Simultaneous, Phenotypic Knockout of VEGF-R2 and Tie-2 with an Intradiabody Enhances Antiangiogenic Effects In Vivo

Abstract

Background: Intracellular antibodies (intrabodies) have been used for the generation of phenotypic knockouts in vivo by surface depletion of extracellular or transmembrane proteins. Intrabodies present an alternative to methods of gene inactivation that target genomic DNA or m-RNA, such as RNA interference. 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 in tumor growth. Methods: In order to generate a precise tool for the simultaneous silencing of two independent signaling pathways essential for angiogenesis, we developed a bispecific, tetravalent endoplasmatic reticulum (ER)-targeted intradiabody, against Tie-2 and VEGF-R2. Results: Using an adenovirus mediated gene delivery system, we achieved the simultaneous downregulation of the two cell surface receptors and demonstrate that the intradiabody is significantly more powerful with respect to efficiency and duration of surface depletion of Tie-2 and VEGF-R2 when compared to scFv intrabodies. In a human melanoma xenograft mouse model, we could show that blockade of both VEGF-R2 and Tie-2 pathways or the VEGF receptor pathway alone resulted in a significant inhibition of tumor growth and tumor angiogenesis (92.2 % and 74.4 %). Conclusion: We demonstrate for the first time that simultaneous inhibition of the VEGF and the Tie-2 receptor pathways result in additive antiangiogenic effects in vitro and in vivo as compared to single VEGF receptor pathway blockade, strengthening the potential of simultaneous targeting of multiple pathways as a therapeutic strategy.

Zusammenfassung

Hintergrund: Intrazelluläre Antikörper (Intrabodies) können durch die Oberflächendepletion von extrazellulären oder transmembranösen Proteinen gezielt die Funktion eines Proteins in der Zelle ausschalten. Intrabodies stellen eine alternative Methode zu Antisense-Oligonukleotiden oder RNA Interference, bezüglich der Gen-Inaktivierung von genomischer DNA oder messenger RNA dar. Studien zeigen, dass der VEGF-Rezeptor-Signalweg und der Tie-2-Signalweg voneinander unabhängige Mediatoren der Angiogenese sind. Dies hat zu der Hypothese geführt, dass die simultane Blockade beider Signalwege in additiven Effekten bezüglich der Inhibition des Tumorwachstums resultieren sollte. Methode: Für die simultane, präzise Blockade beider voneinander unabhängiger Signalwege haben wir einen bispezifischen, tetravalenten Intradiabody gegen VEGF-R2 und Tie-2 entwickelt, der im endoplasmatischen Retikulum (ER) exprimiert wird. Ergebnisse: Mittels rekombinanten Adenoviren, die als Transportvehikel für den Intradiabody eingesetzt wurden, erreichten wir die simultane Blockade von zwei Zelloberflächen-Rezeptoren und zeigten, dass der bispezifische Intradiabody einem herkömmlichen monospezifischen scFv-Intrabody bezüglich Effizienz und Dauer der Oberflächendepletion von VEGF-R2 und Tie-2 deutlich überlegen ist. In einem humanen Melanom-Mausmodell resultierte die Blockade von VEGF-R2 und Tie-2 in einer signifikanten Reduktion von Tumorwachstum und Angiogenese (92,2 %), während die alleinige Blockade von VEGF-R2 nur zu einer Inhibition von 74,4 % führte. Schlussfolgerung: Wir konnten zum ersten Mal zeigen, dass die simultane Inhibition des VEGF-R2 und Tie-2 Rezeptor-Signalwegs zu additiven Effekten bezüglich der Inhibition des Tumorwachstums in vitro und in vivo führt, verglichen mit der alleinigen Blockade des VEGF-Rezeptor-Signalwegs. Dieses Ergebniss unterstreicht das Potential des simultanen Knockouts von verschiedenen Pathways der Angiogenese als therapeutische Strategie.

Key words

antiangiogenesis - VEGF-R2 - Tie-2 - intrabodies - adenovirus

Schlüsselwörter

Antiangiogenese - VEGF-R2 - Tie-2 - Intrabodies - Adenovirus

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