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.

Citation Format: Stacie A. Chvatal, Meghan T. Logun, Denise D. Sullivan, Heather B. Hayes, Daniel Millard, Mueller P. Katie, Nicole J. Piscopo, Amritava Das, Kris Saha, Daniel J. Brat, Lohitash Karumbaiah. GD2 CAR-T cells engineered using retroviral transduction or CRISPR editing exhibit strong cytolytic potency against glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2817.