Manufacturing
chimeric antigen receptor (CAR) T cell
therapies is complex, with limited understanding of how medium composition impacts T cell phenotypes. CRISPR-Cas9
ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem cell memory T cell population, a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 TRAC-CAR T cells using "metabolic priming" (MP), where the cells were activated in
glucose/
glutamine-low medium and then expanded in
glucose/
glutamine-high medium. T cell products were evaluated using spectral flow cytometry, metabolic assays,
cytokine production, cytotoxicity assays in vitro, and potency against human GD2+ xenograft
neuroblastoma models in vivo. Compared with standard TRAC-CAR T cells, MP TRAC-CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound
NAD(P)H activity, and reduced IFN-γ,
IL-2, IP-10, IL-1β,
IL-17, and TGF-β production at the end of manufacturing ex vivo, with increased central memory CAR T cells and better persistence observed in vivo. MP with medium during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo, which could lead to better responses against solid
tumors in vivo.