Quantification of CAR-T potency and proliferation using a high-throughput benchtop image cytometry assay

Chimeric antigen receptor modified T cell (CAR-T) therapy has transformed treatment of a subset of hematological malignancies, driving development of CAR-T products that require robust potency and functional assays. Existing chromium-51, bioluminescence, impedance and flow cytometry assays are either costly, laborious, potentially hazardous and/or lack multiplexing capability. We developed a rapid 3-color image cytometry assay that measures target cell cytotoxicity and CAR-T proliferation, providing an affordable, high-throughput, benchtop alternative to other assays. This novel image cytometry assay and a standard flow cytometry assay demonstrated comparable performance under multiple different conditions when assessing target cell cytotoxicity. Antigen-dependent and -independent CAR-T proliferation and survival were assessed, demonstrating inverse association between antigen-dependent proliferation and the effector:target (E:T) ratio. Compared to a standard flow cytometry assay, preparation and acquisition of 24 samples using this image cytometry assay was markedly faster (164min vs 692 min; 4.2-fold); and labor and materials costs were 25.6% lower than flow cytometry ($265.40 AUD vs $1038.49 AUD). We present a readily available benchtop image cytometry assay that measures CAR-T cytotoxicity and proliferation, and is a more affordable, high-throughput alternative to flow cytometry.