Use of cellular FAD autofluorescence as a label-free cellular attribute for the production of chimeric antigen receptor T cells.

Chimeric antigen receptor (CAR) T-cell therapy has become an attractive approach for treating hematologic malignancies. However, the accessibility of this therapy is limited by factors such as complex manufacturing processes, limited capacity of manufacturing facilities, and the requirement of a highly skilled workforce for the manual steps of CAR T-cell production. To minimize manual processes, the CAR T-cell manufacturing field is shifting towards closed and automated systems, including analytical tools that offer intermittent monitoring of cells in production. Therefore, label-free technologies for closely monitoring CAR T-cells in closed systems are needed. NADH and FAD are autofluorescent molecules that can serve as label-free biomarkers that correlate with T cell activation. Here, we evaluate the use of a flow cytometer equipped with a 405-nm violet laser for investigating the nicotinamide adenine dinucleotide reduced (NADH) and flavin adenine dinucleotide (FAD) autofluorescence in T cells. Our results revealed that the increase of NADH and FAD autofluorescence was significantly correlated with the upregulation of T-cell activation marker CD25 and the increase of extracellular lactate in spent media in the first 3 days after T-cell activation. We demonstrate the potential use of FAD for determining the endpoint of CAR T-cell manufacture by establishing a relationship between the rate of change in the mean fluorescence intensity (MFI) of FAD in CAR T-cells and the rate of change in T-cell proliferation using a G-Rex bioreactor. Collectively, these findings suggest that autofluorescence, particularly FAD autofluorescence, can serve as a label-free biomarker (cellular attribute) for monitoring T-cell activation and expansion during CAR T-cell production. The use of 405-nm visible light to substitute for the genotoxic ultraviolet wavelengths for assessing NADH and FAD autofluorescence paves the way to incorporate autofluorescence measurements into closed and automated systems for in-process monitoring of CAR T-cell manufacturing.