Label free metabolic imaging to enhance the efficacy of chimeric antigen receptor T cell therapy

Abstract

Chimeric antigen receptor (CAR) T cell therapy for solid tumors is challenging not only because of the immunosuppressive tumor microenvironment, but also because of a complex manufacturing process that is difficult to monitor. Manufacturing directly impacts CAR T cell yield, phenotype, and metabolism, which correlate with in vivo potency and persistence. In particular, though metabolic fitness is a critical quality attribute, how T cell metabolic requirements vary throughout manufacturing remains unexplored. Here, we address this limitation with optical metabolic imaging (OMI), a non-invasive, label-free method to evaluate single-cell metabolism based on autofluorescent metabolic coenzymes NAD(P)H and FAD. Using OMI, we identified the dominating impacts of media composition over the selection of antibody stimulation and/or cytokines on anti-GD2 CAR T cell metabolism, activation strength and kinetics, and phenotype. We demonstrated that OMI parameters can indicate cell cycle stage and optimal gene transfer conditions for both viral transduction and electroporation-based CRISPR/Cas9. Notably, in a virus-free CRISPR-edited anti-GD2 CAR T cell model, OMI measurements allowed accurate prediction of an oxidative metabolic phenotype that yielded higher in vivo potency against neuroblastoma. Our data supports OMI’s potential as a robust, sensitive analytical tool to identify optimal manufacturing conditions and monitor cell metabolism throughout manufacturing for increased CAR T cell yield and metabolic fitness.