Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence

Abstract

Manufacturing chimeric antigen receptor (CAR) Tcell therapies is complex, with limited understanding of how medium composition impacts Tcell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous Tcell receptor alpha constant (TRAC) gene resulting in TRAC-CAR Tcells with an enriched stem cell memory Tcell population, a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 TRAC-CAR Tcells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine-low medium and then expanded in glucose/glutamine-high medium. Tcell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays invitro, and potency against human GD2+ xenograft neuroblastoma models invivo. Compared with standard TRAC-CAR Tcells, MP TRAC-CAR Tcells 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 exvivo, with increased central memory CAR Tcells and better persistence observed invivo. MP with medium during CAR Tcell biomanufacturing can minimize glycolysis and enrich memory phenotypes exvivo, which could lead to better responses against solid tumors invivo.