We have cloned a novel high affinity novel thrombospondin-1 (TSP-1) binding protein from a prostate cancer cell library. The recombinant protein expressed in bacteria, termed angiocidin, inhibited endothelial cell viability and tube formation in vitro and prevented the growth of tumors in vivo. When injected intravenously into mice bearing Lewis Lung carcinoma, the protein inhibited the growth of the tumor by more than 500 percent as compared to the buffer control. As shown by immunohistochemical staining of human breast tumors, angiocidin was expressed in malignant ductal epithelium and tumor microvasculature while no significant expression was observed in normal and benign tissues. Our preliminary data indicate that angiocidin is posttranslationally modified in eukaryotic cells. Post-translational modification such as glycosylation are known to greatly affect the half-life of proteins in circulation and may therefore increase the bioavailability and half-life of angiocidin injected systemically. Thus, these post-translational changes may increase the anti-tumor activity of angiocidin as well as produce protein with no endotoxin contamination suitable for clinical development. In this supplemental proposal, we will evaluate which protein expression system is optimal for production of angiocidin suitable for administration in human clinical cancer trials. To accomplish this goal, the anti-tumor activity of recombinant angiocidin expressed in bacteria with angiocidin expressed in yeast, insect cells, and Chinese hamster ovary cells will be compared. Anti-tumor activity of the recombinant proteins expressed in the three expression systems will be evaluated in two animal models, an orthotopic and a syngeneic mouse model, as well as in an in vitro angiogenesis model. Criteria for choosing a protein expression will include specific anti-tumor activity, and protein yield. These experiments will develop the necessary protocols needed for commercial production of protein under GMP (good manufacturing practices) conditions suitable for use in clinical trials ? ?
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