Simultaneous Silencing of Two Independent Signaling Pathways Essential for Angiogenesis Using Bispecific, Tetravalent Intra-Diabodies.

Vascular endothelial growth factor and its receptors (VEGF-R) have been implicated in promoting tumor growth and metastasis. Emerging evidence suggest that angiogenesis also play an important role in the growth of leukemias and lymphomas. Antiangiogenic therapies that interfere with the VEGF/VEGF-R pathways represent novel approaches to effective treatment for certain leukemia, such as myeloid leukemias. But not all types of tumors respond to interruption of the VEGF/VEGF-R pathway. This suggest that alternative pathways for vascular growth exist and can drive tumor angiogenesis. Intrabodies have been used to specifically target intracellular proteins and manipulate biological processes,as well as for the generation of phenotypic knockouts in vivo by surface depletion of extracellular or transmembrane proteins. In order to generate a precise tool for the simultaneous silencing ot two independent signaling pathways essential for angiogenesis, we developed an endoplasmatic-reticulum (ER) targeted bispecific, tetravalent intrabody format, targeting VEGF-R2 and Tie-2. Comparison of the ER-targeted intradiabody with the corresponding conventional ER-targeted single-chain antibody fragment (scFv) intrabodies showed that the intradiabody is significantly more efficient with respect to efficiency and duration of surface depletion of VEGF-R2 and Tie-2. In vitro and in vivo data demonstrate the development of a very effective antiangiogenic intrabody format for the simultaneous functional knockout of two cell surface receptors using an adenovirus-mediated gene delivery system. Our findings suggest that simultaneous interference with the VEGF and the Tie-2 receptor pathway results in at least additive antiangiogenic effects. Our study provides a novel antiangiogenic therapeutic tool for the silencing of two independent signaling pathways involved in angiogenesis.