) The overall goal of this project is to develop a commercially viable bacterial toxin-VEGF (vascular endothelial growth factor) fusion protein for selective targeting of endothelial cells at sites of angiogenesis, primarily in solid tumors and growing metastases. VEGF interacts with the endothelial cell specific KDR/flk-1 receptor that is overexpressed in endothelial cells at the sites of angiogenesis relative to its expression on quiescent endothelium. In Phase I of this project we have constructed, expressed and purified two novel fusion proteins, SLT-VEGF/L and SLT-VEGF/S which contain, respectively, The A subunit of Shiga-like toxin I (SLT) and a functionally active fragment of the A subunit. SLT causes hemorrhagic colitis and hemolytic uremic syndrome by damaging endothelial cells suggesting that endothelial cells are particularly sensitive to SLT . SLT-VEGF/L and SLT-VEGF/S proteins selectively inhibit growth of endothelial cells overexpressing KDR/flk-1 receptors with IC-50 of 0.15 nM. At low nanomolar concentrations SLT-VEGF/L is cytotoxic for endothelial cells overexpressing KDR/flk-l receptors. However, these proteins do not affect growing endothelial cells with low numbers of KDR/flk-l receptors, quiescent endothelial cells, and growing non-endothelial cells. These results provide a rationale for Phase II in vivo testing of SLT-VEGF fusion proteins as highly selective inhibitors of angiogenesis. We will also test whether SLT-VEGF proteins may synergize with other antitumor drugs by enhancing delivery through damaged tumor endothelium or by creating hypoxic conditions for bioreductive therapeutics. Finally, we will optimize SLT-VEGF fusion proteins to achieve high levels of expression, activity, and stability that are necessary for preclinical and clinical trials.
A brief summary of the potential commercial applications of the research: VEGF-toxin fusion proteins that inhibit angiogenesis but do not affect normal endothelium or normal cells will be commercial products for therapy of angioaenesis-dependent pathologies.
