Abstract 339: Anti-doppel monoclonal antibody as a tumor endothelial cell-specific angiogenesis inhibitor

Introduction: Approved angiogenesis inhibitors generally target certain growth factors or their receptors, which exist in both normal and cancerous cells; this results in suppression of cell-signaling pathways throughout the body and causes adverse effects. For this reason, there is a need for a new angiogenesis inhibitor that can specifically target the tumor vasculature. In our previous study, we revealed that doppel, a prion-like protein, was overexpressed specifically in the tumor vasculatures, but not in normal endothelium, and its expression enhanced blood vessel formation. To develop our new anti-angiogenic agent, we produced monoclonal antibodies which target doppel as the antigen. Methods: We evaluated whether the doppel antibody inhibits angiogenesis by a spheroid assay. We cultured human colonic tumor-associated endothelial cells (HCTEC) and collected the cells by a hanging drop method. After producing compact aggregates, the spheroids were collected and embedded into collagen-based 3D cultures. The culture media/growth factor/doppel antibody mixture was added to each well that contained 3D spheroid cultures. After 24 hours, the number of sprouts was counted and analyzed. Further, HCTECs were incubated with culture media, growth factor, and doppel antibodies for investigating the underlying mechanisms. Each well was lysed, and we conducted western blot and microarray experiments using the lysates. In addition, we injected fluorescent dye-conjugated doppel antibody into tumor xenograft mouse models and visualized the tumor-targeting efficacy using in vivo imaging system (IVIS). Results: In spheroid assays, we found that doppel antibody SNU-H01 and SNU-H02 inhibit angiogenesis (by 32% and 29%, respectively) compared to control. In anti-phosphorylation assays using western blot, the amount of phosphorylated VEGFR2 and phosphorylated FGFR1 was decreased in doppel antibody SNU-H01 and SNU-H02-treated groups. Also, when SNU-H02-treated lysate was added to the microarray kit, STAT5A and beta-catenin were inhibited. Inhibition of STAT5A may promote apoptosis and increase sensitivity to anticancer drugs, and inhibition of beta-catenin may further increase suppression of angiogenesis. In addition, doppel antibody SNU-H04 accumulates in the tumor significantly higher compared to IgG injected mouse group. Conclusion: Doppel antibodies in development, SNU-H01, -H02, -H03, -H04, will be screened and selected for their anti-angiogenic efficacy and favorable ph