? Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes progressive joint destruction, deformities and disability. One of the earliest observed features of RA is the development of a new vascular network within the synovium that allows for the delivery of cells and nutrients to the invading pannus. This formation of new blood vessels (also known as angiogenesis) is a highly regulated process under the influence of the host microenvironment and a number of secreted mediators. Many proangiogeneic mediators are expressed in RA. One of these mediators in particular, vascular endothelial growth factor (VEGF) appears to play a critical role in the differentiation of endothelial cells and the development of the vascular system in the synovial lining of the joints and therefore, is a reasonable target for therapeutic intervention in RA patients. Our overall goals are to create a long acting VEGF inhibitor and test its efficacy in animal models of RA. Using the published structural information for soluble VEGF Receptor I (also known as sFlt-1), we will rationally design polyethylene glycol (PEG)-sFlt-1 conjugates using cysteine-reactive PEGs. We will introduce a new """"""""free"""""""" cysteine using site-directed mutagenesis in regions of sFlt-1 that are believed to be non-essential for biological activity. The """"""""free"""""""" cysteine residue will serve as the site for the covalent modification of the protein using a thiol-reactive PEG. This technology allows for the creation of novel, fully active PEG-Cys-sFlt-1 analogues of defined structure and overcomes the problems of reduced bioactivity and heterogeneity when proteins are modified using standard amine-reactive PEGs. During Phase I we will identify sites in sFlt-1 that can be modified without affecting the protein's in vitro bioactivity. We will also perform a pharmacokinetic study to verify that PEGylation extends the circulating half-life of sFlt-1. During Phase II, we develop a cost effective manufacturing process and produce sufficient quantities of PEGylated recombinant sFlt-1 for testing in animal disease models and biochemical characterization. ? ?
