Abstract 3248: Metabolic rewiring shifts engineered TCR T cell bioenergetics to enhance cytotoxicity

Abstract Background: Adoptive T cell-based transfer has shown tremendous success in hematological cancers. However, an important obstacle to using cell and gene therapy for solid tumors is the aggressive tumor microenvironment (TME). To significantly improve prognosis of subjects undergoing immunotherapy, it is essential to incorporate unique methods to drive T cell energy production and metabolism towards empowering T cells to dismantle the suppressive TME. This can promote anti-tumor effects and persistence of the transfused engineered T cells. In the present study we present a simple metabolic preconditioning process that improves T cell performance and bioenergetics. Methods: Using the Agilent xCELLigence RTCA eSight, and Seahorse XF Analyzer we assessed the killing efficiency, and bioenergetics of engineered T-cells after pretreating with Arginine. Briefly, engineered TCR T-cells against MART-1 were generated by transducing CD3+ T-cells (Hemacare, Seattle, WA) with retrovirus SAMEN-DMF5 with a CD34 marker gene, against MART-1. The TCR T cells were pre-conditioned in 6mM Arginine for 7 days, followed by a killing assay using MART-1 expressing melanoma cell line as target cells (624. 38) at the E: T ratio of 5: 1. Target melanoma cells, 624. 38 cells, were engineered to express a red-fluorescent nuclear protein. The comparison was made with transduced T-cells grown in RPMI (no added amino acid supplementation, and non-transduced T cells. The cytotoxicity assay was assessed using real-time impedance and live cell imaging data collected on xCELLigence RTCA eSight. Concurrently, the metabolic profiling of the engineered T-cells was examined by the XF96 Analyzer. Results: Supplementing the growth medium with 6 mM Arginine significantly increased the potency of MART-1 engineered T cells (up to 6-fold) and bioenergetic studies revealed increased spare respiratory capacity, basal respiration, and ATP production via mitochondrial respiration. Conclusions: Arginine pre-conditioning improved TCR T cell potency through metabolic rewiring favoring mitochondrial respiration. For Research Use Only. Not for use in diagnostic procedures RA45236. 3483333333 Citation Format: Rashmi Pillai, Xiaoyu Zhang, Yama Abassi. Metabolic rewiring shifts engineered TCR T cell bioenergetics to enhance cytotoxicity abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 3248.