Immunogenetic metabolomics revealed key enzymes that modulate CAR-T metabolism and function [DNA-edit]
Immune evasion is a critical step of cancer progression that remains a major obstacle for current T cell-based immunotherapies. Hence, we seek to genetically reprogram T cells to exploit a common tumor-intrinsic evasion mechanism, whereby cancer cells suppress T cell function by generating a metabolically unfavorable tumor microenvironment (TME). Specifically, we use an in silico screen to identify ADA and PDK1 as metabolic regulators, in which gene overexpression (OE) enhances the cytolysis of CD19-specific CD8 CAR-T cells against cognate leukemia cells, and conversely, ADA or PDK1 deficiency dampens such effect. Additionally, ADA-OE in CAR-T cells improves cancer cytolysis under high concentrations of adenosine, the ADA substrate and an immunosuppressive metabolite in the TME. High-throughput transcriptomics and metabolomics in these CAR-Ts reveal alterations of global gene expression and metabolic signatures in both ADA- and PDK1- engineered CAR-T cells. Functional and immunological analyses demonstrate that ADA-OE increases proliferation and decreases exhaustion in a-CD19 and a-HER2 CAR-T cells. We then show ADA-OE improves tumor infiltration and clearance by a-HER2 CD4 and CD8 CAR-T with an in vivo colorectal cancer model.  Collectively, these data unveil systematic knowledge of metabolic reprogramming directly in CAR-T cells, and reveal potential targets for improving CAR-T based cell therapy. Overall design: Genetic perturbations from CRISPR-RNP were quantified from a genomic locus of interest. For each comparison, indels were quantified from samples treated with target or control gRNA (n=3 per condition for eah comparison)