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

Abstract

Chimeric antigen receptor (CAR) T cell therapy for solid tumors remains challenging due to the complex manufacturing process and the immunosuppressive tumor microenvironment. The manufacturing condition directly impacts CAR T cell yield, phenotype, and metabolism, which correlate within vivopotency and persistence. Optical metabolic imaging (OMI) is a non-invasive, label-free method to evaluate single cell metabolism based on autofluorescent metabolic coenzymes NAD(P)H and FAD. Using OMI, we identified the dominating impacts of media composition over the selection of antibody stimulation and/or cytokines on anti-GD2 CAR T cell metabolism, activation strength and kinetics, and phenotype. We demonstrated that OMI parameters were indicative of cell cycle stage and optimal gene transfer conditions for both viral transduction and electroporation-based CRISPR/Cas9. Notably, OMI accurately predicted oxidative metabolic phenotype of virus-free CRISPR-edited anti-GD2 CAR T cells that correlated to higherin vivopotency against neuroblastoma. Our data supports OMI’s potential as a robust, sensitive analytical tool that enables dynamic and optimal manufacturing conditions for increased CAR T cell yield and metabolic fitness.One sentence summaryAutofluorescence imaging informs manufacturing conditions that enhance yield and metabolic fitness of CAR T cells for neuroblastoma.

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

“…These label-free, single-cell analyses confirm the presence of more primed cells with increased NAD(P)H binding activity within the MP TRAC -CAR T cell products and are consistent with lifetime imaging of similar TRAC- CAR products. 42 …”

“…TexMACS medium is associated with attenuated cellular activation, possibly due to its lower glucose/glutamine content relative to Immunocult XF. 17 , 42 The result is that MP TRAC -CAR T cells are less reliant on glycolysis, exhibit more stem cell memory phenotypes, and consume less glucose than their non-primed counterparts. Furthermore, these MP TRAC -CAR T cells can adopt a central memory phenotype following exposure to solid tumors in neuroblastoma xenograft models.…”

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

“…S3). 42 Activating T-cells in TexMACs rewires their metabolism away from glycolysis (Fig. S1) and it may be possible that factors in Immunocult XF can uniquely enforce this phenotype post-EP.…”

“…Additionally, while the media choice during TRAC- CAR T cell culture seems to impact metabolism more than the choice of cytokines, priming anti-GD2 TRAC- CAR T cells with IL-2 could produce similar stem cell memory phenotypes with enhanced in vivo performance. 42 Additional strategies will need to be explored to determine if these effects can be enhanced even further. For example, treating T-cells during activation with N-acetylcysteine, a well-known antioxidant, can promote expression of stem cell memory markers and lower glycolysis.…”

“…TexMACS media is associated with attenuated cellular activation, possibly due to its lower glucose/glutamine content relative to Immunocult XF. 37, 42 The result is that MP TRAC -CAR T cells are less reliant on glycolysis, exhibit more stem cell memory phenotypes, and consume less glucose than their non-primed counterparts. Furthermore, these MP TRAC -CAR T cells can adopt a central memory phenotype following exposure to solid tumors in neuroblastoma xenograft models.…”