TARGETING TOXIN POLYPEPTIDES TO TUMOR VASCULATURE

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For a sustained growth beyond the size of 2 - 3 mm in diameter, solid tumors are dependent on the establishment of new blood supply. Tumor cells secrete a number of angiogenic growth factors to induce localized vascular sprouting from pre-existing host vasculature (neovascularization). Recent studies have shown that Vascular endothelial growth factor (VEGF)/Vascular permeability factor (VPF) is an important mediator of tumor neovascularization and stroma development. VEGF binds to receptor tyrosine kinases (flt-1 and KDR/flk-1) which are differentially overexpressed on the endothelial cells of tumor vasculature when compared to blood vessels of surrounding normal tissues. Since each microvessel is a life line for a large number of tumor cells, targeting the tumor vasculature will have a profound effect on tumor growth. On the basis of these studies, we prepared a cytotoxic conjugate by chemically linking a splice variant of VEGF and a truncated form of diphtheria toxin. VEGF-toxin conjugate selectively inhibited the proliferation of endothelial cell lines in vitro and experimental angiogenesis in vivo. Receptor negative cell lines such as tumor cells were not affected by the conjugate treatment. Following these studies, we investigated the effect of VEGF-toxin conjugate on the growth of two solid tumors in experimental animal models. Intraperitoneal administration of VEGF-toxin conjugate 7 days after tumor cell transplantation delayed tumor appearance and subsequent growth. Preliminary histopathological examination showed VEGF- toxin selectively affected the tumor vasculature and induced necrosis. Normal tissues obtained from the same animals showed no vascular damage/hemorrhage or necrosis in liver, kidney or lung. These data support that VEGF-toxin can be safely administered. The differential effect of VEGF-toxin conjugate correlated with the proliferative status of endothelium. Quiescent normal endothelium is resistant to VEGF-toxin conjugate. In the current proposal we will critically evaluate and improve the anti-tumor activity of VEGF-toxin conjugate by optimizing the dose, route of administration and by improving the composition of the construct with genetically engineered toxin molecules. In addition, we will identify the limitations of this approach by extensive toxicological studies and develop strategies to overcome the problems. Finally, VEGF- fusion toxins will be prepared to facilitate further preclinical development.