Characterization and Functional Analysis of scFv-based Chimeric Antigen Receptors to Redirect T Cells to IL13Rα2-positive Glioma

Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13R alpha 2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13R alpha 2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13R alpha 2, they also recognize IL13R alpha 1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13R alpha 2-specific CARs that contain the IL13R alpha 2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.zeta (IL13R alpha 2-CARs). IL13R alpha 2-CAR T cells recognized IL13R alpha 2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13R alpha 1. However, only IL13R alpha 2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13R alpha 2-CARs with short spacer regions and CD28.zeta, 41BB.zeta, and CD28.OX40.zeta endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13R alpha 2-CAR T cells hold the promise to improve current IL13R alpha 2-targeted immunotherapy approaches for GBM and other IL13R alpha 2-positive malignancies.