Background Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for several cancers. However, CAR T cell therapy is currently hampered by off-tumor toxicity that can manifest in multiple forms and can offset improvements in patient condition from tumor elimination. One such class of CAR T cell side effects, termed on-target, off-tumor toxicity, involves direct engagement of the target antigen by CAR T cells on non-cancerous cells. Following engagement, CAR T cells may kill these non-cancerous cells and cause damage to healthy tissue.

Methods To study these on-target, off-tumor side effects in vitro, we developed a model using HER2-CAR T cells and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) on the Maestro Pro MEA platform. The iPSC-CMs (50K) were seeded in each well of a 24-well CytoView MEA plate and dosed with increasing effector:target (E:T) ratios (1:80 to 1:1) of HER2-CAR T cells. Both MEA viability (resistance) and electrophysiology measurements were taken each day for up to 7 days using the Maestro Pro.

Results We confirmed HER2 expression in iPSC-CMs using flow cytometry and saw robust activation and cluster formation of HER2-CAR T cells following co-culture with the iPSC-CMs. We found a dose-dependent decrease in resistance detected from iPSC-CMs dosed with increasing E:T ratios of HER2-CAR T cells and detected significant killing (55.6% decrease in resistance) by the 1:1 CAR T group as early as Day 1 post-dose. By comparison, we observed no difference in resistance between HER2-negative iPSC-derived neuron control wells and those treated with HER2-CAR T cells. Further, we dosed the iPSC-CMs with non-transduced T cells and saw a minimal (5.7%) decrease in iPSC-CM resistance at Day 4 compared to dosing with CAR T cells at the same E:T ratio (71.8% decrease). These results confirm the specificity of the HER2-CAR T cells’ killing of iPSC-CMs. Finally, we measured the values of electrophysiological parameters including Fridericia-corrected field potential duration (FPDc), beat period, and spike amplitude of our iPSC-CMs dosed with CAR T cells at increasing E:T ratios. Interestingly, large decreases in all three metrics were apparent at 1 hour post-dose in the 1:1 CAR T dose group, illustrating that these electrophysiological changes may signal on-target, off-tumor effects even earlier than cytotoxicity data.

Conclusions In summary, the Maestro Pro can be used to measure cytotoxicity and electrophysiological changes in models of CAR T cell on-target, off-tumor effects.