Intrabody-based strategies for inhibition of vascular endothelial growth factor receptor-2: effects on apoptosis, cell growth, and angiogenesis

Abstract

VEGF, an endothelial-specific mitogen, is an important tumor angiogenesis growth factor. The major receptor for VEGF on endothelial cells is KDR. We hypothesized that an intrabody could bind newly synthesized KDR and block receptor transport to the cell surface, thereby inhibiting 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 human umbilical vein endothelial cells (HUVECs), producing marked (80%) apoptosis at 48 h postinfection. 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, because Ad-HAK infection of cell lines with minimal or no KDR had little effect on cell viability. Infected HUVECs 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.