Identification of amino acids essential for the antiangiogenic activity of tumstatin and its use in combination antitumor activity

Tumstatin is an angiogenesis inhibitor that binds to alpha v beta 3 integrin and suppresses tumor growth. Previous deletion mutagenesis studies identified a 25-aa fragment of tumstatin (tumstatin peptide) with in vitro antiangiogenic activity. Here, we demonstrate that systemic administration of this tumstatin peptide inhibits tumor growth and angiogenesis. Site-directed mutagenesis identified amino acids L, V, and D as essential for the antiangiogenic activity of tumstatin. The tumstatin peptide binds to alpha v beta 3 integrin on proliferating endothelial cells and localizes to select tumor endothelium in vivo. Using 3D molecular modeling, we identify a putative interaction interface for tumstatin peptide on alpha v beta 3 integrin. The antitumor activity of the tumstatin peptide, in combination with bevacizumab (anti-VEGF antibody), displays significant improvement in efficacy against human renal cell carcinoma xeno-grafts when compared with the single-agent use. Collectively, our results demonstrate that tumstatin peptide binds specifically to the tumor endothelium, and its antiangiogenic action is mediated by alpha v beta 3 integrin, and, in combination with an anti-VEGF antibody it exhibits enhanced tumor suppression of renal cell carcinoma.