Abstract Background: Clinical resistance to immune checkpoint blockade (ICB) is prevalent across solid malignancies, thus prompting the need for novel therapies. Interleukin-12 (IL-12) is an important cytokine for cancer immunotherapy due to its ability to bridge innate and adaptive immunity. However, the narrow therapeutic index and toxicity associated with systemic IL-12 administration has hampered its clinical development. ANK-101 is an alum-anchored human IL-12 providing tumor retention upon intra-tumoral delivery. In this study, we investigated the anti-tumor activity and mechanism of action of murine ANK-101 (mANK-101) delivered intra-tumorally in combination with the class I histone deacetylase (HDAC) inhibitor entinostat, in different ICB-refractory murine tumor models, including CT26 (colorectal) and MOC-1 (HPV16neg head and neck). Methods: Mice receiving Entinostat diet and/or mANK-101 were monitored for anti-tumor activity, survival and protective memory. The contribution of CD8+, CD4+, and NK lymphocytes to antitumor effects were investigated via immune depletion. Analysis of tumor-specific T cell responses and comprehensive proteomic, transcriptomic, and tumor architecture analysis of the immunome was performed in MOC-1 tumors, tumor-draining lymph node (tdLN), and spleen. In addition, functional analysis of CD8+T cells in the periphery were performed. Results: We demonstrate that mANK-101 synergizes with Entinostat to suppress CT26 (colon, Kras G12Dmut) and MOC-1 (oral, HPV16neg) tumors, resulting in significant anti-tumor effects, survival benefit, and protective memory. Immune depletion studies demonstrated a role for CD8+ T cells, CD4+ T cells, and NK cells in the anti-tumor activity elicited by combination therapy. Mechanistically, combination therapy elicited significant activation of peripheral CD8+ T cells and NK lymphocytes, increased CD8+ T effector memory, tumor-specific T cell responses, and multifunctional IFNγ+TNFα+CD8+ T cells, with concomitant decrease in CD4+ Tregs and increased CD8/Treg ratio. Notably, spatial analysis of tumor architecture indicates the development of B-cell rich stemness hubs exclusive to combination therapy. Ongoing in-depth studies including single cell transcriptomics in the tumor and tDLN, and investigation of the role of immune stemness hubs in the observed anti-tumor activity will allow for an in-depth understanding of the synergistic effect of mANK-101 with HDAC inhibition. Conclusion: Collectively, these findings provide a rationale for the combination of intratumoral delivery of anchored IL-12 with epigenetic modulation for patients with ICB-refractory solid tumors, such as colorectal and HPV16neg head and neck cancers. Citation Format: Asma S. Khelifa, Ainara Meler, Christine M. Minnar, Katherine E. Lothstein, Masaya Miyamoto, Nicholas Roller, Lisa K. Poppe, David Peeney, Sailaja Battula, Howard L. Kaufman, Jeffrey Schlom, Sofia R. Gameiro. Intratumoral IL-12 in combination with HDAC inhibition overcomes checkpoint-refractory tumors 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 1563.
Khelifa et al. (2026) studied this question.