GD2 CAR-T Cells exhibit strong cytolytic potency against glioma stem cells

Abstract Glioblastoma (GBM) is an aggressive brain cancer without effective treatments. CAR-T cells targeted to tumor-associated antigens offer promise for treating GBM. Here, we used cellular impedance assays to compare the cytolytic potency and kinetics of conventional viral vs non-viral CRISPR engineered GD2 CAR-T cells against glioma stem cells (GSC), a subpopulation of glioblastoma cells. Patient-derived N08 GSCs were plated at 50k cells/well on 96-well plates, and impedance was continuously monitored on the Maestro Z impedance platform (Axion BioSystems). GD2 CAR-T cells were engineered using either retroviral transduction (RV) or non-viral CRISPR editing (NV). At 48 hours, GD2 CAR-T cells were added at Effector:Target ratios of 0.1:1, 1:1, and 10:1. Comparisons were made to mCherry T cells (mCh) as a control. Impedance and cytolysis were monitored up to 7 days. RV and NV GD2 CAR-T cells caused decreases in impedance consistent with T cell-mediated lysis of GSCs, whereas mCh T cells induced little change. NV CAR-T cells exhibited faster killing kinetics compared to RV CAR-T cells. The time to 50% cytolysis (KT50) was significantly shorter for NV vs RV CAR-T cells at 1:1 and 10:1 E:T ratios. Cytotoxic function was validated with flow cytometry and cytokine analysis at 7 days. All T cells exhibited chronic activation measured by CD69 and CD137 upregulation. Importantly, NV CAR-T cells exhibited less exhaustion, as measured by PD1 and LAG3 expression. Both RV and NV GD2 CAR-T cells effectively cytolyzed GSCs, with NV CAR-T cells exhibiting more potent and efficient killing. The high potency, fast kinetics, and reduced exhaustion of NV CRISPR GD2 CAR-T cells offer great clinical promise for treating GBM.