Metabolic Rewiring of GPC3-Targeted CAR T Cells Expressing IL-15 and HER2-Targeted CAR T Cells via the ADA1–CD26 Axis Promotes Tumor Infiltration and Limits Systemic Toxicities

Background: Chimeric antigen receptor (CAR) T-cell therapy for solid tumors is limited by inadequate T-cell persistence and tumor infiltration, as well as treatment-associated cytokine release syndrome (CRS). To address these challenges, we developed a metabolically rewired CAR T-cell (MRCAR) platform by co-expressing adenosine deaminase 1 (ADA1) and CD26 in CAR T cells. Methods: MRCAR T cells were generated using either GPC3-targeted CARs for hepatocellular carcinoma (HCC) or HER2-targeted CARs for breast cancer. For HCC, we further evaluated constructs with and without enforced IL-15 expression to boost T-cell function. Results: In vitro, IL-15–expressing GPC3-MRCAR T cells exhibited enhanced proliferation, sustained effector function, reduced exhaustion, and superior cytotoxicity following repeated antigen stimulation compared with conventional CAR T cells. CD26 expression significantly enhanced CAR T-cell invasiveness in transwell assays and promoted tumor infiltration in vivo in both tumor models. ADA1 expression enhanced CAR T-cell persistence and was associated with increased frequencies of memory and stem-like T-cell populations in vivo. Notably, although IL15-GPC3-CAR T-cell treatment increased antitumor activity, it also caused high mortality and systemic inflammation in Huh7 tumor-bearing mice, whereas incorporation of ADA1 and CD26 into MRCAR T cells significantly improved survival and reduced serum levels of IL-6, IFN-γ, and TNF-α. Mechanistically, conventional IL-15–expressing GPC3-CAR T cells induced proinflammatory M1-like macrophage activation, whereas MRCAR T cells markedly suppressed this inflammatory response. Conclusions: Collectively, these results indicate that metabolic rewiring via ADA1 and CD26 enhances CAR T-cell expansion, tumor infiltration, persistence, and safety, supporting the MRCAR platform as a promising strategy to improve both efficacy and tolerability of CAR T-cell therapy for solid tumors.