Associated antitumor effects of oncoytic vaccinia virus expressing a tethered IL-12/IL-2 fusion cytokine.

e14557 Background: Cancer immunotherapy has revolutionized cancer treatment, but many solid tumors remain non-inflamed and resistant to current approaches. This necessitates strategies to convert "cold" tumors into "hot" (immune-inflamed) ones. Oncolytic viruses (OVs), especially those engineered to deliver immunostimulatory molecules like cytokines, offer a promising solution. Cytokines, however, face limitations such as short half-lives, high dose requirements, and systemic toxicities in clinical use. Our previous work showed that intratumoral delivery of tethered IL-2 or IL-12 via oncolytic vaccinia viruses (oVV) achieved strong antitumor effects without systemic toxicity. This study hypothesized that an oVV expressing a tethered fusion cytokine of IL-12 and IL-2 could achieve superior antitumor effects compared to single-cytokine viruses. Methods: We engineered two oncolytic vaccinia viruses: one expressing a single-chain IL-12 (vvDD-scIL-12-FG) and another expressing a tethered fusion cytokine of IL-12 and IL-2 (vvDD-scIL-12-R-IL-2-RG) via homologous recombination. Transgene expression was confirmed by RT-qPCR, and membrane association was validated by flow cytometry. We evaluated the antitumor efficacy across multiple murine tumor models and investigated the underlying mechanisms of action using RT-qPCR and flow cytometry. Results: Our data confirm successful expression and membrane tethering of the IL-12/IL-2 fusion cytokine without impairing viral replication or cytotoxicity. Treatment with vvDD-scIL-12-R-IL-2-RG demonstrated robust antitumor activity in Lewis lung carcinoma, renal adenocarcinoma Renca, and murine colon adenocarcinoma MC38 models. Notably, in a late-stage MC38 colon cancer model, 85% of mice achieved complete tumor regression without systemic toxicity. Critically, vvDD-scIL-12-R-IL-2-RG outperformed viruses expressing tethered IL-2 or IL-12 alone. Mice cured by vvDD-scIL-12-R-IL-2-RG resisted rechallenge with MC38 cells but not unrelated B16 melanoma cells, indicating durable, tumor-specific immunity. Mechanistically, vvDD-scIL-12-R-IL-2-RG treatment elevated intratumoral Th1 chemokines and IFN-γ, alongside increased infiltration of IFN-γ⁺ CD8⁺ T cells. Peripheral lymphocyte depletion did not significantly attenuate these superior antitumor effects, suggesting reliance on pre-existing tumor-infiltrating lymphocytes. These changes effectively converted non-inflamed tumors into immune-inflamed ones, driving superior responses. Conclusions: Our findings indicate that an oVV-delivered, tethered IL-12/IL-2 fusion cytokine is both safe and effective, making it a promising candidate for clinical translation in cancer immunotherapy.