Glioblastoma (GBM) is an aggressive form of brain cancer that has no effective treatments and a prognosis of only 12-18 months. Immune effector T cells are a promising therapy due to their innate cytotoxicity. In addition, 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 such T cell therapies in vitro at high throughputs is vital for the preclinical development of these promising 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 not only the potency but also the kinetics of T cell killing. Here, 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 a PEI and laminin-coated CytoView-Z 96-well plates, and their impedance was continuously monitored on the Maestro Z impedance platform (Axion BioSystems). After 48 hours, human naïve 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 naïve activated T Cells after 7 days of exposure. In addition, the kill time 50, defined as the time to reach 50% cytolysis of target cells, was shorter for GD-2 CAR-T Cells compared to naïve 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 (33% of cells), and induction of GrB (70%). Initial exhaustion was suggested by expression of PD1 (80% cells) and LAG3 (35%), but not TIM3.
Overall, both naïve activated T-Cells and GD2 CAR-T Cells were effective for cytolysis of GSCs, with CAR-T exhibiting more efficient killing. CAR-T Cells engineered to target the GD2 antigen exhibited stronger potency and faster kinetics, suggesting greater clinical potential against glioblastoma.
Citation Format: Heather Brant Hayes, Meghan T. Logun, Stacie A. Chvatal, Katie Mueller, Nicole Piscopo, Amritava Das, Krishanu Saha, Daniel C. Millard, Lohitash Karumbaiah. Kinetics and potency of T cell and CAR-T cell mediated cytolysis of glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1552.