(1) Cappabianca D (2) Pham D (3) Forsberg MH (4) Bugel M (5) Tommasi A (6) Lauer A (7) Vidugiriene J (8) Hrdlicka B (9) McHale A (10) Sodji QH (11) Skala MC (12) Capitini CM (13) Saha K

Molecular therapy. Methods & clinical development | Free PMC Article

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-_, IL-2, IP-10, IL-1_, IL-17, and TGF-_ 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.

Author Info: (1) Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA. Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 5

Author Info: (1) Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA. Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA. (2) Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA. Morgridge Institute for Research, University of Wisconsin-Madison, Madison, WI 53715, USA. (3) Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA. (4) Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA. Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA. (5) Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA. Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA. (6) Promega Corporation, Fitchburg, WI 53711, USA. (7) Promega Corporation, Fitchburg, WI 53711, USA. (8) Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA. Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA. (9) Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA. Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA. (10) University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53705, USA. Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA. (11) Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA. Morgridge Institute for Research, University of Wisconsin-Madison, Madison, WI 53715, USA. University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53705, USA. (12) Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA. University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53705, USA. (13) Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA. Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA. University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53705, USA.

Citation: Mol Ther Methods Clin Dev 2024 Jun 13 32:101249 Epub04/10/2024

Link to PUBMED: http://www.ncbi.nlm.nih.gov/pubmed/38699288

Tags: