(23g) Extracellular Domains of CARs Reprogram T-Cell Metabolism without Antigen Stimulation

Metabolism is a network of biochemical reactions that supports T cells’ proliferation and anti-cancer activity by supplying energy and biochemical building blocks. Chimeric antigen receptors (CARs) enable T cells to recognize and attack cancer cells. Most successful CAR-T cell therapies to date target B-cell malignancies that express CD19. Designing new CARs involves combinatorial assembly of peptide domains. Specifically, the scFv (single-chain variable fragment), an extracellular CAR domain, determines T cell ligand-binding specificity and directs cytotoxicity against cancer cells presenting cognate antigens. While intracellular co-stimulatory and signaling domains affect CAR-T cells’ metabolism and efficacy, the effects of extracellular non-signaling domains (e.g., scFv) on CAR-T cell function remain unexplored. Here we explored how CARs differentially rewire T-cell metabolism depending on the choice of scFv. Across a panel of human T cells expressing seven CARs differing only in the non-signaling portions, we quantified metabolite concentrations and fluxes. Even in the absence of antigen, all CAR-T cells displayed different metabolic profiles. T cells harboring a 14g2a-based anti-GD2 CAR and a rituximab-based anti-CD20 CAR displayed the most active metabolism with fast glucose and amino acid uptake, while anti-CD19 and other anti-CD20 CAR-T cells displayed minimal alteration in central carbon and nucleotide metabolism compared to control T cells. Interestingly, hyperactive CAR-T cells increased nitrogen uptake to produce excess nucleotides, non-essential amino acids, and ammonia. The inefficient nitrogen metabolism undermined these CAR-T cells’ ability to proliferate and attack cancer cells in a tumor microenvironment containing high levels of nitrogen waste. In mice with Raji and Ramos lymphoma xenografts, the conjunction of modest overflow metabolism of CAR-T cells and metabolic compatibility between cancer and CAR-T cells extended the survival of tumor-bearing mice. Thus, metabolic rewiring serves as an important prognostic marker for streamlined CAR design, and metabolic engineering should be an integral task in translating the fundamental knowledge of immunometabolism into successful CAR-T cell therapies.