Targeting the Tie2–αvβ3 integrin axis with bi-specific reagents for the inhibition of angiogenesis

Background: Increased activity of the receptor tyrosine kinase Tie2 has been implicated in the promotion of pathological angiogenesis. This activity is mainly mediated through angiopoietin (Ang)1- and Ang2-dependent activation of integrins by Tie2, rendering the Ang/Tie2/integrin axis an attractive putative target for cancer therapeutics.

Results: To target this axis, we developed single domain, non-immunoglobulin high-affinity bi-specific protein inhibitors against both Tie2 and α_vβ₃ integrin. We have previously engineered the Ang2-binding domain of Tie2 (Ang2-BD) as a Tie2 inhibitor. Here, we engineered an exposed loop in Ang2-BD to generate variants that include an integrin-binding Arg–Gly–Asp (RGD) motif and used flow cytometry screening of a yeast-displayed Ang2-BD RGD loop library to identify the integrin antagonists. The bi-specific antagonists targeting both Tie2 and α_vβ₃ integrin inhibited adhesion and proliferation of endothelial cells cultured together with the α_vβ₃ integrin ligand vitronectin, as well as endothelial cell invasion and tube formation. The bi-specific reagents inhibited downstream signaling by Tie2 intracellularly in response to its agonist Ang1 more effectively than the wild-type Ang2 BD that binds Tie2 alone.

Conclusions: Collectively, this study—the first to describe inhibitors targeting all the known functions resulting from Tie2/integrin α_vβ₃ cross-talk—has created new tools for studying Tie2- and integrin α_vβ₃-dependent molecular pathways and provides the basis for the rational and combinatorial engineering of ligand–Tie2 and ligand–integrin α_vβ₃ receptor interactions. Given the roles of these pathways in cancer angiogenesis and metastasis, this proof of principle study paves the route to create novel Tie2/integrin α_vβ₃-targeting proteins for clinical use as imaging and therapeutic agents.