| VEGF, an endothelial specific mitogen, is an important tumor angiogenesis growth factor. The major receptor for VEGF on endothelial cells is KDR.{09}We hypothesized that an intrabody could bind newly synthesized KDR, block receptor transport to the cell surface, and thereby inhibit important VEGF effects. We expressed a single chain antibody (p3S5) to KDR with or without the endoplasmic reticulum (ER) retention signal (KDEL), using either a plasmid (p3S5-HAK) or a tet-off adenoviral system (Ad-HAK). Plasmid mediated expression of the tethered intrabody significantly reduced KDR expression (from 82.5 ± 12.5% to 27.9 ± 13.6% of cells; p < 0.01) and thymidine incorporation in successfully transfected cells. Ad-HAK infection resulted in intrabody expression in >90% of HUVEC, producing marked (80%) apoptosis at 48 hours post-infection. The intrabody was essential for these effects, as confirmed by inhibiting its expression with doxycycline or by expressing irrelevant genes (lacZ, GFP). Cell death was dependent on KDR, since Ad-HAK infection of cell lines with minimal or no KDR, had little effect on cell viability. Infected HUVEC were unable to form tubes on Engelbreth Holm-Swarm (EHS) tumor gel matrix. These results demonstrate the potential for development of an intrabody-based strategy to block angiogenesis and prevent tumor growth. |
