Entinostat Enhances Antigen-Specific CD8 T-Cell Response to Immunotherapies in Lung Cancer Models

Background: Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Although immune checkpoint inhibitors (ICIs) have significantly improved clinical outcomes, most patients do not respond to treatment or fail to achieve durable responses. Histone deacetylase inhibitors (HDACi) have emerged as promising immunomodulatory agents, with the potential to sensitize tumors to ICIs. We investigated the immunomodulatory effects of entinostat, a class I HDACi, in combination with dual ICI (anti-PD-1 and anti-CTLA-4) as well as with T-cell receptor (TCR) engineered T cells in preclinical NSCLC models. Methods: We employed human NSCLC cell lines and the immunogenic KRASG12D/p53-mutant KPN1.1 murine NSCLC cell line. In vitro, we assessed entinostat-induced changes in MHC class I and PD-L1 expression. In addition, we evaluated the effects of entinostat on a KRASG12D-specific TCR. In vivo, therapeutic efficacy and immune modulation were assessed by transplanting KPN1.1 cells subcutaneously and orthotopically into immunocompetent mice, followed by treatment with dual ICI, with or without entinostat. Immune populations in the spleen and blood were subsequently analyzed. Results: In vitro, entinostat induced the upregulation of MHC-I and PD-L1 expression in both human and murine NSCLC cell lines. In addition, entinostat treatment significantly enhanced antigen-specific tumor recognition and killing by T cells engineered to express a KRASG12D-specific TCR. In vivo, the addition of entinostat to dual immune checkpoint inhibition showed an incremental trend toward improved tumor growth control. Notably, entinostat plus dual ICI enhanced systemic immune activation, increasing circulating and splenic T-cell populations and significantly expanding both antigen-specific and overall effector CD8⁺ T cells. Consistently, the ex vivo co-culture of splenocytes from KPN1.1-bearing mice with KPN1.1 tumor cells demonstrated enhanced CD8⁺ antigen-specific T-cell recognition. Conclusions: In human and murine NSCLC models, entinostat potentiates TCR- and ICI-mediated tumor recognition through tumor-intrinsic and systemic immune modulation. These effects were reflected by increased MHC-I expression, expansion of antigen-specific effector CD8⁺ T cells, and enhanced CD8⁺ T-cell tumor recognition. These findings support a further evaluation of entinostat as a strategy to improve immunotherapy efficacy in NSCLC.