716 TOMM20 overexpression enhances T-cell mitochondrial fitness and improves adoptive cell therapy in sarcoma

Background Mitochondrial metabolism plays a context-dependent role in cancer and anticancer immunity. In cancer cells, high oxidative phosphorylation (OXPHOS) is associated with immune evasion, therapy resistance, and tumor progression. Our cancer-cell studies identify Translocase of Outer Mitochondrial Membrane 20 (TOMM20) as a driver of sarcoma aggressiveness by enhancing OXPHOS, increasing complex I abundance, maintaining a reduced redox state, reducing reactive oxygen species, and promoting resistance to apoptosis and tumor growth. Although TOMM20-driven mitochondrial fitness supports tumor progression in cancer cells, the same metabolic program may benefit therapeutic T cells, where durable antitumor function requires OXPHOS, metabolic flexibility, persistence, and resistance to exhaustion. We hypothesized that TOMM20 overexpression enhances T-cell OXPHOS capacity and metabolic resilience under suppressive sarcoma conditions.Methods Syngeneic murine CD3 + T cells were activated with anti-CD3/CD28 beads and retrovirally transduced with TOMM20 or control vector. Oxygen consumption rate (OCR) and substrate utilization were assessed by Seahorse extracellular flux analysis under glucose, glutamine, and lactate conditions. Complex I, BCL-2, and MYC expression were evaluated by Western blotting. Exhaustion and effector function were analyzed by flow cytometry using PD-1, LAG-3, TIGIT, IFN-γ, TNF-α, Granzyme B, and perforin. Cytotoxicity was measured using a flow-based assay with CellTrace Violet-labeled MCA-205-hgp100 sarcoma cells. In vivo antitumor activity and persistence were evaluated in a syngeneic MCA-205-hgp100 fibrosarcoma adoptive cell transfer model.Results TOMM20-overexpressing CD3 + T cells showed increased OCR across glucose, glutamine, and lactate conditions, indicating enhanced mitochondrial respiration and substrate flexibility. TOMM20 upregulated the expression of Complex I, BCL-2, and MYC, supporting improved mitochondrial fitness, survival capacity, and metabolic programming. TOMM20-overexpressing T cells demonstrated reduced inhibitory/exhaustion phenotypes, including approximately 46% lower PD-1+ and 44% lower TIGIT+ CD3+ T-cell frequencies, with reduced LAG-3 expression. These cells also decreased effector/cytotoxic markers, including IFN-γ and Granzyme B, and mediated approximately 1.9-fold higher killing of fibrosarcoma cells. In vivo, TOMM20-overexpressing CD3+ T cells improved tumor control, with approximately 75% lower tumor volume, 33% tumor-free mice at day 28 versus 0% in controls, and extended median survival from 8 to 19 days (p = 0.0006).Conclusions TOMM20 overexpression promotes CD3 + T-cell mitochondrial respiratory fitness, effector function, and antitumor activity following adoptive transfer while reducing exhaustion marker expression. These data support TOMM20 engineering as a potential strategy to improve adoptive T-cell therapy for sarcoma.