Background Immunotherapy (IMT) has shown tremendous success in hematological cancers. However, an important obstacle to using IMT for solid tumors is the aggressive tumor microenvironment (TME). To significantly improve clinical prognosis of patients undergoing IMT, it is essential to integrate novel strategies to improve T cell bioenergetics and metabolically prepare them to bypass the hurdles posed by the TME.

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 glutaminase inhibitor BPTES and Arginine respectively. Briefly, two engineered T-cells were used in the study, CAR T cells targeting EpCAM (EpCAM-CAR T-Cells) and TCR T-cells against MART-1. EpCAM-CAR T cells were pre-treated with BPTES (3 and 6 µM) for 4 days before performing the cytotoxicity assay using T-47D as target cells at the effector to target cell ratio (E:T) of 0.5:1. 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. Both target cells, T-47D and 624.38 cells, were engineered to express a red-fluorescent nuclear protein. The CAR/TCR T-cell killing against the tumor cells was determined from real-time impedance and live cell imaging data collected on xCELLigence RTCA eSight. Concurrently, the metabolic profiling of the engineered T-cells was determined by the XF96 Analyzer.

Results 1. Pre-treating EpCAM targeted CAR T cells with 3 and 6 µM of BPTES significantly enhanced cytotoxicity effects by 42% and 30% (two tailed student t-test p <0.001) respectively compared to no- treatment control where the E:T was 0.5. A shift in metabolism was revealed in a parallel T cell metabolic profiling assay. 2. 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 The BPTES treated EpCAM CAR T cells shifted metabolism resulting in enhanced cytotoxicity towards breast cancer cells T-47D.Arginine pre-conditioning improved TCR T cell potency through metabolic rewiring favoring mitochondrial respiration.

For Research Use Only Not for use in diagnostic procedures.

RA45166.5737384259