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

Manufacturing Chimeric Antigen Receptor (CAR) T cell therapies is complex, with limited understanding of how media composition impact 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 media 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 media and then expanded in glucose/glutamine high media. 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 to 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-{gamma}, IL-2, IP-10, IL-1{beta}, IL-17, and TGF{beta} production at the end of manufacturing ex vivo, with increased central memory CAR T cells and better persistence observed in vivo. Metabolic priming with media 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.