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.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
Statistics from Altmetric.com
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.