Angiogenesis is important for the growth and metastasis of cancer. Inhibition of angiogenesis can therefore complement adjuvant chemotherapy of ovarian and breast cancer. Two major groups of endogenous angiogenesis inhibitors are currently investigated; proteolytic cleavage products of collagens and coagulation related proteins. Endostatin is a 20 kDa-C-terminal fragment of the non-collagenous domain (NC1) of collagen type XVIII. Endostatin treatment had resulted in varying degree of tumor growth inhibition in a number of preclinical tumor models. One of the reasons for the inconsistencies in response is due to protein folding which is affected by the type of expression system used to prepare the antiangiogenic molecule. We have identified and characterized a mutant endostatin containing a substitution of proline to alanine residue at position 125. P125A-endostatin showed increased binding to endothelial cells and inhibited angiogenesis better than the native molecule. Independent studies at NCI and in our laboratory confirmed that the mutant endostatin is better in inhibiting tumor growth than the native protein in three different tumor model systems. Therefore, it is important to understand the functional importance of substitutions at this position. Polymorphism in the NC1 domain of collagen XVIII mostly involves truncations and leads to vascular problems associated with Knobloch syndrome. The only other known mutation is D104N which however does not affect the biological activity of endostatin. No polymorphism has been yet identified at P125.
In specific aim 1 we propose to systematically generate other P125 substitutions and investigate the relative antiangiogenic potency of the mutants. Mechanistic studies will be carried out to identify mutation specific changes in the biological activity of endostatin.
In specific aim 2 we propose to evaluate the therapeutic and pharmacotoxicologic properties of mutant endostatins expressed in mammalian cells. Initial studies will focus on comparing the efficacy of protein therapy using Alzet pumps and AAV- mediated gene therapy. Based on these results we will critically evaluate a selected mutant endostatin in clinically relevant ovarian and breast cancer models. Recent studies have shown improved inhibition of tumor growth when chemotherapy was combined with mutant endostatin treatment. The mechanism of synergy between these two methods will be investigated so as to facilitate expedited clinical development of this approach, facilitate expedited clinical development of this approach.
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