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.

Objectives: Despite advancements in molecular-targeted therapies and immune checkpoint inhibitors, the survival rate of patients with advanced lung cancer remains unsatisfactory. Therefore, new and effective treatment strategies are urgently needed. Both mesenchymal-epithelial transition (MET) inhibitors and oncolytic viruses exhibit immunomodulatory properties along with direct antitumor effects. Materials and Methods: The antitumor effects of a combination therapy using MDRVV, a modified vaccinia virus for oncolytic virus therapy, and tepotinib, a MET inhibitor, were evaluated in vitro and in vivo using lung cancer models. Results: The combination therapy demonstrated additive cytotoxic effects on various lung cancer cell lines in vitro and significantly suppressed tumor growth in an immunocompetent mouse model. MDRVV triggered immunogenic cell death, evidenced by the release of adenosine triphosphate (ATP) and high-mobility group box-1 (HMGB-1). Additionally, the combination therapy enhanced CD4+ and CD+ T-cell infiltration more effectively than either agent alone. MDRVV exhibited antitumor effects not only in the inoculated tumors but also in distant tumors, with the most pronounced effect observed when combined with tepotinib. Conclusions: These findings suggest that combining a MET inhibitor with oncolytic vaccinia virus represents a promising and effective strategy for improving lung cancer treatment by targeting both tumor cells and the tumor microenvironment.

PDF file - 43K, Inhibition of CAR expression of A549 cells by siRNA transduction.

PDF file - 415K, In vivo toxicological assays including histological and serum biochemical analysis of mice administered with CVB3.

Supplementary Figure 2 from Tumor and Vascular Targeting of a Novel Oncolytic Measles Virus Retargeted against the Urokinase Receptor

PDF file - 96K, Remarkable CVB3 induced suppression of H1299 human tumor growth in mice.

Oncolytic viruses have two anticancer functions: direct oncolysis and elicitation of antitumor immunity. We previously developed a novel fusogenic oncolytic vaccinia virus (FUVAC) from a non-fusogenic vaccinia virus (VV) and, by remodeling the tumor immune microenvironment, we demonstrated that FUVAC induced stronger oncolysis and antitumor immune responses compared with non-fusogenic VV. These functions depend strongly on cell-cell fusion induction. However, FUVAC tends to have decreased fusion activity in cells with low virus replication efficacy. Therefore, another combination strategy was required to increase cell-cell fusion in these cells. Histone deacetylase (HDAC) inhibitors suppress the host virus defense response and promote viral replication. Therefore, in this study, we selected an HDAC inhibitor, trichostatin A (TSA), as the combination agent for FUVAC to enhance its fusion-based antitumor potential. TSA was added prior to FUVAC treatment of murine tumor B16-F10 and CT26 cells. TSA increased the replication of both FUVAC and parental non-fusogenic VV. Moreover, TSA enhanced cell-cell fusion and FUVAC cytotoxicity in these tumor cells in a dose-dependent manner. Transcriptome analysis revealed that TSA-treated tumors showed altered expression of cellular component-related genes, which may affect fusion tolerance. In a bilateral tumor-bearing mouse model, combination treatment of TSA and FUVAC significantly prolonged mouse survival compared with either treatment alone or in combination with non-fusogenic VV. Our findings demonstrate that TSA is a potent enhancer of cell-cell fusion efficacy of FUVAC.

Supplementary Figure Legend from Retargeted Oncolytic Measles Strains Entering via the EGFRvIII Receptor Maintain Significant Antitumor Activity against Gliomas with Increased Tumor Specificity

Supplementary Table, and Figure Legends 1-3 from Tumor and Vascular Targeting of a Novel Oncolytic Measles Virus Retargeted against the Urokinase Receptor

PDF file - 62K, In vitro large-scale screening of 28 enteroviruses for promising oncolytic virus candidates (MOI = 0.01, 0.1).

PDF file - 120K, In vitro and in vivo oncolytic effects of CVB3 against TC-1 mouse lung cancer cells.

Supplementary Figure 1 from Tumor and Vascular Targeting of a Novel Oncolytic Measles Virus Retargeted against the Urokinase Receptor