Abstract
<title>Abstract</title> <p>Background/Objectives: Esophageal adenocarcinoma (EAC) is a highly lethal malignancy with limited response to immune checkpoint blockade (ICB) despite its benefits in select patients. Oncolytic viruses (OVs) offer a promising immunotherapy platform by inducing tumor lysis and stimulating anti-tumor immunity. We previously demonstrated that a genetically engineered oncolytic vaccinia virus expressing GPI-anchored, membrane-bound interleukin-2 (vvDD-IL-2-RG) safely reprogrammed the cancer-immune set point and showed synergistic effects with PD-1/PD-L1 blockade in transplantable syngeneic models. The objective of this study was to evaluate vvDD-IL-2-RG in combination with PD-1 pathway inhibition in a clinically relevant de novo EAC model. Methods Viral infectivity, tumor selectivity, and IL-2 bioactivity of vvDD-IL-2-RG were validated in vitro using human EAC cell lines. Anti-tumor efficacy was assessed in vivo using the modified Levrat rat model, which recapitulates reflux-induced EAC in immunocompetent animals. Animals were treated with vvDD-IL-2-RG, a PD-1 inhibitor, or a combination, via oral gavage or tail vein injection. Tumor burden was monitored by serial MRI, and tumor microenvironment analysis was performed to assess immune cell infiltration and gene expression. Results In vitro, vvDD-IL-2-RG demonstrated selective tumor replication and robust membrane-bound IL-2 expression while preserving viral cytotoxicity. In vivo, combination therapy significantly reduced tumor burden compared to monotherapy or untreated controls, with serial MRI confirming substantial tumor regression. Tumor microenvironment analysis revealed increased CD4 + and CD8 + T-cell infiltration, indicating enhanced anti-tumor immune responses, particularly with localized transoral delivery. These findings support localized IL-2-expressing oncolytic virus delivery as a promising strategy. Conclusions Localized delivery of the IL-2-expressing oncolytic vaccinia virus (vvDD-IL-2-RG) significantly enhances PD-1 pathway inhibition in a de novo EAC model, leading to substantial tumor control, increased intratumoral T-cell infiltration, and improved tumor control. This approach represents a promising strategy to overcome resistance to immunotherapy in EAC and warrants further investigation for clinical translation.</p>