Developing Bispecific Antagonists with High Affinity to Both Tie2 and α_vβ₃ Integrin for Cancer Imaging

The upregulation and overexpression of the receptor tyrosine kinase Tie2 and α_vβ₃ integrin correspond with poor prognosis in many cases of human cancer. The aforementioned receptors are involved in mediating tumor proliferation, migration and angiogenesis, and therefore stand as attractive targets for therapeutic purposes. Alongside of cancer therapeutic agents, there is a great need for molecular imaging agents that can be utilized for early detection and diagnosis of the disease. In that aspect, bispecific proteins that could bind both Tie2 and α_vβ₃ integrin are very promising. In a previous work, we have successfully engineered degenerate mutants of the natural ligand of the Tie2 receptor, Angiopoietin-2 (Ang2), which could bind to the receptor without the ability to activate it. Furthermore, the mutants were introduced to the RGD motif, enabling them to bind to α_vβ₃ integrin as well, and therefore possess bispecific capabilities. We hypothesis that targeting both Tie2 and α_vβ₃ integrin receptors with bispecific proteins will result in increased affinity and selectivity properties that would enhance their efficacy as in vivo imaging agents. Our current work is focused on the production, purification and evaluation of some of our most potent bispecific variants. We conducted several binding assays to both endothelial and cancer cell lines, proceeded with the most potent variant, tested and showed its potential as a cancer imaging agent in in-vivo xenograft models. We envision that the protein variants engineered in these efforts will pave the way to the development of next-generation cancer therapeutic and imaging agents.