The endothelial cell receptor-tyrosine kinases, VEGF receptor 2 (VEGF-R2) and Tie-2, and their ligands, vascular endothelial growth factor (VEGF) and angiopoietins 1 and 2, respectively, play key roles in tumor angiogenesis. Several studies suggest that the VEGF receptor pathway and the Tie-2 pathway are independent and essential mediators of angiogenesis, leading to the hypothesis that simultaneous interference with both pathways should result in additive effects on tumor growth. In this study, a human melanoma xenograft model (M21) was used to analyze the effects of simultaneous intradiabody depletion of vascular endothelial growth receptor-R2 and Tie-2 on tumor angiogenesis and tumor xenograft growth. The intradiabodies were expressed from recombinant adenovirus delivered through subtumoral injection. Blockade of both VEGF-R2 and Tie-2 pathways simultaneously or the VEGF receptor pathway alone resulted in a significant inhibition of tumor growth and tumor angiogenesis (92.2% and 74.4%, respectively). In addition, immunohistochemical staining of intradiabody-treated tumors demonstrated a decreased number of tumor-associated blood vessels versus control treatment. Previous studies with intrabodies had demonstrated that the Tie-2 receptor pathway was essential for tumor growth. The simultaneous blockade of the VEGF and Tie-2 pathways resulted in effective inhibition of tumor growth and demonstrated the potential of simultaneous targeting of multiple pathways as a therapeutic strategy

The specific and high affinity binding properties of intracellular antibodies (intrabodies), combined with their ability to be stably expressed in defined organelles, provides powerful tools with a wide range of applications in the field of functional genomics and gene therapy. Intrabodies have been used to specifically target intracellular proteins, manipulate biological processes, and contribute to the understanding of their functions as well as for the generation of phenotypic knockouts in vivo by surface depletion of extracellular or transmembrane proteins. In order to study the biological consequences of knocking down two receptortyrosine kinases, we developed a novel intrabody-based strategy. Here we describe the design, engineering, and characterization of a bispecific, tetravalent endoplasmic reticulum (ER)-targeted intradiabody for simultaneous surface depletion of two endothelial transmembrane receptors, Tie-2 and vascular endothelial growth factor receptor 2 (VEGF-R2). Comparison of the ER-targeted intradiabody with the corresponding conventional ER-targeted single-chain antibody fragment (scFv) intrabodies demonstrated that the intradiabody is significantly more efficient with respect to efficiency and duration of surface depletion of Tie-2 and VEGF-R2. In vitro endothelial cell tube formation assays suggest that the bispecific intradiabody exhibits strong antiangiogenic activity, whereas the effect of the monospecific scFv intrabodies was weaker. These findings suggest that simultaneous interference with the VEGF and the Tie-2 receptor pathways results in at least additive antiangiogenic effects, which may have implications for future drug developments. In conclusion, we have identified a highly effective ER-targeted intrabody format for the simultaneous functional knockout of two cell surface receptors.Antibodies can bind almost any molecule with high specificity and affinity, providing powerful biotechnological tools for diagnostic and therapeutic applications. Advances in recombinant DNA technology have facilitated the manipulation of the antibody genes, so that design, cloning, expression, and use of single-chain antibodies have become routine procedures in protein engineering. The potential of single-chain antibody fragments (scFv) 1 for intracellular applications, termed "intrabodies," has been exploited in a number of laboratories (1-8). To date, intrabodies have been utilized for targeting proteins in a singular fashion. Bispecific and tetravalent antibody fragments could improve and expand the inhibitory potential of intrabodies by exhibiting increased apparent affinity for their antigen and by being more efficient at inhibiting protein function or intracellular trafficking (9). Intrabodies present a potent alternative to methods of gene inactivation that target at the level of DNA or mRNA, such as antisense (10), zinc finger proteins (11), targeted gene disruption, or the relatively new RNA interference (12). Operating at the posttranslational level, intrabodies can be directed to relevant...