Acute coronary and cerebrovascular accidents are currently the first death cause in the world. In addition, the global incidence of recurrence and death in the 6 month post-treatment period after an acute coronary syndrome is still 8-15%. In the case of acute coronary syndrome with ST segment elevation, mechanical treatment with coronary angioplasty and introduction of a stent is highly efficient to urgently restore coronary artery flow, but does not prevent morbidity/mortality for about 15% of patients in the next 6 months.
Thrombolytic treatments, which are based on long term fibrinolytic, anticoagulant and anti-aggregating drugs associations, give even less encouraging results. Indeed, despite improvements in medical treatment of thrombosis, morbidity/mortality at 6 months is similar to that observed for acute coronary syndrome without segment ST elevation.
Concerning cerebrovascular ischemic accidents, treatments are still very limited due to the generally late caring of most patients and to the hemorrhagic risk of currently available anti-thrombotic treatments.
There is thus a real pressing clinical need for improving treatments for cardiovascular diseases, and especially for new molecules with improved features compared to available molecules, in particular for molecules with a reduced hemorrhagic effect.
Platelets-collagen interactions are critical in the appearance of acute arterial thrombosis and post-thrombotic vascular remodeling. Glycoprotein VI (GPVI), the main receptor for platelets activation by collagen, has been demonstrated in animals to play a role in experimental thrombosis, vascular remodeling, atherothrombosis and acute myocardial ischemia.
Contrary to WIND integrin antagonists, which are currently used in thrombosis treatment and inhibit platelets final activation phase, GPVI is implicated into platelets initial activation phase, and GPVI antagonists should thus prevent not only platelet aggregation, but also secondary agonists liberation as well a growth factors and cytokines secretion resulting into vascular lesions development. In addition, GPVI deficit is not associated with a high hemorrhagic risk, which is a crucial feature for patient's safety. Finally, GPVI expression is limited to platelets, and thus represents a perfectly specific target for anti-thrombosis treatment.
GPVI antagonists should thus be efficient for specifically and efficiently preventing primary or secondary thrombosis, while involving only a low hemorrhagic risk.
Various kinds of potential GPVI antagonists have been generated. In one approach, a soluble GPVI recombinant protein has been generated, which is a fusion protein between GPVI extracellular domain and human Ig Fc domain (see for instance WO 01/00810 (1) and WO 03/008454 (2)). Thus soluble recombinant GPVI protein competes with platelet GPVI for binding collagen. Encouraging results were first obtained with this soluble GPVI protein in a thrombosis murine model (3), but these results were not confirmed (4). In addition, this approach involves structural, functional and pharmacological disadvantages. First, this compound is a high molecular weight protein (˜160 kDa) the half life of which is expected to be short. Since GPVI contains at least one cleavage site for proteases, the hydrolysis of the soluble recombinant GPVI-Fc protein has to be envisaged. When bound to collagen, the protein will expose its Fc domain to the blood stream. Human platelets (but not mice platelets) and leucocytes express the low affinity Fc receptor (FcγRIIA) at their surface. Cross-linking of the platelet FcγRIIA by immobilized GPVI-Fc is susceptible to activate platelets and thus to have an opposite effect to the one expected. The timing and the dose to which the protein should be administrated also cause problem. Once bound to collagen platelets are rapidly and irreversibly activated. Thus, to be effective GPVI-Fc should be administrated before platelet activation that is before the thrombotic event, a situation rare in current medicine. Furthermore, the amount of protein that should be administrated will vary as a function of the size and the nature of the vascular lesion, a parameter impossible to predict.
Many others have tried to develop neutralizing monoclonal antibodies directed against human GPVI. For instance, EP 1224942 (5) and EP 1228768 (6) disclose a monoclonal anti-GPVI antibody JAQ1, which specifically binds to mouse GPVI, for the treatment of thrombotic disease. JAQ1 antibody induces irreversible internalization of the GPVI receptor on mouse platelets.
EP1538165 (7) describes another monoclonal anti-GPVI antibody hGP 5C4, which Fab fragment was found to have marked inhibitory effects on the main physiological functions of platelets induced by collagen stimulation: stimulation of collagen-mediated physiological activation parameters PAC-1 and CD 62P-Selectin was completely prevented by hGP 5C4 Fab, and hGP 5C4 Fab potently inhibited human platelet aggregation ex vivo without any intrinsic activity.
WO 2005/111083 (8) describes 4 monoclonal anti-GPVI antibodies OM1, OM2, OM3 and OM4, that were found to inhibit GPVI binding to collagen, collagen-induced secretion and thromboxane A2 (TXA2) formation in vitro, as well as ex vivo collagen-induced platelet aggregation after intravenous injection to Cynomolgus monkeys. OM4 also appears to inhibit thrombus formation in a rat thrombosis model.
WO 01/00810 (1) also describes various monoclonal anti-GPVI antibodies named 7I20.2, 8M14.3, 3F8.1, 9E18.3, 3J24.2, 6E12.3, IP10.2, 4L7.3, 7H4.6, 9O12.2, 7H14.1, and 9E18.2, as well as several scFv fragments named A9, A10, C9, A4, C10, B4, C3 and D11. Some of these antibodies and scFv fragments were found to inhibit GPVI binding to collagen, including antibodies 8M14.3, 3F8.1, 9E18.3, 3J24.2, 6E12.3, IP10.2, 4L7.3, 7H4.6, and 9O12.2, and scFv fragments A10, A4, C10, B4, C3 and D11.
In addition, 9O12.2 Fab fragments were found to completely block collagen-induced platelet aggregation and secretion, to inhibit the procoagulant activity of collagen-stimulated platelets and platelet adhesion to collagen in static conditions, to impair platelet adhesion, and to prevent thrombi formation under arterial flow conditions (9).
However, none of the currently known anti-GPVI antibodies have proven really efficient in vivo for preventing and/or treating cardiovascular diseases implicating platelet aggregation such as arterial and venous thrombosis, restenosis, acute coronary syndrome, and cerebrovascular accidents due to atheroscleroris. Until recently the different anti-GPVI antibodies that have been reported appeared not fitted for the development of an antithrombotic for medical use in human. Only few antibodies have been reported to have inhibitory properties. This is the case of JAQ1 that is directed to mouse GPVI and does not cross react with human GPVI (10). Human scFvs directed to human GPVI have also been reported to be inhibitory (11,12) but their affinity appears to be low. Very recently, new inhibitory antibodies with a good affinity for human GPVI have been characterised (13) and proposed to be developed as therapeutic tools.
However, cross-linking of GPVI at the platelet surface by a divalent or multivalent ligand results in platelet activation. This is the case of the 9O12.2 IgGs that activate platelets via GPVI dimerisation and via cross-linking of GPVI to the low affinity Fc receptor (FcγRIIA) (9). Fab′2 also activate platelets via GPVI dimerisation (9). In contrast, monovalent 9O12.2 Fab fragments are inhibitory. However these fragments could not be used in therapeutic due to their size and their animal origin which makes them immunogenic in human patients.
There is thus still a need for an efficient neutralizing GPVI antagonist, which would inhibit with high efficiency the initial phase of platelets aggregation, with a low hemorrhagic risk, as well as a low immunogenic effect.