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

Dan Cappabianca; Dan Pham; Matthew H Forsberg; Madison Bugel; Anna Tommasi; Anthony Lauer; Jolanta Vidugiriene; Brookelyn Hrdlicka; Alexandria McHale; Quaovi H Sodji; Melissa C Skala; Christian M Capitini; Krishanu Saha

doi:10.1016/j.omtm.2024.101249

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

Mol Ther Methods Clin Dev. 2024 Apr 10;32(2):101249. doi: 10.1016/j.omtm.2024.101249. eCollection 2024 Jun 13.

Authors

Dan Cappabianca^{1

2}, Dan Pham^{2

3}, Matthew H Forsberg⁴, Madison Bugel^{1

2}, Anna Tommasi^{1

2}, Anthony Lauer⁵, Jolanta Vidugiriene⁵, Brookelyn Hrdlicka^{1

2}, Alexandria McHale^{1

2}, Quaovi H Sodji^{6

7}, Melissa C Skala^{2

3

6}, Christian M Capitini^{4

6}, Krishanu Saha^{1

2

6}

Affiliations

¹ Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA.
² Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA.
³ Morgridge Institute for Research, University of Wisconsin-Madison, Madison, WI 53715, USA.
⁴ Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA.
⁵ Promega Corporation, Fitchburg, WI 53711, USA.
⁶ University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53705, USA.
⁷ Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA.

Abstract

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.

Keywords: CAR T cells; CRISPR; GMP-compatible; biomanufacturing; cancer; gene editing; metabolism; pre-clinical; scale-up; stem cell memory.

Abstract

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