Abstract
High grade gliomas (HGG) remain some of the most difficult to treat pediatric malignancies resulting in dismal outcomes. As even the best treatments currently available show poor efficacy and high toxicity, there is an urgent need to develop new treatment approaches. Immunotherapy with T cells expressing chimeric antigen receptors (CARs) specific for the glioma antigen IL-13Rα2 is an attractive approach with high potential for improving outcomes. We recently demonstrated that IL-13Rα2 CAR T cells had potent anti-tumor activity in preclinical glioma models. Genetic modification of these CAR T cells further enhanced this efficacy both in vitro and in vivo. However, despite these modifications, brain tumors eventually recurred. Mechanistic studies revealed that limited CAR T-cell function was the primary cause of therapeutic failure. The goal of this project is to enhance the effector function of IL-13Rα2-CARs through CRISPR/Cas9-mediated silencing of SHP-1, a potent negative T cell regulator. We generated IL-13Rα2.SHP-1 KO CAR T cells by electroporation of IL13Rα2-CARs with SHP-1 targeted Cas9-sgRNA complexes which resulted in highly efficient SHP-1 knockout. Importantly, this method did not affect CAR expression level, T-cell viability, or phenotype. IL-13Rα2.SHP-1 KO CAR T cells showed enhanced in vitro proliferation in response to T cell receptor stimulation and were as efficient as IL-13Rα2 CAR T cells in killing IL-13Rα2-positive gliomas in vitro. Treatment of intracranial and subcutaneous U373 glioma-bearing mice with IL-13Ra2.SHP-1-KO CAR T cells resulted in a significant delay in tumor growth and significant survival advantage when compared to mice receiving control IL-13Rα2 CAR T cells. In conclusion, we demonstrate that silencing negative T cell regulators can enhance the in vivo anti-tumor activity of IL-13Ra2 CAR T cells. This study highlights the potential for CRISPR-based gene editing as a powerful approach for improving the therapeutic efficacy of CAR T cells for HGG.