Targeting the Tie2-αvß3 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 αvß3 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 αvß3 integrin inhibited adhesion and proliferation of endothelial cells cultured together with the αvß3 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 αvß3 cross-talk-has created new tools for studying Tie2- and integrin αvß3-dependent molecular pathways and provides the basis for the rational and combinatorial engineering of ligand-Tie2 and ligand-integrin αvß3 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 αvß3-targeting proteins for clinical use as imaging and therapeutic agents.

A bi-specific inhibitor targeting IL-17A and MMP-9 reduces invasion and motility in MDA-MB-231 cells.

The cytokine IL-17A is associated with the progression of various cancers, but little is known about the molecular cross-talk between IL-17A and other tumor-promoting factors. Previous studies have shown that the IL-17A-mediated invasion of breast cancer cells can be inhibited by selective antagonists of the matrix metalloproteinase 9 (MMP-9), suggesting that the cross-talk between IL-17A and MMP-9 may promote cancer invasiveness and metastasis. Here, we present a novel strategy for developing cancer therapeutics, based on the simultaneous binding and inhibition of both IL-17A and MMP-9. To this end, we use a bi-specific heterodimeric fusion protein, comprising a natural inhibitor of MMPs (N-TIMP2) fused with an engineered extracellular domain (V3) of the IL-17A receptor. We show that, as compared with the mono-specific inhibitors of IL-17A (V3) and MMP-9 (N-TIMP2), the engineered bi-specific fusion protein inhibits both MMP-9 activation and IL-17A-induced cytokine secretion from fibroblasts and exhibits a synergistic inhibition of both the migration and invasion of breast cancer cells. Our findings demonstrate, for the first time, that dual targeting of inflammatory (IL-17A) and extracellular matrix remodeling (MMP) pathways can potentially be used as a novel therapeutic approach against cancer. Moreover, the platform developed here for generating the bi-specific IL-17A/MMP-9 inhibitor can be utilized for generating bi-specific inhibitors for other cytokines and MMPs.