Article Text

Download PDFPDF

380 Development and characterization of a CAR T cell potency assay with 3D cancer spheroid models
  1. Omari Weems,
  2. Denise Sullivan,
  3. Austin Passaro,
  4. Stacie Chvatal and
  5. Daniel Millard
  1. Axion Biosystems, Atlanta, GA, USA


Background The tumor microenvironment poses a significant challenge to immune therapies, like CAR T cells, for solid tumor indications. Development of in vitro 3D models may provide a more accurate assessment of CAR T potency. Here, we developed a real-time, label-free potency assay for evaluation of CAR T cell-mediated cytotoxicity of 3D cancer spheroids.

Methods In this study, the Maestro Z impedance system was used to develop a real-time, label-free potency assay for immune-cell mediated killing of 3D cancer spheroids. Cancer spheroids were produced with SKOV3 cells in ultra-low attachment U-bottom microplates for four days and then transferred to the CytoView-Z plate. The attachment and growth of SKOV3 spheroids was monitored using the resistance measurement of the Maestro Z. After 24 hours, the SKOV3 spheroid and monolayer groups were treated with HER2-specific CAR T cells at matched effector-to-target cell ratios and cytolysis was computed for the following 72 hours of real-time measurement.

Results The spheroid potency assay was used to compare CAR T cell-mediated cytolysis of SKOV3 spheroids and monolayer cultures with HER2-specific CAR T cells. The steady increase in resistance for the untreated group indicated continued spheroid growth after transfer to the CytoView-Z 96-well plate. CAR T effector cells were added at five E:T ratios at 24 hours post-plating of the target cells. The monolayer groups (gray) had higher rates of cytolysis than their respective spheroid groups (orange) for the same E:T ratios (figure 1A). Cytolysis was compared at 72 hours post-addition of the CAR T effector cells (figure 1B) and potency was quantified via a dose response regression. The EC50 E:T ratio of the HER2-specific CAR T cells was 1:3 for the monolayer target cells and 1:1 for the spheroid target cells. These results suggest a decreased potency of HER2-specific CAR T cells against a 3D tumor model in vitro. CAR T potency was also evaluated via the KT50, a measure of the kinetics of immune cell-mediated killing (figure 1C). There was a dose dependance of CAR T killing within the monolayer and spheroid groups, with monolayer groups having shorter KT50s than the matched spheroid groups.

Conclusions These results suggest that 3D cancer spheroid had a higher resistance to CAR T killing than monolayer cultures, which may reflect an improved representation of the tumor microenvironment for an in vitro potency assay.

Abstract 380 Figure 1

CAR T cells exhibit reduced cytolysis against spheroids(A) Cytolysis over time of 10k SKOV3 spheroids and monolayers treated with 4-1BB (HER2 targeting) CAR T cells at effector-target ratios of 1:10, 1:5, 1:2, 1:1, and 5:1. (B) Cytolysis between spheroids and monolayer in respect to their effector-target ratios. EC50 measured the 4-1BB CAR T ratio required to achieve 50% cytolysis. (C) KT50 between spheroids and monolayer groups, displaying both dose dependance for both groups and differences in duration to achieve 50% cytolysis between groups. *indicated 1:2 spheroid group did not reach a KT50.

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.