Metabolic priming of GD2TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence

Abstract

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 inTRAC-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-GD2TRAC-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 assaysin vitroand potency against human GD2+ xenograft neuroblastoma modelsin vivo. Compared to standardTRAC-CAR T cells, MPTRAC-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 TGFβ production at the end of manufacturingex vivo, with increased central memory CAR T cells and better persistence observedin vivo. Metabolic priming with media during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypesex vivo, which could lead to better responses against solid tumorsin vivo.

Label free metabolic imaging to enhance the efficacy of Chimeric Antigen Receptor T cell therapy

“…Therefore, we next characterized whether OMI features could inform CRISPR genome editing efficiency. Following 12-to-72 hour activation by either Imm or Tex methods, T cells were imaged with OMI and electroporated to generate CRISPR-edited anti-GD2 CAR T cells as previously described ( 41 ). Notably, across 3 donors, the timeframe that yielded optimal genome editing efficiency, quantified as percent CAR positivity post expansion (Fig 3B-C) , aligned with the timeframe of maximal decrease in NAD(P)H τ m and increase in NAD(P)H α 1 (Fig 2C-D) .…”

“…T cells from 3 donors were activated with either Imm or Tex method prior to EP with CRISPR/Cas9 machinery to express anti-GD2 CAR as previously described ( 9, 41 ). Following EP, CAR T cells were expanded in either ImmunoCult XF or TexMACS media for 7 days in a 37C, 5% CO 2 humidified incubator.…”

“…CAR T cell phenotypes and metabolism were analyzed with flow cytometry and OMI after 7 days of expansion. Flow cytometry was performed on an Aurora spectral cytometer (Cytek) as previously described ( 9, 41 ). Briefly, T cells were stained and analyzed for expression of 16 markers (anti-GD2 CAR, TCR αβ , CD4, CD8, CD45RA, CD45RO, CD62L, CCR7, Human CD45, PD-1, LAG3, TIM3, CD39, TIGIT, CD27, CD5) with specific clones and concentrations as previously described ( 41 ).…”

“…Following EP, Imm and Tex activated anti-GD2 CAR T cells were expanded for 7 days in ImmunoCult XF T cell expansion media supplemented with 500U/mL IL-2 or TexMACS media supplemented with 10ng IL-7, respectively. After expansion, CAR T cells were harvested; percent CAR positivity was quantified via flow cytometry of 1A7 anti-14G2A antibody (National Cancer Institute, Biological Resources Branch) conjugated to APC using a Lightning Link APC Antibody Labeling kit (Novus Biologicals) to evaluate genome editing efficiency as previously described (Fig S1) (9,41). Virus-free anti-GD2 CAR T cell expansion and flow cytometry characterization T cells from 3 donors were activated with either Imm or Tex method prior to EP with CRISPR/Cas9 machinery to express anti-GD2 CAR as previously described (9,41)…”

“…CD5) with specific clones and concentrations as previously described(41). An expression histogram of each surface marker was generated based on single-cell MFIs normalized by donor-matched fluorescence-minus-one (FMO) control.…”