EXTH-85 - EphA3 CART: Dual Targeting of Glioblastoma and Tumor Microenvironment

BACKGROUND In GBM, effective chimeric antigen receptor T-cell (CART) therapy remains a challenge; immunosuppressive components within the tumor microenvironment (TME) inhibit CART cells. EphA3 is a receptor tyrosine kinase with low expression in adult tissues but is highly expressed in tumor neovasculature and tumor stromal cells in GBM and other solid tumors. METHODS We performed RNA sequencing and Immunohistochemical staining for of EphA3 in a clinically annotated GBM PDX biobank and M01 M1 and M2 polarized macrophages. We then investigated the suppressive capability of M2 macrophage on EphA3-CART in an SNB-19 GBM cell co-culture. We tested the antiantitumor potential of EphA3-CART against luciferase⁺ SNB-19 GBM cells in vivo with flank and orthotopically engrafted NOD-SCID-ɣ^-/- (NSG) mice. Engraftment was confirmed by bioluminescent imaging (BLI) and vernier caliber (flank) for 7-14 days post-injection. Mice were randomized to be treated with EphA3-CART or untransduced T-cells (UTD). Subsequently tumor size and mouse survival was monitored to determine the antitumor effect. RESULTS EphA3 was highly expressed on GBM PDX models as well as M2 macrophages. Compared to M0 and M1 macrophages, M2 macrophages significantly suppressed EphA3-CART However, despite the suppressive activity of the M2 macrophage, EphA3-CART exhibited significant antitumor activity . In both flank and orthotopically implanted mouse models, there was an observed reduction in tumor volume with EphA3-CART treated model compared to the UTD counterpart. EphA3-CART treatment resulted in significantly improved survival of the treated models compared to UTD models (hazard ratio = 0.053 **p=0.005). CONCLUSION We demonstrate that EphA3 is expressed on GBM cells as well as on M2 macrophages. EphA3 CART is able to overcome and ameliorate the effect of a major component of the tumor microenvironment (TME), M2 macrophages. EphA3 CART appears effective in flank and orthotopic mouse models of GBM.