Cell-cell fusion and immunostimulatory cytokines significantly impact oncolytic vaccinia virus immunotherapeutic potential.

Oncolytic viruses are therapeutic agents that induce local oncolysis and systemic antitumor immunity. The fusogenic oncolytic vaccinia virus (FUVAC) exhibits strong immunotherapeutic potential via tumor immune microenvironment remodeling following fusion-enhanced oncolysis. However, relying on systemic antitumor immunity to treat distant (non-virally injected) tumors, such as metastases, is insufficient owing to the lack of direct viral oncolysis. To improve the systemic antitumor immune response, we engineered an FUVAC with two immunostimulatory cytokines, interleukin (IL)-12 and C-C motif chemokine ligand 21 (CCL21). FUVAC-IL-12/CCL21 increased distant tumor regression compared to that by a non-armed FUVAC or cytokine-armed non-fusogenic virus and achieved a 72% complete response rate with a single unilateral injection in a bilateral murine tumor model. Single-cell transcriptome analysis revealed that FUVAC-IL-12/CCL21 increased the number of effector-memory CD8+ T cells and decreased the number of exhausted CD8+ T cells in virus-untreated tumors. Cell-cell fusion stimulation by FUVAC-IL-12/CCL21 increased T cell receptor diversity on infiltrating effector-memory CD8+ T cells. The conversion of immune cold into hot tumors by FUVAC-IL-12/CCL21 enhanced the response to programmed cell death protein-1 blockade. The novel dual cytokine-armed FUVAC systemically stimulates the tumor immune microenvironment through cell-cell fusion and cytokine activation, potentially overcoming virotherapy resistance in metastatic tumors.