A Bispecific Inhibitor Targeting the Tie2–α_vβ₃ Integrin Axis as a Potential Therapy for Angiogenesis

Primarily known for its clinical applicability as a tumor biomarker, human Tie2 has recently developed into a major signaling molecule with therapeutic potential. It is now known that its activity is mainly mediated through the downstream angiopoetin-1 (Ang1) - and Ang2-dependent, Tie2 activation of α_vβ₃ and α₅β₁ integrins, rendering this Ang1/Tie2/integrins axis as an attractive putative target for therapeutic interventions. Despite these recent exciting developments on the pathophysiological roles of this axis in angiogenesis, the development of inhibitors that target this axis remains an unfulfilled task. To address this gap, we established the first Ang-based multi-target therapy for angiogenesis, which display increased inhibition of Tie2/integrins activity. The development of the Tie2 inhibitor was based on the engineering of Ang2 binding domain (Ang2-BD), with high affinity towards Tie2 on one hand and high affinity towards integrin on the other. The high affinity Ang2-BD variants were developed through flow cytometry screening of yeast-displayed Ang2-BD mutant libraries.

On the basis of this screening, we generated single domain proteins that have high affinity towards Tie2 and others that contain bivalent binding epitopes to Tie2 and integrin. These high affinity dual-specific proteins inhibited Ang1-mediated Tie2/Akt phosphorylation, proliferation, invasion and tube formation of endothelial cells. These assays enabled us to show the superiority of the combined target effects of the inhibitors and to generate new tools for studying Tie2- and α_vβ₃ integrin-dependent molecular mechanisms mediating angiogenesis and other important biological processes. Based on the new Tie2/α_vβ₃ integrin binding epitopes, a new combination variant was designed and tested for in vitro and in vivo anti-cancer applications.