IMMU-17. TRANSGENIC EXPRESSION OF IL15 IMPROVES ANTIGLIOMA ACTIVITY OF IL13RΑ2-CAR T CELLS

Abstract

Background: Diffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are the most aggressive primary brain tumors in children. Immunotherapy with T cells expressing chimeric antigen receptors (CARs) specific for the DIPG and GBM antigens is an attractive approach to improve outcomes. IL13Rα2 is expressed at a high frequency in DIPG and GBM but not in normal brain, making it a promising target for CAR T-cell immunotherapy. We recently generated the first scFv-based CAR that is specific for IL13Rα2 and demonstrated that it had potent anti-GBM activity in preclinical models. However, CAR T-cell persistence was limited, resulting in recurrence of IL13Rα2-positive GBMs. Objectives: The goal of this project is to evaluate if transgenic expression of IL-15, a cytokine that is critical for T-cell proliferation and survival, enhances persistence and anti-tumor activity of IL13Rα2-CAR.CD28.ζ T cells. Design/Method: We generated IL13Rα2-CAR.CD28.ζ T cells expressing IL-15 (IL13Rα2- CAR.IL15 T cells) by double transducing T cells with retroviruses containing expression cassettes encoding i) IL13Rα2-CAR.CD28.ζ or ii) IL-15, tNGFR, and iC9 separated by 2A sequences. We determined the effector function of IL13Rα2-CAR.IL15 T cells in vitro using standard assays, and in the U373 GBM xenograft model. Results: Double transduction of CD3/CD28-activated T cells resulted in T-cell lines that expressed both transgenes in 45-50% of T cells. At a baseline, IL13Rα2-CAR.IL15 T cells produced on average 69.5 pg/ml of IL15. Production was significantly increased after CD3 or antigen-specific T-cell stimulation (176.7 pg/ml; n = 6; p<0.001). IL13Rα2-CAR.IL15 T cells were as efficient as IL13Rα2-CAR T cells in killing IL13Rα2-positive GBMs in vitro. After intratumoral injection into U373 glioma-bearing mice IL13Rα2-CAR.IL15 T cells persisted significantly longer than IL13Rα2-CAR T cells (p<0.05). This resulted in a significant increase in progression free (84 vs 49 days; p = 0.008) and overall survival (p = 0.02) of treated mice. Up to date, 4/10 IL13Rα2-CAR.IL15 T-cell treated mice remain glioma free with a follow up of at least 80 days. 3/5 examined, recurring U373 gliomas post IL13Rα2-CAR.IL15 T-cell therapy had downregulated IL13Rα2 expression, indicating immune escape. While all recurring GBMs expressed the glioma-associated antigen EphA2, 1/5 GBMs had also downregulated the expression of the glioma-associated antigen HER2. Conclusion: Here we demonstrate that transgenic expression of IL15 enhances the in vivo persistence of IL13Rα2-CAR T cells resulting in improved anti-glioma activity. However, enhanced T-cell persistence resulted in the development of antigen-specific and -unspecific loss variants highlighting the need to target multiple glioma-associated antigens in tumors with heterogeneous antigen expression such as GBM and DIPG.