Fc-fused IL-2(C125S) improves MSLN-targeted CAR-T cell persistence, metabolic fitness, and antitumor activity

Mesothelin (MSLN)-targeted chimeric antigen receptor T-cell (CAR-T) therapy is a promising strategy for pancreatic ductal adenocarcinoma (PDAC), but its efficacy is limited by insufficient persistence, progressive exhaustion, and metabolic dysfunction within the tumor microenvironment. We investigated whether CAR-T-cell-derived Fc-fused IL-2(C125S) could enhance the functional persistence, metabolic fitness, and antitumor activity of MSLN-targeted CAR-T cells. Second-generation MSLN CAR-T cells were engineered to secrete an IgG1 Fc-fused IL-2(C125S) variant (MSLN-IL2m CAR-T cells). CAR-T cells generated from five independent healthy donors were evaluated using PDAC cell lines with different levels of MSLN expression, serial antigen-stimulation assays, suppressive-stress models, phenotypic and functional analyses, and metabolic-flux assays. The contribution of IL-2Rα–STAT5 signaling was assessed using pharmacological STAT5 inhibition and IL-2Rα blockade. Antitumor efficacy and tolerability were further evaluated in an AsPC-1 xenograft model. MSLN-IL2m CAR-T cells showed enhanced expansion and STAT5 activation while maintaining comparable CAR expression and viability. Compared with conventional MSLN CAR-T cells, they exhibited stronger antigen-dependent cytotoxicity, proliferation, cytokine production, and functional persistence during repeated antigen stimulation, together with reduced expression of exhaustion-associated markers and preservation of memory-associated phenotypes. Under PDAC-associated suppressive conditions, MSLN-IL2m CAR-T cells maintained greater mitochondrial membrane potential, spare respiratory capacity, ATP production, and expression of PPARGC1A and CPT1A, while displaying a lower mitochondrial superoxide-associated signal. STAT5 inhibition or IL-2Rα blockade partially attenuated these functional, phenotypic, and metabolic advantages, supporting an important but non-exclusive contribution of IL-2Rα–STAT5 signaling. In vivo, MSLN-IL2m CAR-T cells showed greater intratumoral accumulation, improved tumor control, reduced bioluminescent tumor burden, and prolonged survival compared with conventional MSLN CAR-T cells, without major abnormalities in the evaluated hepatic, renal, hematologic, or body-weight parameters. CAR-encoded Fc-fused IL-2(C125S) enhanced the persistence, metabolic fitness, and antitumor activity of MSLN-targeted CAR-T cells in preclinical PDAC models. These findings provide proof-of-concept support for localized cytokine delivery as a strategy to improve CAR-T-cell efficacy in solid tumors, while further validation in clinically relevant models and comprehensive safety assessment are required.