Abstract

Glioblastoma is an aggressive form of brain cancer with a prognosis of 12-18 months and no effective treatments. Immune effector T cells are a promising therapy due to their innate cytotoxicity; engineering chimeric antigen receptors (CAR) to target tumor-associated or neo-antigens can lend high specificity. The ability to assess the efficacy and potency of T cell therapies in vitro at high throughput is vital for the development of such therapies. Cellular impedance assays offer a sensitive, non-destructive, and label-free method to continuously monitor cancer cell proliferation and immune cell-mediated cytotoxicity in real-time, revealing the potency and the kinetics of T cell killing. We used co-culture to compare the cytolytic potency and kinetics of naïve activated T Cells and targeted GD2 CAR-T cells against glioma stem cells (GSC), a subpopulation of glioblastoma cells.

Patient-derived N08 GSCs were plated at 50k cells per well on CytoView-Z 96 plates; their impedance was continuously monitored with the Maestro Z impedance platform. After 48 hours, human naive activated T Cells (ImmunoCult CD3/CD28 activation media) or GD2 CAR-T cells were added at varying effector:target ratios ranging from 0.1:1 to 10:1. Impedance and cytolysis were subsequently monitored for up to 7 days.

The addition of activated T Cells or GD2 CAR-T Cells resulted in cell swelling, followed by a decrease in impedance consistent with T cell-mediated lysis of GSCs. GD-2 CAR-T cells resulted in a significantly higher percent cytolysis of GSCs compared to naive activated T Cells after 7 days of exposure. In addition, the kill time 50 was shorter for GD-2 CAR-T Cells compared to naive activated T Cells.

Cytotoxic function was validated with subsequent flow cytometry and cytokine analysis. After 7 days in co-culture, GD2 CAR-T cells exhibited markers of chronic activation, including greater CD8 expression than CD4, upregulation of CD69