NON-INVASIVE EARLY DETECTION OF CANCER: DEVELOPING BI-SPECIFIC ANGIOPOETIN-2 FRAGMENT THAT BINDS TO BOTH αVβ3 INTEGRIN AND TIE2

The dysregulation and over-expression of αvβ3 integrin and Tie2 receptor tyrosine kinase correlate with poor prognosis in many human tumors, making these proteins attractive targets for therapeutic intervention. Numerous papers have demonstrated the cross-talk between biological processes mediated by αvβ3 integrins, Tie2 and their ligands, particularly pathways responsible for angiogenesis. Along with the development of therapeutics, it is critical to develop selective noninvasive molecular imaging agents that can identify patients who would benefit most from the above-described targeted treatments and that can be used to monitor treatment and disease progression. In that aspect, bispecific proteins that bind to both αvβ3 integrins and Tie2 and that have the potential to modulate both receptors, are particularly promising.

The innovative approach proposed here entails engineering multispecificity into angiopoietin-2 (Ang2), the natural ligand of Tie2, a method that we predict will form a major part of a highly effective strategy for creating ligand-based multispecific molecular tools for receptor recognition.

Our combinatorial strategy to engineer the mutated ligand for higher affinity binding for αvβ3 integrin and Tie2 simultaneously involves using directed evolution methods for screening random mutagenesis libraries against both Tie2 and αvβ3 integrin and selecting the mutants with the highest affinities in increasingly stringent sorts. We are currently in the process of fully characterizing the binding of the purified selected variants by employing in vitro and cell-based binding assays. Our studies will generate a technology that could dissect and monitor the expression profile, in real time and in living animals, of multiple receptors that are involved in cancer progression from its very early stages to highly invasive and metastatic carcinomas.