Abstract 4065: Entinostat synergizes with the IL-15 superagonist N-803 and anti-PD-1 to engage cDC1 and stem-like TCF-1+ CD8+ T and NK cells to overcome colorectal tumors refractory to anti-PD-1 therapy

Abstract Background: Immune checkpoint blockade (ICB) has limited efficacy in colorectal carcinoma (CRC), where ~85% of patients harbor microsatellite stability (MSS). A minor subset of CRC patients (~15%) harbor microsatellite instability (MSI) and demonstrate improved responses to ICB ranging from 30-50%. Combination therapy has the potential to increase the response rate. Entinostat, a class I HDAC inhibitor, has demonstrated potential to enhance ICB efficacy, such as αPD-1 and immunostimulatory cytokines. N-803, a heterodimeric IL-15 superagonist that enhances proliferation and activation of CD8+ and NK cells has demonstrated to enhance the clinical benefit of Nivolumab in non-small cell lung cancer patients that were relapsed or refractory to αPD-1 monotherapy. Here, we investigated the antitumor efficacy and mode of action of combination therapy comprising αPD1, Entinostat, and N-803 in αPD1-refractory CT26 and Jak1-deleted MC38 CRC tumor models. Methods: Entinostat, N-803, and αPD1 alone or in combination were administered to mice bearing established CT26 or Jak1-deleted MC38 tumors. Antitumor efficacy, survival, and protective memory upon tumor rechallenge were monitored. Comprehensive flow cytometric profiling of the tumor microenvironment (TME), tumor-draining lymph node (tdLN), and spleen cell populations was conducted. CT26 tumor and tdLN immunomes were examined by single-cell transcriptomics. Cytokines and chemokines were examined in the TME and sera. Antigen-specific IFNg T cell responses were evaluated by ELISpot. Results: We demonstrated that combination therapy with Entinostat, N-803 and αPD1 elicits potent suppression of CT26 and Jak1-deleted MC38 tumors, with median overall survival increasing 33% to not reached. These effects were associated with increased activation, infiltration, cytokine production, and cytolytic function of tumor CD8+ T and NK cells, and increased sera IFNg and TNFα. Tumor resolution and protective memory were associated with development of p15e-specific T cell responses. Increased migration of CD8+ T cells and cross-presenting dendritic cells (cDC1) to the tdLN, as well as significant enrichment of cross-presentation genes in tumor cDC1s was observed. Transcriptomic and phenotypic analyses demonstrated increased stem-like TCF-1+ CD8+ T cell accumulation in the tdLNs and TME of mice treated with triple combination. Conclusions: Combination therapy with Entinostat plus N-803 and αPD1 synergized to promote a sustained antitumor response and prolonged survival. We hypothesize the antitumor effects were due to collaborative efforts of CD8+ T and NK cells, and cDC1 cells in tdLN and TME. These results provide a rationale for the clinical testing of αPD1, Entinostat and N-803 combination for patients with CRC, including those with acquired resistance to ICB. Citation Format: Christine M. Minnar, Masaya Miyamoto, Asma S. Khelifa, Grace Lui, Katherine L. Lothstein, Paul L. Chariou, Thomas J. Meyer, Alexei Lobanov, Margaret Cam, Jeffrey Schlom, Sofia R. Gameiro. Entinostat synergizes with the IL-15 superagonist N-803 and anti-PD-1 to engage cDC1 and stem-like TCF-1+ CD8+ T and NK cells to overcome colorectal tumors refractory to anti-PD-1 therapy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 4065.