Abstract 2849: A novel solution to assess T cell metabolic phenotype for the optimization of antitumor T cell therapy products

Abstract In recent years, chimeric antigen receptor T (CAR-T) cells have shown significant promise in treating a range of cancers, with a number of approved products currently on the market. This has led to significant interest in optimizing cell therapy design and manufacturing. Traditionally, CAR-T cells are transfected expanded, and partially characterized before infusing into patients. However, the ability to predict in vivo efficacy is still challenging. Bioenergetic metabolism has been extensively linked to immune cell function and therefore represents a key attribute to be assessed during T cell therapy optimization and production. In addition, metabolic reprogramming can represent a mechanism to drive a desired immune phenotype. Agilent Seahorse XF T cell Metabolic Profiling kit is a robust new solution that combined with the Seahorse XF analyzer, allows for the simultaneous measurement of basal metabolic requirements, metabolic poise and mitochondrial bioenergetic capacity in T cells. These parameters have been shown to correlate with increased T cell persistence and improved metabolic fitness in nutrient restricted environments. This assay incorporates an improved uncoupler (BAM15) to allow more robust measurements of mitochondrial T cell bioenergetic capacity (measured as spare respiratory capacity or maximum respiration) with minimum optimization compared to uncouplers previously utilized. The XF T cell Metabolic Profiling kit has been used to evaluate the influence of expansion media composition on the metabolic phenotype of expanded human T cells. Indeed, when human T cells were activated and expanded up to 22 days in different commercial culture media formulations supplemented with different cytokine combinations, it resulted in T cell populations with differential bioenergetic profiles and mitochondrial spare capacities. These different metabolic phenotypes also correlated with different functional markers that have previously been associated with increased persistence in vivo. Similarly, the XF T cell Metabolic Profiling kit was used to assess metabolic poise and bioenergetic capacity of activated T cells under altered or restricted metabolic conditions like the ones present in the tumor microenvironment (low glucose, low glutamine), therefore allowing for better understanding of the effect of carbon source depletion during T cell stimulation. In addition, this assay coupled to the existing XF Hu T cell activation assay allowed for the assessment of T cell ‘fitness’ after re-stimulation in nutrient limited conditions. Taken together, the XF T cell Metabolic Profiling kit is a novel solution that provides a complete insight into the metabolic state of T cells in real-time, facilitating the evaluation of cell therapy expansion conditions and metabolic fitness of T cells under limited nutrient availability, which can be used to better design T cell therapies. Citation Format: Jessica Walls, Yoonseok Kam, Natalia Romero. A novel solution to assess T cell metabolic phenotype for the optimization of antitumor T cell therapy products [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2849.