Glycoprotein vi fusion proteins

This application is a divisional application of U.S. application Ser. No. 10/483,810, filed on Jan. 15, 2004, which is the National Stage application under §371 of PCT/EP02/07796, filed Jul. 12, 2002.

The present invention relates to Glycoprotein VI (GPVI) fusion proteins (GPVI-fusion proteins) comprising a tag like myc, GST, HA, FLAG, STREP but preferably a immunoglobulin molecule (Ig), more preferably a Fc portion of said Ig and a protein or oligopeptide having the biological activity of GPVI (GPVI-like protein) which is binding to collagen.

Procedures for production and purification of said fusion proteins are disclosed. Methods and kits for the screening of potential agonists or antagonists for GPVI-collagen and/or platelet-collagen interactions are provided.

The GPVI-fusion proteins are useful for the treatment of thrombotic and cardiovascular events and disorders related to GPVI-collagen and/or platelet-collagen interactions. Furthermore the fusion proteins are useful for coating natural or artificial surfaces in order to render them nonadhesive for cells and prevent the activation of cells.

The adhesion and activation of resting, circulating platelets at a site of vascular injury is the first step in a process leading to the formation of a thrombus, which is converted into a hemostatic plug. Collagen is one of the major components of the vessel wall responsible for platelet activation. Many types of collagen exist, and seven of these are found in the subendothelial layers. Several different receptors for collagen have been identified on platelets including CD36 (Matsuno, et al., Br. J. Haematol. 92, 960-967, 1996) and a p65 collagen type I specific receptor (Chiang et al., J. Clin. Invest. 100, 514-521, 1997), but the major ones are now considered to be integrin α₂β₁ and the nonintegrin GPVI. It was determined about 20 years ago that GPVI is a major platelet glycoprotein with a molecular mass in the 60-65-kDa range and an acid pl (Clemetson et al., J. Clin. Invest. 70, 304-311, 1982) which forms a complex together with the Fcγ common subunit. The GPVI subunit contains the collagen binding site and the Fcγ subunit is responsible for signalling. The complex forms one of the major collagen receptors on the platelet surface, critical for platelet activation in response to collagen. Its role as a putative collagen receptor was established following the identification of a patient in Japan with a mild bleeding disorder whose platelets had a specific defect in response to collagen and lacked this receptor (Moroi et al., J. Clin. Invest. 84, 1440-1445, 1989). This patient had also developed autoantibodies to the deficient receptor, and these were used to characterize the molecule further (Sugiyama et al., Blood 69, 1712-1720, 1987). It was also demonstrated that the recognition sequence on collagen for GPVI is a repeated Gly-Pro-Hyp (Hyp=hydroxyproline) triplet within the collagen triple helical structure and that synthetic peptides based on this structure could be used as specific GPVI-directed agonists (Morton et al., Thromb. Res. 72, 367-372, 1993). The GPVI/FCγ complex was shown to signal to the platelet interior by an immune receptor-like mechanism, involving activation of p72^syk and leading by a cascade of kinase/phosphatase/adaptor protein interactions to activation of PLCγ2 and hence to release of granules and platelet aggregation.

Thus, it is clear from the prior art that GPVI-like proteins are very interesting compounds either as tool for the screening of potential agonists or antagonists of the GPVI-collagen interaction or as active principle.

In WO 00/68377 DNA coding for GPVI or biologically fragments thereof, recombinant human GPVI and pharmaceutical compositions thereof are disclosed. Further, the application describes the use of recombinant GPVI as a screening tool for detecting specific inhibitors or activators of platelet-collagen interactions. However, because of its transmembrane domain the recombinant protein is not secreted in the extracellular medium and so it is difficult to purify. Therefore the whole cells expressing this protein have to be used in a screening assay. Further, in view of GPVI as active agent, it is known that recombinant proteins often have a reduced circulating half-life, which leads to a need of frequent application of the drug and therefore results in high costs for the therapy.

Therefore, it was the goal of the present invention to provide molecules with the biological activity of GPVI having the following improved properties: high expression level in the host cell, secretion into the extracellular medium and easy purification, enhanced circulating half-life and, with respect to the use as screening tool, easy detectability by commercially available antibodies.

An object of the present invention is therefore to provide fusion proteins and their use as active agent and as screening tools for detecting specific inhibitors or activators of GPVI-collagen interaction and of platelet-collagen interactions.

Another object of the invention is to provide DNA molecules encoding said fusion proteins, vectors comprising said DNA molecules, host cells comprising said vectors and methods for producing said fusion proteins.

Another object of the invention is to provide kits and methods comprising such fusion proteins for the screening of agonists or antagonists of GPVI-collagen interaction and/or of platelet-collagen interactions.

A further object of the invention is to provide said fusion proteins for the use in the treatment of thrombotic and cardiovascular events and disorders related to GPVI-collagen interaction and/or to platelet-collagen interactions and medicaments and pharmaceutical packs comprising said fusion proteins.

A further object of the invention is to provide said fusion proteins for coating natural or artificial surfaces in order to render them nonadhesive for cells and prevent the activation of cells and for modifying intraocular lenses in order to lessen the thrombogenicity of the lens material.

Other objects of the present invention are apparent for a skilled person on the basis of the following detailed description.

These objects are achieved on the basis of the finding that fusion proteins comprising a tag like myc, GST, HA, FLAG, STREP, but preferably an Immunoglobulin molecule (Ig), more preferably a Fc portion of said Ig and a protein or oligopeptide having the biological activity of GPVI are expressed in high amounts in the host cells, are secreted in the extracellular medium and are therefore easy to purify. The disclosed molecules possess enhanced circulating half-life and are easily detectable by commercially available antibodies.

Due to their easy producibility they can be used for the screening of agonists or antagonists of GPVI-collagen and/or platelet-collagen interactions. Methods and kits for the screening of agonists and antagonists comprising a fusion protein as defined above and below are provided.

The fusion proteins of the present invention are useful in the prevention, prophylaxis, therapy and treatment of thrombotic and cardiovascular events and disorders related to GPVI-collagen and/or platelet-collagen interactions including increased platelet activation with collagen, such as atherosclerotic plaque rupture, unstable angina or, during surgical treatment such as percutaneous transluminal coronary angioplasty (PTCA). Pharmaceutical compositions comprising said fusion proteins for the treatment of thrombotic and cardiovascular events and disorders related to GPVI-collagen and/or platelet-collagen interactions are provided.

Furthermore, the fusion proteins can be used for coating natural or artificial surfaces coming in contact with body fluids, for example protheses, artificial organs, ocular lenses, sutures, artificial vascular segments, catheters, dialysers, tubes and vessels carrying blood, in order to render them nonadhesive for cells and prevent the activation of cells.