Targeting the Tie2-α_vβ₃ integrin axis with bi-specific reagents for the inhibition of angiogenesis

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.

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.

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.