Tempered signal strength via low-dose MEK inhibition optimizes therapeutic performance of engineered T cells

Background Optimizing T cell activation strength is emerging as a critical factor in improving adoptive cellular therapy (ACT). We previously reported that neoantigen-specific T cell receptor (TCR) clonotypes from a patient with metastatic melanoma exhibited enhanced resilience to repeated stimulation when initially activated at moderate levels. Methods Building on these observations, we applied transient, low-dose MEK inhibition (MEKi) to fine-tune T cell signal strength during early activation. We evaluated this combinatorial strategy in vitro using co-cultures of CD8+ T cells engineered with patient-derived neoantigen-specific TCRs, alongside chimeric antigen receptor-T cells, bispecific T cell engagers, and non-engineered tumor-infiltrating lymphocytes (TILs). In vivo efficacy was evaluated in a xenograft model with intravenous TCR-T cell transfer and systemic low-dose MEKi. Results MEKi co-treatment induced a more tempered activation profile that enhanced T cell proliferation, fitness, and persistence under strong stimulation. These effects were consistent across various in vitro and in vivo models for engineered T cells as well as primary melanoma-derived TILs. MEKi dampened the pro-inflammatory T cell activation profile, most notably diminishing tumor necrosis factor (TNF) secretion, mechanistically driven by coordinated and selective disruption of the key transcriptional regulators nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and nuclear factor of activated T cells (NFAT) while partly preserving activator protein 1 (AP-1) activity. Conclusion These findings highlight moderate activation as a critical determinant of engineered T cell long-term performance. Low-dose MEKi offers a therapeutic tool for fine-tuning T cell activation and enhancing ACT efficacy.