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. During Phase I, we used the published structural information for soluble VEGF Receptor I (also known as sFlt-1) to rationally design polyethylene glycol (PEG)-sFlt-1 conjugates using cysteine-reactive PEGs. We introduced a new """"""""free"""""""" cysteine using site-directed mutagenesis in regions of sFlt-1 that were believed to be non-essential for biological activity. The """"""""free"""""""" cysteine residue served 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 identified sites in sFlt-1 that can be modified without affecting the protein's in vitro bioactivity. We also performed a pharmacokinetic study to verify that PEGylation extends the circulating half-life of sFlt-1. During Phase II, we will develop a cost effective manufacturing process and produce sufficient quantities of PEGylated recombinant sFlt-1 for testing in animal disease models of RA. Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes progressive joint destruction, deformities and disability. Vascular endothelial growth factor (VEGF) appears to play a critical role in this inflammatory process and therefore, is a reasonable target for therapeutic intervention in RA patients. VEGF inhibitors have been tested in humans and have been shown to delay the progression of the disease. Our overall goal is to create a long acting VEGF inhibitor that can be dosed once a week or once every two weeks. ? ? ?