A56-anchored IL-15Rα arms a fusogenic oncolytic vaccinia virus to reprogram the tumor microenvironment and enhance antitumor immunity

Oncolytic viruses (OVs) represent a promising class of cancer immunotherapies that selectively infect and lyse tumor cells while stimulating antitumor immune responses. Vaccinia virus (VV) is an attractive OV platform that can be engineered to deliver immunomodulatory molecules to the tumor microenvironment (TME). Interleukin-15 (IL-15) is a potent cytokine that promotes the activation and persistence of cytotoxic lymphocytes through trans-presentation by its high-affinity receptor, IL-15Rα. To enhance local IL-15 trans-presentation within tumors, we engineered a novel fusogenic oncolytic VV (IL15Rα-VV) expressing IL-15 and membrane-anchored IL-15Rα by replacing the extracellular domain of the VV fusion regulatory protein A56 with the extracellular domain of IL-15Rα. This strategy was designed to promote localized IL-15 trans-presentation while enhancing fusogenic oncolysis. In vitro, IL15Rα-VV induced surface expression of IL-15Rα on infected tumor cells, enhanced cytotoxicity and cell fusion, and maintained replication kinetics. In bilateral subcutaneous pancreatic tumor models, intratumoral administration of IL15Rα-VV significantly suppressed the growth of both injected and non-injected tumors compared with parental VV and IL-15-secreting VV. Mechanistically, IL15Rα-VV remodeled the TME by increasing infiltration of natural killer cells and activated CD8 + T-cells while reducing regulatory T-cells and M2-like macrophages. In addition, activated, effector, central memory, and progenitor exhausted (Tpex) CD8 + T-cells, as well as dendritic cells, were expanded in tumor-draining lymph nodes. Despite increased PD-L1 expression within tumors, IL15Rα-VV reduced the frequency of exhausted CD8 + T-cells and exhibited enhanced antitumor efficacy when combined with PD-L1 blockade. Collectively, these findings demonstrate that A56-mediated surface display of IL-15Rα represents a novel strategy to couple fusogenic oncolysis with localized IL-15 trans-presentation, thereby amplifying OV-mediated antitumor immunity.