Kinetics and potency of GD2 CAR-T cell-mediated cytolysis of gioblastoma

 

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Welcome to our poster: Kinetics and potency of GD2 CAR-T cell-mediated cytolysis of glioblastoma. Glioblastoma is an aggressive form of brain cancer with no effective treatments and a short prognosis. Immune cell therapies offer promise and targeting tumor-specific antigens with chimeric antigen receptors can lead to high specificity.

Axion BioSystems Maestro Z platform is an impedance-based system for real-time, label-free continuous monitoring of cell viability and cytolysis. Impedance is measured from electrodes embedded in the bottom of each well of a 96- or 384- well plate and as cells attach the impedance increases and then if cells die or detach, in response to dosing, impedance decreases.

Here we use the Maestro Z to assess the potency and kinetics of immune cell-mediated killing of glioma stem cells, a subpopulation of glioblastoma, by GD2 targeted CAR T-cells over the course of seven days. First patient derived NO8 glioma stem cells were assessed for CAR T targets using flow cytometry. Glioma stem cells exhibited high expression of the antigen GD2 compared to other popular CAR T targets. High GD2 expression was also seen in rodent xenograph tumors and human patient tissue arrays compared to healthy tissues. Next patient-derived NO8 glioma stem cells were plated at 50k cells per well on CytoView-Z 96-well plates and their impedance was continuously monitored on the Maestro Z. As the glioma stem cells attached, their impedance increased, while media only wells, shown here in red, showed little change. At 48 hours GD2 targeted CAR T-cells were added at effector to target ratios ranging from 0.1 up to 10:1.

Impedance and cytolysis were then subsequently monitored for up to seven days. In addition, some wells were left untreated, usually glioma stem cells alone, shown here in teal, to serve as a no treatment control while others receive 50k CAR T cells alone, shown here in pink, and because these don't attack they show little change in impedance. All ratios of GD2 CAR T-cells shown here in green, purple, and blue, induced a decrease in impedance that indicates glioma stem cell death. Higher ratios of GD2 CAR T-cells cause an initial increase in impedance that's likely related to cell swelling or inflammation before then inducing rapid GSC cell death.

Because impedance is non-destructive and label-free, impedance can also be used to measure not only endpoint potency but the kinetics of cell-mediated killing as well.

Up here we present percent cytolysis. Zero percent cytolysis is equivalent to the impedance of glioma stem cells alone so untreated cells, whereas a hundred percent cytolysis is equivalent to full cell death. We compared the cytolysis induced by naive activated T-cells compared to targeted GD2 CAR T-cells. As you can see targeted GD2 CAR T-cells showed greater potency for killing glioma stem cells with all ratios reaching greater than 50 cytolysis.

Interestingly while the final percent cytolysis did not differ across ratios of GD2 CAR T-cells, the kinetics did and that's shown here over time. The blue, the 10 to 1, the highest ratio, actually showed a delayed cytolysis again likely due to that initial swelling and a slightly longer time to reach 50 percent cytolysis. Finally GD2 CAR T-cells were evaluated for activation and early exhaustion after two and seven days in co-culture. After seven days in coculture GD2 CAR T-cells exhibited markers of chronic activation, including high CD69 and increasing CD137. Early exhaustion of GD2 CAR T-cells was also suggested by expression of PD1 and LAG3.

Overall, the Maestro Z platform enabled continuous, dynamic, label-free quantification of the potency, efficiency, and kinetics of immune-cell mediated cytolysis of glioblastoma. Our results suggest that GD2 CAR T-cells are a promising treatment for glioblastoma.