ATPS-81SINGLE-AGENT TARGETING OF Eph RECEPTORS A2, A3, B2 AND INTERLUKIN-13 RECEPTOR ALPHA 2 IN GLIOBLASTOMA

Abstract

Glioblastoma (GBM), is the most common and aggressive primary tumor of the central nervous system, with few treatment options and poor patient outcome. We and others demonstrated that A2, A3 and B2 receptors of the Eph family of protein tyrosine kinase receptors and Interlukin-13 receptor alpha 2 (IL-13RA2), are overexpressed in GBM tumors, but not normal brain. These receptors play important roles in GBM tumor progression and resistance to therapies involving both initiating and differentiating tumor cells, infiltrating tumor cells, tumor-associated macrophages (TAMs), and neovasculature. To this end, we have designed and constructed a bi-valent protein which would recognize all these four receptors using a human IgG1 scaffold. The bi-valent protein (eA5-Fc-IL-13.E13K) consists of ephrin-A5 (eA5, which acts as a ligand for the three Eph receptors), the Fc region of IgG1 followed by a modified IL-13, IL-13.E13K. The bi-valent protein was expressed in Sf9 insect cells using the baculovirus expression system and purified from the cell media to homogeneity using affinity Protein G column. The non-reduced and reduced SDS-PAGE showed the expected size bands of 125 and 65 kDa, respectively. The bi-valent binding and function of protein was tested in GBM cells. The bi-valent protein specifically bound, internalized and down-regulated EphA2 receptor in GBM cells, mimicking the action of eA5 alone. Using the PMS/MTS cell viability assay, we determined that the bi-valent protein effectively prevented an IL-13.E13K based cytotoxin from binding to and killing GBM cells by competing for the cytokine binding sites. Thus, we have generated a bi-valent compound capable of recognizing four receptor targets: IL-13RA2, EphA2, EphA3 and EphB2 that are over-expressed in most of the pathobiologically important tumor compartments in all GBM patients. Therefore, the protein under investigation represents a novel therapeutic route to be exploited in the treatment of patients with a heterogeneous disease like GBM.