Endostatin is a 20 kDa non-glycosylated protein that is a specific inhibitor of endothelial proliferation and a potent anti-angiogenesis agent. The therapeutic effects of endostatin have been difficult to evaluate for several reasons. Soluble preparations of endostatin quickly lose activity and are plagued by technical difficulties related to storage, handling, and purification methods. In addition, because of its relatively short serum half-life, large quantities of protein are needed to see significant bioactivity in vivo. The goal of our research is to create novel endostatin proteins with both improved physical characteristics and enhanced in vitro and in vivo activities. Our approach towards accomplishing this task involves modifying endostatin by the site-specific covalent addition of a polyethylene glycol (PEG) moiety. By using the known structure of endostatin, we have been able to engineer into the protein, specific cysteine mutations to serve as attachment sites for thiol-reacfive PEGs. This strategy allows for the rational design of fully active protein analogues of defined structure and overcomes the problem of loss of bioactivity and product heterogeneity when proteins are modified using standard lysine-reactive PEG reagents. During the Phase II portion of this grant we will evaluate a number of these PEGylated endostatin muteins both in vitro and in vivo. Based upon our experience and literature reports, PEGylated human endostatin should have enhanced stability, greater potency and lower dose requirements. This will allow a larger number of patients access to the drug since it is estimated that more than 9 million cancer patients may benefit from anti-angiogenesis therapy.
