglycerides with a chain length of about 6C to about 24C); mixtures of fatty acids are split from the natural oil (for example coconut oil palm kernel oil, babassu oil, or the like) and are refined. In some embodiments, about 8C to about 12C fatty acid medium chain triglycerides are useful.
  • medium to large chain fatty acids e.g. capric acid and/or caprylic acid
  • esters thereof such as fatty acid triglycerides with a chain length of about 6C to about 24C
  • mixtures of fatty acids are split from the natural oil (for example coconut oil palm kernel oil, babassu oil, or the like) and are refined.
  • about 8C to about 12C fatty acid medium chain triglycerides are useful.
  • non-aqueous carriers include, but are not limited to, saturated coconut oil (which typically includes a mixture of lauric, myristic, palmitic, capric and capric acids), including those sold under the Miglyo trademark from HuIs and bearing trade designations 810, 812, 829, and 840). Also noted are the NeoBee products sold by Drew Chemicals. Isopropyl myristate is another example of a non-aqueous carrier.
  • Synthetic oils examples of which include triglycerides, and propylene glycol diesters of saturated or unsaturated fatty acids having from 6 to 24 carbon atoms such as, for example hexanoic acid, octanoic (caprylic), nonanoic (pelargonic), decanoic (capric), undecanoic, lauric, tridecanoic, tetradecanoic (myristic), pentadecanoic, hexadecanoic (palmitic), heptadecanoic, octadecanoic (stearic), nonadecanoic, heptadecanoic, eicosanoic, heneicosanoic, docosanoic, and lignoceric acids, and the like,
  • Unsaturated carboxylic acids examples of which include oleic, linoleic, and linolenic acids, and the like.
  • non-oil for example polyethylene glycol.
  • non-aqueous carrier can comprise the mono-, di-, and triglyceryl esters of fatty acids or mixed glycerides and/or propylene glycol diesters wherein at least one molecule of glycerol has been esterified with fatty acids of varying carbon atom length
  • the injectable formulation may be in an aqueous carrier or a non-aqueous carrier.
  • Formulations comprising as aqueous and nonaqueous carriers, diluents, solvents or vehicles water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, for example), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • coating materials such as lecithin
  • surfactants The features of this paragraph may be applied to the formulations of any of preceding paragraphs 1, 2, 3, 4, 5, 6, 8, 9, 10 and 11.
  • Formulations comprising an acid, e.g. formulations with pH greater than 1.8, for example at least 2, e.g. at least 3, as in the case of formulations having a pH of at least 4. Often acidic formulations have a pH of at least 5.
  • Formulations comprising a base, e.g. formulations with pH of less than 14 for example less than 13, e.g. no more than 12, as in the case of formulations having a pH of no more than 11.
  • a base e.g. formulations with pH of less than 14 for example less than 13, e.g. no more than 12, as in the case of formulations having a pH of no more than 11.
  • acidic formulations have a pH of no more than 10, particularly no more than 9.
  • the pH is no more than 8.
  • Cremophor EL aqueous carrier
  • Cremophor EL is also known asPolyoxyl 35 Castor Oil.
  • Cremophor EL is a non-ionic solubilizer and emulsifier obtained by causing ethylene oxide to react with castor oil of German Pharmacopoeia (DAB 8) quality in a molar ratio of 35 moles to 1 mole. Cremophor EL forms clear solutions in water.
  • Formulations comprising dextrose, e.g. up to 5%, in an aqueous solvent, for example water or saline.
  • Formulations comprising ethanol, e.g. up to 15% and optionally up to 5%, in an aqueous solvent, for example water or saline.
  • Formulations comprising glycerol, e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising sorbitol, e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising phosphate buffer.
  • Formulations comprising polyethylene glycol, e.g. PEG 300 or 400, neat or e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising propylene glycol or a propylene glycol derivative, for example propylene glycol alginate, neat or or e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising a sugar, e.g. lactose, sucrose or glucose, whether as a solid or in solution, e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • a sugar e.g. lactose, sucrose or glucose
  • an alcoholic or aqueous solvent for example water or saline.
  • Formulations comprising an antioxidant, e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising an amino acid additive, e.g. in an alcoholic or aqueous solvent, for example water or saline.
  • Formulations comprising a lipid, e.g. a phospholipid.
  • Formulations comprising a polyoxyethylenesorbitan ester surfactant, e.g. Tween 20 (polyoxyethylenesorbitan monolaurate), Tween 40 (polyoxyethylenesorbitan monopalmitate), Tween 60 (polyoxyethylenesorbitan monostearate), Tween 80 (polyoxyethylenesorbitan monooleate ) or Tween 85 (polyoxyethylenesorbitan trioleate).
  • a polyoxyethylenesorbitan ester surfactant e.g. Tween 20 (polyoxyethylenesorbitan monolaurate), Tween 40 (polyoxyethylenesorbitan monopalmitate), Tween 60 (polyoxyethylenesorbitan monostearate), Tween 80 (polyoxyethylenesorbitan monooleate ) or Tween 85 (polyoxyethylenesorbitan trioleate).
  • Formulations comprising a poloxamer (poly(oxyethylene)-poly(oxypropylene) block copolymer).
  • Formulations comprising a Solutol, for example Solutol HS 15 (Polyethylene glycol-15- hydroxystearate), e.g. up to 30% in water
  • Formulations comprising a microsphere-based delivery system, e.g. composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide-co-glycolide (PLG).
  • a microsphere-based delivery system e.g. composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide-co-glycolide (PLG).
  • Formulations comprising lipophilic carbohydrate excipients, termed oligosaccharide ester derivatives (OEDs), which have been used to modify pharmacokinetic profiles of drugs (SoliDoseTM technology, Elan Pharmaceuticals).
  • OEDs oligosaccharide ester derivatives
  • Intravenous and other parenteral compositions provided as lyophilisates or dry powders for reconstitution prior to administration.
  • a subcutaneous implant 35. A subcutaneous implant.
  • transdermal formulation for example in the form of matrices or membranes, or as fluid or viscous formulations in oil or hydrogels or as a compressed powder pellet. See above for further details.
  • Injectable depot forms e.g. comprising microencapsule matrices of the drug in biodegradable polymers, for example a polylactide-polyglycolide, poly(orthoester) and poly(anhydride), or comprising the drug entrapped in liposomes or microemulsions which are compatible with body tissues.
  • biodegradable polymers for example a polylactide-polyglycolide, poly(orthoester) and poly(anhydride
  • Formulations comprising a biodegradable polymer, e.g. one mentioned previously.
  • dosage forms and formulations of the present invention may be one of, or any permissible combination of, features(s) of any of the above paragraphs 1 to 38.
  • the compounds of the invention are orally active, have rapid onset of activity and low toxicity.
  • the compounds of the invention have the advantage that they may be more efficacious, be less toxic, be longer acting, have a broader range of activity, be more potent, produce fewer side effects, be more easily absorbed than, or that they may have other useful pharmacological properties over, compounds known in the prior art.
  • the invention also includes a method for the long term or chronic prevention of atherosclerosis and/or arterial thrombosis, comprising administering to a subject an effective amount of an agent of the disclosure.
  • Example 8 contains data indicating that the agents disclosed herein will prevent or retard atherogenesis, in patients without active atherosclerotic lesions (or at least substantially free of observable lesions) as well as in patients with active atherosclerotic lesions.
  • the data indicate that the agents will be useful to treat or prevent vascular disorders caused by or associated with dysfunctional endothelium (e.g. atherogenesis, ischemia, pro-thrombotic state, thrombosis, pro- inflammatory state, inflammation, atherosclerotic lesion formation, plaque activation and rupture, vasospasm, impaired vasomotion).
  • the data further indicate that the agents will be useful for the treatment or prevention of stroke, myocardial infarction and atherosclerosis as well as other cardiovascular diseases. Included in the disclosure therefore are methods for performing the following treatments by administering a therapeutically effective amount of an agent disclosed herein:
  • treatment e.g. prevention or retardation of, atherogenesis in patients or arterial sites without active atherosclerotic lesions
  • treatment e.g. prevention or retardation of, atherogenesis in patients or arterial sites with active atherosclerotic lesions
  • treatment e.g. prevention or retardation of, atheroprogression in patients or arterial sites without active atherosclerotic lesions
  • treatment e.g. prevention or retardation of, atheroprogression in patients or arterial sites with active atherosclerotic lesions
  • treatment e.g. prevention or retardation of, vascular disorders caused by or associated with dysfunctional endothelium in patients or arterial sites without active atherosclerotic lesions
  • treatment e.g. prevention or retardation of, vascular disorders caused by or associated with dysfunctional endothelium in patients or arterial sites with active atherosclerotic lesions
  • treatment e.g. prevention or retardation of, stroke, myocardial infarction and atherosclerosis as well as other cardiovascular diseases in patients or arterial sites without active atherosclerotic lesions
  • treatment e.g. prevention or retardation of, stroke, myocardial infarction and atherosclerosis as well as other cardiovascular diseases in patients or arterial sites with active atherosclerotic lesions
  • treatment e.g. prevention or retardation of, vascular disorders in patients or arterial sites with dysfunctional endothelium
  • treatment e.g. prevention or retardation of, vascular disorders in patients or arterial sites without dysfunctional endothelium.
  • Methods of treatment described herein may be chronic or acute.
  • the agents described herein may be administered over a period of at least one month, e.g. two, four, six or twelve months or more.
  • chronic treatment is performed by oral administration, for example of an oral formulation as described herein.
  • oral formulations are those containing humanised antibodies (e.g. humanised Fabs) and single domain antibodies, e.g. VHH's.
  • Acute treatment may be administered parenterally, e.g. by intravenous injection and/or infusion.
  • compositions whether oral or parenteral, comprise an active agent which binds to vitronectin or fibronectin (or, and this is more usual, both) and blocks (that is, blocks to a therapeutically useful extent) binding of the vitronectin or fibronectin to GPVI, thereby inhibiting platelet/endothelium adhesion, including amongst others in patients or arterial sites substantially free of observable active atherosclerotic lesions.
  • active agent which binds to vitronectin or fibronectin (or, and this is more usual, both) and blocks (that is, blocks to a therapeutically useful extent) binding of the vitronectin or fibronectin to GPVI, thereby inhibiting platelet/endothelium adhesion, including amongst others in patients or arterial sites substantially free of observable active atherosclerotic lesions.
  • blocks that is, blocks to a therapeutically useful extent binding of the vitronectin or fibronectin to GPVI, thereby inhibiting platelet/end
  • GPVI-Fc fusion protein comprising the extracellular domain of GPVI or a vitronectin-binding or fibronectin-binding amino acid sequence contained within said domain; in either case a natural sequence may be modified by one or more amino acid deletions, substitutions or insertions (e.g. conservative substitutions).
  • variants of natural sequences particularly those derived from GPVI, and polypeptides having the vitronectin-binding and/or fibronectin-binding characteristics of GPVI (but not necessarily its quantitative degree of affinity for vitronectin or, as the case may be, fibronectin, or both).
  • a GPVI-Fc fusion protein having the sequence disclosed in Figure 7 of WO03/104282 (PCT Application No. PCT/EP03/05929) in the uses and treatments disclosed above.
  • the activity of an agent of the present disclosure can also be analyzed by a platelet adhesion assay.
  • the adhesion assay is performed as follows: 5'Cr-labeled platelets are incubated in microtiter plates that have collagen, convulxin or BSA immobilized to the surface of the wells, the cells are washed, 2% SDS is added to each well, and the number of adherent platelets is determined by counts for 5'Cr using a scintillation counter (see, e.g., Jandrot-Perrus et al., 1997, 3. of Biol. Chem. 272: 27035-27041).
  • the activity of an agent can be analyzed by platelet aggregation assays or secretion assays known to those of skill in the art (see, e.g., Moroi et al., 1989, J. Clin. Invest. 84: 1440-1445and Poole et al., 1997, EMBO J. 16 (9): 2333-2341).
  • the platelet aggregation isperformed as follows: platelets are incubated with collagen or convulxin in a cuvette at 37 C while being stirred, and the cell suspension is monitored by a lumiaggregometer.
  • Such assays may be utilized as part of GPVI, collagen, fibronectin or vitronectin diagnostic assays.
  • such assays may be utilized as part of screening methods for identifying compounds that modulate the activity and/or expression and/or interactions of GPVI, collagen, fibronectin or vitronectin.
  • the disclosure includes a method (also referred to herein as a "screening assay") for identifying agents, i.
  • the disclosure provides a method of identifying an agent which binds to GPVI, fibronectin and/or vitronectin comprising contacting a candidate agent with GPVI, fibronectin and/or vitronectin or fragments thereof.
  • assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of polypeptides, for example GPVI, fibronectin or vitronectin, or biologically active portions thereof.
  • the agents of the present disclosure can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrugDes.
  • an assay is a cell-based assay in which a cell which expresses a membrane- bound form of polypeptides of the disclosure, for example GPVI, fibronectin and/or vitronectin, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to the polypeptide determined.
  • the cell for example, can be a yeast cell or a cell of mammalian origin.
  • Determining the ability of the test compound to bind to the polypeptide can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the polypeptide or biologically active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled withl25I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting a cell which expresses a membrane-bound form of the polypeptides, for example GPVI, fibronectin and/or vitronectin, or a biologically active portion thereof, on the cell surface with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with GPVI, fibronectin and/or vitronectin, wherein determining the ability of the test compound to interact with GPVI, fibronectin and/or vitronectin comprises determining the ability of the test compound to preferentially bind to GPVI, fibronectin and/or vitronectin or a biologically active portion thereof as compared to the known compound.
  • an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of the polypeptides, for example GPVI, fibronectin and/or vitronectin, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e. g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof or to interfere with its interaction with other molecules. Determining the ability of the test compound to modulate the activity of GPVI, fibronectin and/or vitronectin or a biologically active portion thereof can be accomplished, for example, by determining the ability of the polypeptide protein to bind to or interact with a target molecule.
  • a target molecule is a molecule with which a selected polypeptide (e.g., GPVI, fibronectin and/or vitronectin) binds or interacts with in nature, for example, a molecule on the surface of a cell which expresses the selected protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • a target molecule can be a polypeptide described herein or some other polypeptide or protein.
  • a target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a polypeptide of the disclosure) through the cell membrane and into the cell or a second intercellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with GPVI, fibronectin and/or vitronectin.
  • an extracellular signal e.g., a signal generated by binding of a compound to a polypeptide of the disclosure
  • a second intercellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with GPVI, fibronectin and/or vitronectin.
  • Determining the ability of the polypeptide, for example, GPVI, collagen, fibronectin and/or vitronectin, to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca2+,diacylglycerol, IP3,etc), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the disclosure operably linked to a nucleic acid encoding a detectable marker, e. g., luciferase), or detecting a cellular response, for example, cellular differentiation, or cell proliferation.
  • a reporter gene e.g., a regulatory element that is responsive to a polypeptide of the disclosure operably linked to a nucleic acid encoding
  • an assay of the present disclosure is a cell-free assay comprising contacting GPVI, collagen, fibronectin and/or vitronectin or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to GPVI, collagen, fibronectin and/or vitronectin or biologically active portion thereof. Binding of the test compound to GPVI, collagen, fibronectin and/or vitronectin can be determined either directly or indirectly as described above.
  • the assay includes contacting GPVI, collagen, fibronectin and/or vitronectin or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or biologically active portion thereof as compared to the known compound.
  • an assay is a cell-free assay comprising contacting a polypeptide of the disclosure for example GPVI, collagen, fibronectin and/or vitronectin or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e. g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished, for example, by determining the ability of the polypeptide to bind to a target molecule by one of the methods described above for determining direct binding.
  • determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished by determining the ability of the polypeptide of the disclosure to further modulate the target molecule.
  • the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
  • the cell-free assay comprises contacting a polypeptide of the disclosure, for example GPVI, vitronectin, collagen and/ or fibronectin or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the polypeptide to preferentially bind to or modulate the activity of a target molecule.
  • the cell-free assays of the present disclosure are amenable to use of both a soluble form or the membrane-bound form of a polypeptide of the disclosure, for example GPVI, collagen, vitronectin and/ or fibronectin.
  • a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution.
  • non-ionic detergents such as n-octylglucoside,
  • binding of a test compound to consider whether it is an agent according to the present disclosure, to the polypeptide, or interaction of the polypeptide with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase fusion proteins or glutathione-S- transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or a polypeptide of the disclosure, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of binding or activity of the polypeptide of the disclosure can be determined using standard techniques.
  • Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the disclosure.
  • either the polypeptide of the disclosure, for example GPVI, collagen, vitronectin and/or fibronectin or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polypeptide of the disclosure or target molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques well known in the art (e. g., biotinylation kit, Pierce Chemicals; Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals; Rockford, IL
  • streptavidin-coated 96 well plates Piereptavidin-coated 96 well plates
  • antibodies reactive with the polypeptides of the disclosure or target molecules but which do not interfere with binding of the polypeptide of the disclosure to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the disclosure trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the polypeptide(s) of the disclosure or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the polypeptide of the disclosure, for example, GPVI, fibronectin, vitronectin and/or collagen or target molecule.
  • modulators of expression of a polypeptide of the disclosure are identified in a method in which a cell is contacted with a candidate compound, to consider whether it is an agent of the present disclosure and the expression of the selected mRNA or protein (i. e., the mRNA or protein corresponding to a polypeptide or nucleic acid of the disclosure) in the cell is determined.
  • the level of expression of the selected mRNA or protein in the presence of the candidate compound is compared to the level of expression of the selected mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of expression of the polypeptide of the disclosure based on this comparison. For example, when expression of the selected mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of the selected.
  • the disclosure provides for the use of fibronectin and/or vitronectin or fragments thereof in a binding assay for identifying an agent capable of binding GPVI, preferably an agent capable of inhibiting platelet aggregation, more preferably an agent capable of inhibiting platelet aggregation by fibronectin and/or vitronectin interaction with platelet-bound GPVI.
  • an agent capable of binding GPVI preferably an agent capable of inhibiting platelet aggregation, more preferably an agent capable of inhibiting platelet aggregation by fibronectin and/or vitronectin interaction with platelet-bound GPVI.
  • the present invention considers the use of fibronectin or fragments thereof having GPVI binding activity in a binding assay for identifying an agent capable of inhibiting interaction of GPVI and fibronectin.
  • the agent is capable of inhibiting binding of GPVI to fibronectin.
  • vitronectin or fragments thereof having GPVI binding activity in a binding assay for identifying an agent capable of inhibiting interaction of GPVI and vitronectin.
  • the agent may be capable of inhibiting binding of GPVI to fibronectin.
  • Example 1 The following methods and examples represent a way of producing or obtaining an agent of the present invention. It will be apparent to the skilled person that alternative methods are available to obtain the agent of the invention.
  • Example 1
  • the extra-cellular domain of human GPVI was cloned and fused to the human immunoglobin Fc-domain.
  • the Fc was amplified from a human heart cDNA library (Clonetech, Palo Alto, CA) by PCR using the forward primer 5'-cgcggggcggccgcgagt- ccaaatcttgtgacaaaac-3' and the reverse primer 5'-gcgggaagctttcatttacccggagacagggag-3'.
  • the PCR reaction was performed at 58°C annealing temperature and 20 cycles with the Expand High Fidelity
  • PCR System (Roche Molecular Biochemicals, Mannheim, Germany). The PCR fragment was cloned in the plasmid pADTrack CMV with Notl/Hindlll and the sequence was checked by sequencing (MediGenomix, Martinsried, Germany).
  • RNA from cultured megakaryocytes was isolated (RNeasy Mini Kit; Qiagen, Hilden, Germany) according to the manufacturer's protocol and reverse transcription was performed (Omniscript RT Kit; Qiagen) with 2 ⁇ g RNA at 37 0 C overnight. 100 ng of the reaction was used as a template in PCR amplification of the hGPVI with the primer 5'- gcggggagatctaccaccatgtctccatcccccgacc-3' and 5'-cgcggggcggccgcgttgcccttggtgtagtac-3'.
  • the PCR reaction was performed at 54 0 C annealing temperature and 24 cycles with the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Mannheim, Germany).
  • the PCR fragment was cloned in the plasmid pADTrack CMV Fc with Bglll/Notl and the sequence was checked by sequencing.
  • the human Fc-GPVI-nt was amplified from the plasmid pADTrackCMV human Fc-GPVI-nt by PCR using the forward primer 5'- gcgggggctagcaccaccatgtctccatccccgac -3' and the reverse primer 5'- cgcgggggatcctcatttacccggagacagggag -3'.
  • the PCR reaction was performed at 58°C annealing temperature and 24 cycles with the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Mannheim, Germany).
  • PCR fragment was cloned in the plasmid pREP4 (Invitrogen, Carlsbad, CA) with Nhel/BamHI and the sequence of the resulting plasmid pREP4 human Fc-GPVI-nt was checked by sequencing (MediGenomix, Martinsried, Germany).
  • CHO Kl cells DSMZ, Braunschweig, Germany
  • effectene transfection reagent Qiagen, Hilden, Germany
  • Monoclonal antibodies were generated essentially as described (Kremmer, E., Kranz, B. R., HiIIe, A., Klein, K., Eulitz, M., Hoffmann-Fezer, G., Feiden, W., Herrmann, K., Delecluse, H. J,, Delsol, G., Bomkamm, G. W., Mueller-Lantzsch, N., Grassert, F. A. (1995) Rat monoclonal antibodies differentiating between the Epstein-Barr virus nuclear antigens 2A (EBNA2A) and 2B (EBNA2B). Virology 208, 336-342).
  • Lou/C rats were immunized with human dimeric Fc-GPVI-nt fusion protein (PR-15) as disclosed in WO03/104282). Screening of hybridoma supernatants was performed in a solid-phase immunoassay using (PR-15) dimeric Fc-GPVI-nt or an Fc portion lacking the GPVI domain. Screening identified the supernatant of hybridoma different antibodies to bind specifically to dimeric Fc-GPVI-nt but not to Fc lacking the external GPVI domain. The immunoglobulin type was determined with rat Ig class (anti-IgM) and IgG subclass-specific mouse mAbs. The monoclonal antibodies were purified using Protein G-Sepharose columns.
  • hGP 5C4 Antibody specificity of monoclonal antibody (specific to GPVI) hGP 5C4 was verified by immunoblotting against dimericFc-GPVI-nt and control Fc. hGP 5C4 monoclonal antibody detected recombinant dimeric Fc-GPVI-nt but not control Fc (data not shown). Furthermore, hGP 5C4 binds specifically to the surface of human platelets (data not shown).
  • IgG antibodies were digested to generate Fab-fragments of anti-GPVI antibodies with ImmunoPure Fab Kit (Pierce Biotechnology, Inc., Rockford, IL, USA) according to the manufacturer's instructions. Accordingly, IgG molecules were digested into Fab fragments and Fc fragments by using immobilized papain. After digestion, the fragments were purified on an immobilized Protein A column. Detailed instructions allow for flexibility in the protocol for hard to digest antibodies. The success of Fab-fragment generation was tested by comparing molecular size of both antibody formats in SDS gels and staining with Coomassie blue (data not shown).
  • Example 5 Confluent human umbilical vein endothelial cells (HUVEC) were activated with TNF- ⁇ (50 ng/ml) and INF- ⁇ (20 ng/ml) for 16 hours. Thereafter, the cells were pre-treated with a soluble form of recombinant dimeric human GPVI (100 ⁇ g/ml) fused to the human immunoglobulin Fc domain via a specific hinge region (hGPVI-Fc) (PR-15) 11 ' 12 or with equimolar amount of Fc control for 60 min. Recombinant soluble dimeric hGPVI-Fc exhibits high affinity for collagen and inhibits collagen-induced platelet aggregation 11 ' 12 .
  • hGPVI-Fc specific hinge region
  • Activated endothelial cells express a variety of adhesion receptors (e.g. ⁇ 3 -integrin, P-selectin, ICAM- I) 13"21 and molecules (e.g. von Willebrand factor (vWF), vitronectin (Vn), fibronectin (Fn)) 22"24 that all have been shown to play a role in platelet/endothelium adhesion. Most of these adhesion molecules are presented in substantial amounts on the luminal aspect of endothelial cells ⁇ Fig. 19c) and thereby are potential candidates for endothelial GPVI ligands.
  • adhesion receptors e.g. ⁇ 3 -integrin, P-selectin, ICAM- I
  • molecules e.g. von Willebrand factor (vWF), vitronectin (Vn), fibronectin (Fn) 22"24 that all have been shown to play a role in platelet/endothelium adhesion.
  • CHO cells stably transfected with human GPVI 12 showed substantially enhanced adhesion to immobilized Vn and Fn ⁇ Fig. 20b). This finding demonstrates that GPVI alone is sufficient to mediate cellular adhesion on Fn and Vn and establishes a novel role of GPVI for platelet physiology.
  • Example 7 To assess, the role of GPVI for platelet adhesion in the early stage of atherogenesis, platelet adhesion to the carotid endothelium was studied using intravital video fluorescence microscopy in apoE ⁇ " mice that were on cholesterol-enriched diet for 6 weeks. At this stage, significant platelet adhesion occurs at lesion-prone sites, but no lesion formation is detectable by histochemistry 27 .
  • ApoE " f ⁇ mice received fluorescence-tagged syngeneic platelets preincubated with IgG or Fab fragments of a blocking monoclonal antibody (JAQl) that is directed against the murine GPVI receptor 28 ' 29 . As control, a Fab fragment of the mAb JON/ A 27 that is directed against murine GPIIb-IIIa (not shown) or an unspecific IgG ⁇ Fig. 20c) were tested.
  • JAQl blocking monoclonal antibody
  • GPVI directly binds to the intact arterial wall, sections of the common carotid artery of 6-weeks old apoE '1' mice were stained with either hGPVI-Fc (10 ⁇ g/ml) or control Fc. Most strikingly, it was found specific hGPVI-Fc binding to the endothelial monolayer of the common carotid artery ⁇ Fig. 2Od) confirming the in vitro observation that GPVI binds to a ligand present on endothelium. Furthermore, two color immunofluorescence studies showed that binding of hGPVI co-localizes with Fn presented on the luminal surface of the atherosclerotic carotid artery ⁇ Fig. 2Oe). Because the present disclosure
  • mice 27 platelet adhesion to endothelium is critical for atherogenesis in mice 27 .
  • 6-weeks old apoE 1' mice were treated with the anti-GPVI mAb JAQl 28 ' 29 or control IgG over a period of 4 weeks by intraperitoneal injection twice a week of 50 ⁇ g.
  • Treatment of mice with JAQl results in specific and sustained loss of GPVI from circulating platelets and, consequently, abolished collagen responses to those cells 29 .
  • the degree of lesion formation was assessed in the carotid artery and the aortic arch thereafter at the age of 10, 13 and 16 weeks, respectively.
  • mice showed substantial decreases in lesion formation in the aortic arch (27 % and 36 % reduction in 13- and 16-wk-old apoE '1' , respectively, p ⁇ 0.01) ⁇ Fig. 21a and 21b).
  • Platelet adhesion to endothelial cells plays a critical role in a variety of disease states such as reperfusion and cardiovascular diseases including stroke, myocardial infarction, and atherosclerosis 1 ' 2 ' 4 .
  • Defining the relative importance of adhesion receptors has been a central goal in order to understand the molecular requirements of platelet/endothelium adhesion.
  • the present disclosure indicates an unexpected role of the platelet receptor GPVI for platelet-endothelium adhesion and vascular remodelling.
  • collagen receptor 7 the present specification discloses for the first time the identification of identified novel ligands for GPVI that are presented on the apical surface of dysfunctional endothelium 23 ' 24 .
  • CHO-hGPVI stable cell line were generated as described 12 .
  • HUVEC Flow cytometry Confluent monolayers of HUVEC were stimulated with TNF- ⁇ (50 ng/ml) and INF- ⁇ (20 ng/ ml) for 16 hours. Thereafter, HUVEC were incubated with 10 ⁇ g/ml with hGPVI-Fc or Fc, respectively. After two steps of gentle washings FITC-labeled anti-human Fc mAb was added for 30 min. Endothelial cells were detached with trypsin/EDTA and analysed on a FACScalibur (Becton Dickinson, Heidelberg, Germany). HUVEC were gated by FSC/SSC characteristics 2 .
  • non-stimulated or TNF- ⁇ /II ⁇ IF- ⁇ -activated HUVEC were incubated for 30 min with fluorochrome-conjugated mAbs as indicated at saturating concentrations before flow cytometric analysis.
  • hGPVI-Fc binding was fused to the human Fc domain as described 11 .
  • ELISA plates Immulon2 HB, Dynx Technologies, Chantilly, VA
  • 1 ⁇ g collagen type I bovine; BD Bioscience, Bedford, MA
  • 2 ⁇ g vitronectin both human, Becton Dickinson, Heidelberg, Germany
  • 2 ⁇ g vWF human, Calbiochem, Bad Soden, Germany
  • the plates were washed with PBS/0.05 % Tween 20 (PBST) twice and blocked with Roti-Block (Roth, Düsseldorf, Germany) over night. Then, 3.0, 6.0, 12.5, 25.0, 50.0 or 100 ⁇ g/ml GPVI-Fc in PBST was added and the plate was incubated for 1 hr at room temperature. After washing, peroxidase-conjugated goat anti-human IgG antibody Fey fragment specific (Dianova, Hamburg, Germany) was added and incubated for 1 hr at room temperature. After 5 fold washing with 250 ⁇ l PBST 100 ⁇ l detection reagent (BM Blue POD Substrate; Roche, Mannheim, Germany) was added and incubated up to 10 min.
  • PBST PBS/0.05 % Tween 20
  • the reaction was stopped by the addition of 100 ⁇ l 1 M H 2 SO 4 and the plate was measured at 450 nm against reference wavelength 690 nm. .
  • the assay was developed as described 12 .
  • a rat monoclonal antibody hGP 5C4 was prepared against the recombinant soluble human GPVI and found to inhibit collagen- induced platelet aggregation 12 .
  • Two ⁇ g of GPVI were incubated with 5 ⁇ g of Fab 5C4 for 30 min and GPVI binding to ligands was analyzed as described above. Adhesion of GPVI-transfectants to immobilized ligands was evaluated .
  • ELISA plates (Immulon2 HB, Dynx Technologies, Chantilly, VA) were coated over night at 4 0 C with collagen, Fn, or Vn.
  • Single separated CHO cells (CHO or CHO- GPVI 11 ) (IxIO 5 per well) were added to the wells for 30 min. After three rounds of gentle washing with Tyrode's buffer cell, adhesion was quantified using a color reaction based method 11 .
  • a 0.25% cholesterol diet Hard Research diets, 0% cholate
  • Platelet adhesion were monitored in vivo by use of video fluorescence microscopy as described in detail elsewhere 12 ' 30 . Where indicated, fluorescent wild type platelets were preincubated with 50 ⁇ g/ml anti-GPVI (JAQl-Fab) 28 or control IgG for 10 minutes prior to infusion. The number of adherent platelets was assessed by counting the cells that did not move or detach from the endothelial surface within 20 seconds 12 ' 30 .
  • a hybridoma cell line producing hGP 5C4 Fab antibody has been deposited under terms of the Budapest Treaty as hGP 5C4 with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen on November 25, 2003 and has been given Accession No. 2631.
  • GPVT mediates platelet adhesion to human atheromatous plaque and is critical for atheroprogression. Platelets play a critical role in development of atherosclerotic lesions, Ruggeri ZM. Nat Med. Nov 2002;8(ll): 1227-1234; Massberg S et al, J Exp Med. Oct 7 2002;196(7):887-896; Theilmeier G et al, Blood. Jun 15 2002; 99(12) :4486-4493; Huo Y et al, Nat Med. Jan 2003;9(l):61-67. During atherogenesis platelets adhere to the arterial wall and induce inflammatory and proliferative reactions resulting in lesion formation, (Massberg S et al, J Exp Med.
  • Collagen is the main constituent of the plaque organ matrix and the predominant subtypes I and III represent 80-90% of total collagenous protein in atherosclerotic lesions, (Katsuda S, Kaji T. J Atheroscler Thromb. 2003;10(5):267-274). While collagen type I heavily accumulates within the fibrous cap that overlies the lipid-rich core, collagen type III has been described to be predominant at the plaque/thrombus interface in regions of plaque erosion, (Fernandez-Ortiz A et al, J Am Coll Cardiol. Jun 1994;23(7): 1562-1569). Collagen acts as a strong activator of platelets and supports platelet adhesion both in vitro and in vivo.
  • GPVI binds to fibrillar collagen, especially types I and III. Extracellular matrix collagen is exposed in microerosions of the vascular wall during atherogenesis resulting in enhanced transient platelet adhesion at lesions sites. Ligand binding to GPVI induces platelet activation leading to secretion and subsequent activation of integrin receptors that stabilize the platelet interaction with the arterial wall, (Cabeza N et al, Diabetes. Aug 2004;53(8):2117-2121); (Nieswandt B, Watson SP, Blood. JuI 15 2003; 102(2) :449-461).
  • the present invention includes processes, products and uses relating to the molecular mechanisms of platelet adhesion to atherosclerotic plaques and investigates the effect of inhibition on atherosclerotic lesion formation.
  • the anti-GPVI mAb 5C4 and the irrelevant idiotype 2Dl were generated as described previously (Massberg S et al, Faseb J. Feb 2004;18(2):397-399).
  • MAb IB4 was raised in rats against human ⁇ 2 ⁇ l integrin.
  • Mouse-antibodies directed against human collagen type I and III, and IgGl-isotype control were purchased from Rockland Immunochemicals (Gilbertsville, PA, USA). Soluble GPVI, the extracellular domain of human GPVI, was cloned and fused to the human Fc domain as described previously, (Massberg S et al, Faseb J. Feb 2004;18(2):397-399).
  • control Fc and GPVI-Fc were purified as described, (Massberg S et al, Faseb J. Feb 2004;18(2):397- 399).
  • Collagen horn was obtained from Nycomed Pharma (Munich, Germany). All other reagents used were from Sigma.
  • Radio-labeled GPVI 125 IGPVI was generated and performed as described, (Gawaz M et a ⁇ , T ⁇ romb Haemost. May 2005;93(5):910-913).
  • Adhesion experiments were basically performed as previously described, (Massberg S et al, J Exp Med. Jan 6 2003;197(l):41-49). Human whole blood from healthy volunteer donors, who had not taken any medication for at least 10 days before the experiment, was collected into syringes containing 0.5 vol% heparin. Platelets were labelled in whole blood by incubation with the fluorescent dye rhodamine-6G (final concentration 0.2 g/L) for 15 min at 37 0 C. Leukocytes could be readily distinguished from platelets by their larger size and nuclear morphology, red cells were not visualized by rhodamine-6G.
  • Adhesion of platelets to coverslips coated with type I collagen, type III collagen, or human atheromatous plaque was determined in a parallel plate flow chamber in the presence of anti-GPVI (5C4, lO ⁇ g/mL), anti- ⁇ 2 ⁇ l integrin (IB4, lO ⁇ g/mL), an irrelevant control antibody (2Dl, lO ⁇ g/mL), 0.8mg/mL GPVI-Fc, or control Fc.
  • Atheromatous plaque tissue was pre-incubated with function blocking antibodies recognizing human collagen type I or type III, or rabbit control IgG, coated onto the coverslips after Ih incubation and perfused with whole blood as follows, Perfusion was performed for 10 minutes at a wall shear rate of 1000 s '1 , followed by a 4 min perfusion with Tyrode 's solution-HEPES buffer at a shear rate of 1000/s using a pulse-free pump.
  • the flow chamber was mounted on an inverted fluorescence microscope (Axiovert, Zeiss, Jena, Germany) and fluorescent images were recorded from microscope fields (2Ox objectives) using a digital photo camera (Axiocam, Zeiss, Jena, Germany).
  • Platelet aggregation was performed in whole blood by luminometry, (Penz S et al, Faseb J. Jun
  • Collagen type I l ⁇ g/ml or collagen type III l ⁇ g/ml or extracted human atheromatous plaque tissue was added to citrated whole blood and stirred at 1000 rpm at 37 0 C.
  • aggregation was performed in the presence of anti-GPVI mAb 5C4 (5 ⁇ g/ml) or control IgG (5 ⁇ g/ml).
  • mice Specific pathogen-free wild type C57BL6/J mice were obtained from Charles River (Suzfeld, Germany). 4-week-old male ApoE ';" (C57BL/6JApoEtmlUnc) mice (The Jackson Laboratory) consumed a 0.25% cholesterol diet (Harlan Research diets, 0% cholate) for 8 and 12 weeks as indicated.
  • Atherosclerotic tissue specimens were obtained from patients who underwent surgery for high grade carotid artery stenosis as decribed previously, (Penz S et al, Faseb J. Jun 2005;19(8):898-909).
  • the carotid plaque tissue was removed by a technique of intraoperative endarterectomy that preserved the plaque structure en bloc.
  • the specimens containing lipid-rich soft plaques were collected.
  • the atheromatous plaques were carefully dissected from other regions of the atherosclerotic tissue specimens, weighed, homogenized and stored at -20 0 C.
  • the atheromatous plaque concentration was 50 mg/mL wet weight corresponding to protein concentrations of 0.5-2.5 mg/mL.
  • homogenized atheromatous plaque (5 mg/mL) was dissolved in PBS containing 15 mM fatty acid-free albumin, incubated for 16 h at 4°C in PBS-albumin (75 mM) to prevent non-specific binding, and immobilized onto glass coverslips.
  • atheromatous plaque was pre-incubated for 1 h with anti-human collagen type I or type III antibodies or rabbit IgG control and thereafter coated onto coverslips as described.
  • Atheromatous plaques were fixed in 4% paraformaldehyde (pH 7.0) and embedded in paraffin as described. Serial paraffin sections of 3 ⁇ m thickness were deparaffinized and dehydrated. The specimens were microwaved in citrate buffer (pH 6.0) for 20 min. Endogenous peroxidase was blocked by 3% H 2 O 2 , followed by incubation with anti-collagen type I or type III antibodies, or IgG (control) antibody. Collagen- positive structures were identified by using the streptavidin-horseradish peroxidase technique (DakoChem Mate Detection kit; Hamburg, Germany). Specific binding of the antibodies was detected by 0.1% 3'3'-diaminobenzidine. For immunohistochemical localization of GPVI-binding sites in situ, atheromatous plaques were processed as described above and stained with GPVI-Fc.
  • Data represent mean ⁇ s.e.m.. Data were analyzed using one-way ANOVA with a Tukey post-hoc test. A value of P ⁇ 0.05 was regarded as significant.
  • soluble GPVI-Fc binding of soluble GPVI-Fc to atheromatous plaque (AP) material isolated from the core region of human carotid plaque specimen was quantified. Substantial and specific binding activity of GPVI-Fc to atheromatous plaque material was found (data not shown), indicating that GPVI interacts with collagen-containing structures present within the atherosclerotic plaque tissue. Binding of soluble GPVI-Fc was enhanced to immobilized collagen type III compared with collagen type I (data not shown). Thus, it is shown that soluble GPVI preferentially binds to collagen structure within the core region of human atherosclerotic plaque.
  • Platelet adhesion to collagen is mediated both through GPVI and the integrin collagen receptor ⁇ 2 ⁇ i, (Nieswandt B, Watson SP, Blood. JuI 15 2003;102(2):449-461).
  • platelet adhesion under dynamic flow conditions was studied using cover slips coated with extracted AP material or collagen type I and III.
  • Significant platelet adhesion to immobilized collagen type I and type III was found. Platelet adhesion to both collagen type I and III was significantly reduced by inhibition of GPVI with the neutralizing anti-GPVI Ab 5C4 (data not shown).
  • GPVI is a critical receptor for platelet interactionwith human atheromatous plaque material.
  • soluble Fc-GPVI-Fc or control Fc fragment was radio-labelled with iodine ( 125 I) and injected into ApoE-deficient or wild type mice, (Gawaz M et al, Thromb Haemost. May 2005;93(5):910-913).
  • Ex vivo autoradiography of the explanted aortic arch revealed a substantial approximately 1.5-fold increase in 125 I-GPVI binding activity of ApoE-deficient mice compared with wild type after tracer administration (ApoE vs wild type, counts/mm 2 : 356+45 vs 250+10) (p ⁇ 0.01) (data not shown).
  • positron emission tomography (PET) imaging revealed substantial accumulation of radioiodinated GPVI-Fc in the aortic arch of ApoE-deficient mice but not wild type mice (data not shown).
  • PET positron emission tomography
  • radioiodinated GPVI-Fc accumulated to a similar degree in wire-induced lesions of the right carotid artery (data not shown).
  • GPVI binds to atherosclerotic lesions of ApoE-deficient mice in vivo.
  • the present disclosure therefore relates to the following: 1) the platelet collagen GPVI binds to atheromatous plaque tissue in situ and recognizes preferentially collagen structures within the core region of plaque tissue. 2) GPVl but not integrin ⁇ 2 ⁇ i is the predominant platelet collagen receptor that mediates adhesion of platelets to atheromatous plaque material under flow conditions. 3) The soluble GPVI receptor (for example in the form of a GPVI fusion protein) substantially binds to atherosclerotic arteries of ApoE-deficient mice in vivo. 4) Prolonged administration of soluble GPVI but not control Fc attenuated atheroprogression in ApoE-deficient mice. The findings indicate that the GPVI is critical for platelet adhesion to atheromatous arteries and plays a critical role in atheroprogression.
  • Biodistribution of GPVI-Fc fusion protein having the sequence shown in Figure 7 (PR-15) was assessed by using 12S I-radiolabelled GPVI-Fc in mice.
  • collagen binding capacity and affinity of GPVI-Fc were not affected by radiolabelling. Radioactivity was counted 30 min, 2 hours, 6 hours and 24 hours after intravenous application in the respective organs and in the serum. Uptake of free atomic iodine to the thyroid gland was blocked by administration of perchlorate to the animals prior to the experiment.
  • Radioactivity was determined by gamma-counting in the serum, the blood, the liver, the kidney, the spleen, the heart, the lung, the intestines, in skeletal muscle and the brain on a counts per tissue weight/tissue volume basis.
  • the respective animals were sacrificed after the indicated time periods after administration of GPVI-Fc 7 and organs were harvested by necropsy.
  • GPVI-Fc Binding of radiolab led GPVI-Fc to arterial lesions was tested in Apo E-/- mice in vivo, and compared to healthy wild type mice.
  • the aortae and the carotid arteries were dissected from mice 24 hours after intravenous administration of GPVI-Fc .
  • the atherosclerotic arteries were exposed to phospho imagers to detect specific GPVI-Fc signals.
  • GPVI-Fc specifically labelled arterial lesions, as determined by subsequent sudan oil red staining of the arteries.
  • the present invention includes the feasibility methods including local administration and delivery of anti-GPVI agents.
  • the following examples demonstrate an effect of local delivery of, for example, soluble GPVI on thrombus formation at the site of balloon-induced carotid injury in rabbits.
  • New Zealand White rabbits (weight 3.6 ⁇ 0.3 Kg; from Asam, Aretsried, Germany) were kept under constant temperature and humidity with chow ad libidum according to the animal health regulations.
  • the animals are anaesthesised with Propofol (Propofol, 2%, Fresenius) and Fentanyl (Fentanyl- Janssen 0.5 mg, Janssen-Cilag, 0.01 mg /kg). They are intubated and artificially ventilated (AWS, Fa. V ⁇ lker, Germany).
  • Buprenorphin Temgesic ®, Boehringer, 0.01 mg /kg
  • Heparin was induced.
  • femoral artery Left femoral artery was prepared and an introducer for arterial catheterization was placeed (Radiofocus®, 4F, Terumo). Under x-ray guidance a Fogarty catheter (EMB: thru-lumen embolectomy catheter, 3F, Edwards Lifesciences, Guidewire: Stabilizer® , 014 in., Cordis) is placed in the right carotid artery. After inflation with 0.4 ml air the balloon is gently rubbed up and down twice between the second and the sixth cervical vertebrae.
  • EMB thru-lumen embolectomy catheter, 3F, Edwards Lifesciences, Guidewire: Stabilizer® , 014 in., Cordis
  • a 3.0 mm modified double-balloon catheter (ACROSTAK Corp., Winterthur, Switzerland) was used for local GPVI (in the form of GPVI-Fc fusion protein) delivery.
  • This system consists essentially of a percutaneous balloon having a distal and proximal segment with occlusive function and a central segment that allows for homogenous transfer of GPVI to the vessel wall.
  • Three holes in the distal ramp fill the drug depot without additional trauma or hydrojets (see figure 4).
  • the balloon was inflated at a low pressure of 2 atm that allows for distal and proximal occlusion of the vessel without additional trauma while simultaneously forming a central drug depot.
  • FC-GPVI or FC solution 1.3 mg/ml
  • FC-GPVI or FC solution 1.3 mg/ml
  • FC solution 1.3 mg/ml
  • the catheters are removed the femoral artery is ligated and the inguinal wound is sutured by using Vicryl ® (3-0).
  • Antibody binding was visualised with an avidin / biotinylated glucose oxidase (ABC-GO) system according to the manufacturer's instructions (Vectastain ABC-GO Kit; Vector Laboratories Inc; Burlingame; CA 94010).
  • Results in this study were expressed as mean ⁇ SD. Statistical analysis was performed with Student t test or ANOVA where appropriate. A value of P ⁇ 0.05 was considered statistically significant.
  • the present disclosure shows that local delivery of the soluble form of platelet collagen receptor glycoprotein VI (sGPVI) efficiently reduces thrombus formation at sites of arterial injury.
  • sGPVI platelet collagen receptor glycoprotein VI
  • the present disclosure includes a new antithrombotic approach to treat spontaneous or ballon-induced arterial lesions without the necessity of systemic antiplatelet therapy.
  • Thrombotic complications arise from arterial segments that are treated with percutaneous coronary interventions (PCI).
  • PCI percutaneous coronary interventions
  • the introduction of intensified combined antiplatelet therapy has significantly lowered the rate of thrombo-ischemic complications significantly after PCI, however, subacute thrombotic vessel occlusion still occurs in 1 to 4% of patients after coronary stenting.
  • the effect of soluble GPVI in the form of PR-15
  • soluble GPVI can be sufficiently delivered locally at site of vascular injury.
  • localized treatment of injured arterial arteries with sGPVI efficiently reduces thrombus formation.
  • the present disclosure includes a method of localized antithrombotic treatment of thrombogenic vulnerable atherosclerotic plaques or stented artery segments without systemic antiplatelet effects.
  • Figures 25 to 27 show a comparison between a GPVI-Fc fusion protein (PR-15), whose sequence is shown in Figure 7 and a GPVI-Fc fusion protein comprising an Ala-Ala-Ala linker linking the extracellular domain of GPVI and the Fc portion.
  • PR-15 GPVI-Fc fusion protein
  • Figure 7 a GPVI-Fc fusion protein comprising an Ala-Ala-Ala linker linking the extracellular domain of GPVI and the Fc portion.
  • Fc-GPVI PR-15
  • Fc a control protein
  • a competing Fc-GPVI-nt with another specific linker according to the sequence alluded to in WO 01/00810 was cloned according to the same method.
  • a similar construct was generated using the same DNA sequence as descried in Figure 7 of PCT/EP03/05929 except for the Gly-Gly-Arg linker on amino acid positions 270-272, which was replaced by a Ala-Ala- Ala linker, amplified and purified according to the same methods. Aggregation and A TP release
  • the right cervical region was dissected free between the sternohyoid and myohyoid muscle, a clamping and subsequent ligation of the cranial internal carotid artery was carried out. Then, the common carotid and the external carotid were clamped. The right internal carotid artery was incised. A think coronary guiding wire of defined size was introduced into the right common carotid artery and a vascular injury was induced by moving the wire up and down three times. This led to complete loss of endothelial cells at the site of injury.
  • the en face thrombus size was measured by digitalized imaging analysis, which measured the relative thrombus area as a percentage of the total vessel area as spread out on the slide.
  • Figure 25 shows the results of aggregation studies. Aggregation and ATP release measurements of freshly isolated human platelets in response to collagen (l ⁇ g/mL) resulted in strong photometric signals as described in PCT/EP03/05929).
  • Figure 25 shows the aggregation as a percentage of the internal standard of the aggregometric device.
  • the aggregation was slightly but not significantly less in the presence of a control Fc protein as compared to collagen-induced aggregation. Aggregation was inhibited slightly by the GPVI fusion protein containing the triple alanine linker.
  • administration of the GPVl fusion protein containing the Gly-Gly-Arg linker (PR-15) at the same concentration (40 ⁇ g/mL) markedly and highly significantly suppressed aggregation. Shown are results from 4 independent experiments. *p ⁇ 0.05 vs controls. It can be seen from Figure 25 that administration of collagen only induced aggregation of approximately 37 to 38% of the internal standard of the device used.
  • the GPVI-Fc fusion protein containing a Gly-Gly-Arg linker is capable of inhibiting platelet aggregation in response to collagen by at least 50%.
  • the GPVI-Fc fusion protein containing a Gly-Gly-Arg linker is capable of inhibiting platelet aggregation in response to collagen by at least 60%, for example at least 70%, e.g. 75% or more.
  • Figure 26 shows the results of mean ATP release from platelets in response to l ⁇ g/mL of collagen in the presence of either a GPVI-Fc fusion protein containing a triple alanine or a GPVI-Fc fusion protein containing a Gly-Gly-Arg linker.
  • Figure 26 shows mean ATP release from human platelets in response to 1 ⁇ g/mL collagen (expressed as % of the internal standard) in the presence of either construct. ATP release was slightly but not significantly reduced in the presence of the control Fc, and slightly more reduced in the presence of the Fc-GPVI-nt containing the Ala-Ala-Ala linker. In contrast, administration of the Fc-GPVI-nt containing the Gly-Gly-Arg linker at the same concentration (40 ⁇ g/mL) markedly and highly significantly suppressed ATP release. Shown are results from four independent experiments. * ⁇ 0.05 vs. controls.
  • the GPVI-Gly-Gly-Arg- Fc fusion protein is capable of inhibiting collagen-induced ATP release from platelets by at least 65%, optionally by at least 70%, optionally by at least 80% and optionally by at least 90%.
  • Figure 27 shows the adhesion of platelets to endothelial lesions in vivo. A comparison between an Fc portion, the GPVI-Fc fusion protein containing a triple alanine linker and a GPVI-Fc fusion protein containing a Gly-Gly-Arg linker (PR-15) was made.
  • the uninjured left carotid arteries were investigated as controls in all animals and did not show any platelet adhesion or thrombus formation.
  • mice were injected with 2mg/kg of either construct, then endothelial lesions were induced.
  • the right carotid arteries were excised, and prepared as described above.
  • the Figure shows the mean en face thrombus sizes related to the overall surface of the respective vessel and expressed as a percentage of the total vessel area as spread out on the slide. Shown are results form five independent experiments in five independent animals.
  • Figure 27 shows that the administration of an Fc portion to the mice resulted in formation of thrombus of approximately 7.5% in area relative to the total vessel area as spread out on a slide.
  • the GPVI-Fc fusion protein containing the triple alanine linker resulted in the formation of a thrombus of approximately 4% in area relative to the total vessel area as spread out on a slide.
  • Administration of the GPVI-Fc fusion protein containing the Gly-Gly-Arg linker resulted in the formation of a thrombus of negligible area relative to the total vessel area as spread out on a slide. It is therefore considered that the GPVI-Fc fusion protein containing the Gly-Gly-Arg linker is capable of inhibiting thrombus formation caused by induction of arterial lesions in a mouse by at least 70% as compared to a control administered with an Fc portion polypeptide.
  • the GPVI-Fc fusion protein containing the triple alanine linker resulted in the formation of a thrombus of approximately 4% in area relative to the total vessel area as spread out on a slide.
  • Fc fusion protein containing the Gly-Gly-Arg linker is capable of inhibiting the thrombus formation by at least 80% and optionally at least 90% and optionally at least 95%.
  • the agent of paragraph 1 which is capable of inhibiting ATP release from human platelets by at least 70%.
  • the agent of paragraph 1 which is capable of inhibiting ATP release from human platelets by at least 80%, e.g. at least 90%.
  • An agent which is capable of binding to a plurality of GPVI-binding sites, optionally a plurality of types of GPVI-binding sites, of a dysfunctional, inflamed or atherosclerotic blood vessel area the agent being capable of inhibiting thrombus formation caused by induction of arterial lesions in the mouse by at least 70% as compared with a control administered with an Fc polypeptide, the relative inhibitions of thrombus formation being determined by measurement of the en face thrombus size as a percentage of an artery wall area placed on a surface for the purpose of said measurement.
  • agent of any preceding paragraph which is not a fusion protein comprising: a) an extracellular domain of GPVI or a variant thereof that is functional for binding to collagen; and b) an Fc domain of an immunoglobulin or a functional conservative part thereof, the extracellular domain and the Fc domain being fused via a linker characterised by the amino acid sequence Gly-Gly-Arg.
  • agent of any preceding paragraph which comprises an amino acid sequence of or comprised in an extracellular domain of GPVI, or a variant thereof that is functional for binding to collagen and to at least one protein selected from fibronectin and vitronectin.
  • nucleic acid sequence which encodes a wild type GPVI extracellular domain
  • nucleic acid sequence which hybridises to said nucleic acid sequence (i) and which encodes a polypeptide that is capable of binding to a plurality of types of GPVI-binding sites of a dysfunctional, inflamed or atherosclerotic blood vessel area; or
  • nucleic acid sequence which differs from said nucleic acid sequence (i) by virtue of the degeneracy of the genetic code.
  • nucleic acid sequence which encodes a wild type GPVI extracellular domain
  • nucleic acid sequence which hybridises to said nucleic acid sequence (i) and which encodes a polypeptide that is which is capable of inhibiting the binding of GPVI to collagen and to one or both of fibronectin and vitronectin; or
  • nucleic acid sequence which differs from said nucleic acid sequence (i) by virtue of the degeneracy of the genetic code.
  • nucleic acid sequence of bases 1 to 807 of SEQ ID No. 2 (i) a nucleic acid sequence of bases 1 to 807 of SEQ ID No. 2 (figure 8); (ii) a nucleic acid sequence which hybridises to bases 1 to 807 of SEQ ID No. 2 (figure 8) and which encodes a polypeptide that is capable of binding to a plurality of types of GPVI-binding sites of a dysfunctional or atherosclerotic blood vessel area; or
  • nucleic acid sequence which hybridises to bases 1 to 807 of SEQ ID No. 2 (figure 8) and which encodes a polypeptide that is which is capable of inhibiting the binding of GPVI to collagen and to one or both of fibronectin and vitronectin; or (iii) a nucleic acid sequence which differs from bases 1 to 807 of SEQ ID No. 2 (figure 8) by virtue of the degeneracy of the genetic code.
  • the agent of any of paragraphs 1 to 14 which comprises an antibody product selected from antibodies and antibody fragments; a protein; a polypeptide; a fusion protein; an aptamer; or a compound.
  • the agent of paragraph 29 or paragraph 30 wherein the antibody product is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a human antibody or a humanized antibody or a fragment of such an antibody.
  • the protein or polypeptide is a recombinant protein or polypeptide which comprises domains which bind to collagen, fibronectin and vitronectin.
  • An agent which comprises: a first portion capable of binding to an intravascular GPVI-binding domain; and a second portion which is capable of binding to a site other than a GPVI-binding domain and/or comprises a therapeutic or diagnostic moiety which has a direct or indirect therapeutic or diagnostic function or a combination of such agents.
  • GPIa/IIa GPIa/IIa
  • GPIV GPIc/IIa
  • GPIb/IX GPIa/IIa
  • GPIc/IIa GPIb/IX
  • PDGF platelet derived growth factor
  • glycoprotein IBa glycoprotein IBa
  • thrombomodulin
  • the second portion comprises an amino acid sequence derived from platelet derived growth factor (PDGF), a platelet receptor protein (e.g. a platelet-bound glycoprotein) hirudin, thrombomodulin, vascular epidermal growth factor, transforming growth factor- ⁇ l, basic fibroblast growth factor, angiotensin II, factor VIII, von Willebrand factor, tick anticoagulant protein (TAP) or nematode anticoagulant protein (NAP), or a sequence having homology to at least part of such sequence.
  • PDGF platelet derived growth factor
  • a platelet receptor protein e.g. a platelet-bound glycoprotein
  • hirudin hirudin
  • thrombomodulin hirudin
  • vascular epidermal growth factor transforming growth factor- ⁇ l
  • basic fibroblast growth factor transforming growth factor- ⁇ l
  • angiotensin II factor VIII
  • factor VIII von Willebrand factor
  • NAP nematode anticoagulant protein
  • An agent of paragraph 35 wherein the second portion comprises an imaging agent, for example a radio-imaging agent, e.g. Tc ⁇ 9m .
  • An agent of any of paragraphs 35 to 41 which comprises at least one further portion which is capable of binding to a site other than a GPVI-binding domain and/or which comprises a therapeutic or diagnostic moiety.

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Abstract

L'invention concerne des agents interférant avec la liaison de GPVI à divers composants. L'invention concerne également des agents interférant avec l'interaction de GPVI avec la fibronectine et/ou la vironectine ou des séquences de celles-ci. L'invention concerne, en outre, des méthodes de traitement de troubles ou de maladies impliquant une interaction pathologique, dysfonctionnelle ou non pathologique de GPVI avec la fibronectine et/ou la vitronectine. L'invention concerne enfin l'utilisation d'agents dans la prévention ou le traitement de troubles engendrés par l'adhésion et l'agrégation de plaquettes du sang.
PCT/GB2005/004764 2002-06-07 2005-12-12 Procedes, produits et utilisations relatifs a des plaquettes et/ou la vasculature Ceased WO2006061650A2 (fr)

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US11/009,106 US7531178B2 (en) 2002-06-07 2004-12-10 Immunoadhesin comprising a glycoprotein VI domain
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CN108918564A (zh) * 2018-08-09 2018-11-30 陕西延长石油(集团)有限责任公司研究院 一种适用于泥页岩矿物成分定量的分析方法
CN108918564B (zh) * 2018-08-09 2021-02-02 陕西延长石油(集团)有限责任公司研究院 一种适用于泥页岩矿物成分定量的分析方法
CN111134760A (zh) * 2020-02-18 2020-05-12 毛静 一种介入微创手术切口按压止血设备

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