Background Immune effector T-cells are a promising therapy due to their innate cytotoxicity. In particular, CAR T-cell therapy uses genetically engineered T-cells that express a chimeric antigen receptor that binds to a specific antigen on tumor cells. A key factor influencing CAR-T cell efficacy is the density of target antigens on cancer cells, which dictates the cytotoxic response. Human epidermal growth factor receptor 2 (HER2), overexpressed in various cancers like ovarian and lung carcinomas, is a promising target for CAR-T cell therapy. Traditional cell viability and cytotoxicity assays often fail to capture the dynamic responses of target cells to novel cell therapies, as they provide only a static endpoint measurement. Live-cell imaging offers a non-invasive alternative for real-time monitoring of cytoxicity. Here, we describe an in vitro potency assay that uses the Omni automated imaging platform to quantify immune cell-mediated cytotoxicity of cancer target cells by immune effector cells.

Methods SKOV3 (ovarian carcinoma) and A549 (lung adenocarcinoma) cells, both tagged with GFP, were seeded in a 96-well plate and exposed to HER2 CAR-T cells at various Effector : Target (E:T) ratios. Fluorescent images of the target cells were captured by the Omni every hour for 4 days. Image analysis was performed using the Confluency Module in the Axion Portal to view attachment and proliferation and quantify cytolysis of fluorescent target cells. Percent cytolysis of the target cells was calculated by comparing the green fluorescent confluency of treated wells to no treatment control wells.

Results A549-GFP cells showed a dose-dependent decrease in confluency over time that correlated with increasing amounts of CAR T-cells. At 92 hours post HER-2 CAR T-cell addition, the 5:1 ET group demonstrated approximately 91.8% +/- 2.0% cytolysis of A549-GFP cells, while the 1:10 group demonstrated approximately 69.2% +/- 3.0% cytolysis. There was a dose-dependent cytotoxic effect of HER2 CAR-T cells on SKOV3 target cells as well, with SKOV3 cells showing higher sensitivity and earlier response at lower E:T ratios compared to A549 cells.

Conclusions This research highlights the importance of target antigen density in CAR-T cell efficacy. SKOV3 cells, with higher HER2 expression, were more susceptible to CAR-T cell killing compared to A549 cells. Overall, the Omni platform enables continuous quantification of the potency and kinetics of immune cell-mediated cytolysis.