Abstract 2829: Afatinib exerts an inhibitory effect on T cell-mediated cytotoxicity

Abstract Immune checkpoint blockade (ICB) has become a cornerstone of cancer therapy by enhancing antitumor immunity, and combinations of targeted therapies with ICB are widely evaluated across malignancies. However, the biological determinants that predict synergistic or antagonistic effects between targeted agents and ICB remain poorly understood. To identify agents that modulate T-cell effector function, we performed an in vitro inhibitor library screening using WT1-specific cytotoxic T cells (CTLs) generated from induced pluripotent stem cells (iPSCs) (Maeda T et al, Cancer Res. 2016). WT1-specific CTLs were co-cultured with HLA class I matched cancer cells in the presence of exogenous WT1 peptide. In this screening, immunosuppressive agents such as JAK inhibitors (ruxolitinib and tofacitinib) and src inhibitor dasatinib markedly suppressed T cell-mediated killing as expected. We newly identified afatinib, a second-generation EGFR tyrosine kinase inhibitor, as a potent suppressor of T-cell effector function. Afatinib significantly reduced interferon-γ (IFN-γ) secretion and T-cell activation. Notably, this IFN-γ reduction was independent of T-cell proliferation. RNA-seq analysis revealed that afatinib downregulated the T-cell receptor (TCR) pathway signature. RT-qPCR demonstrated a dose-dependent suppression of IFNG mRNA expression in afatinib-treated T cells. Furthermore, afatinib impaired tumor rejection in an immunological memory mouse model which mice had been previously cured with anti-PD-L1 therapy, demonstrating functional suppression of T cell mediated antitumor immunity. Collectively, these findings identify afatinib as a negative regulator of T cell effector function and raise the possibility that EGFR-TKI-induced immunosuppression in T cells may contribute to the limited clinical efficacy of EGFR-TKI plus ICI combinations observed in EGFR-mutant NSCLC. Our screen platform provides a useful framework for predicting drug-ICI interactions and guiding rational combination strategies. Citation Format: Masaru Yokomura, Seiji Nagano, Hiroshi Kawamoto, Takahiro Asakage, Ryohei Katayama. Afatinib exerts an inhibitory effect on T cell-mediated cytotoxicity abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2829.