TIM-3 blockade reverses oncolytic vaccinia virus-induced DCs inactivation and T cells exhaustion to improve antitumor immunity and therapeutic efficacy.

Oncolytic vaccinia viruses (OVV) demonstrate the capacity to inflame the tumor microenvironment (TME) and elicit infiltrating tumor-specific T cells responses. However, OVV treatment paradoxically alters the cancer-immune equilibrium within tumors, attenuating anti-tumor immunity and necessitating a deeper understanding of the viral-induced immune landscape to optimize therapeutic potential. This study investigates the impact of single-domain antibody-armed OVs targeting TIM-3 on TME remodeling, aiming to overcome localized immunosuppression and enhance tumor responsiveness to immunotherapeutic interventions. We engineered a tumor-selective OVV vector encoding single-domain antibodies against murine (mNbTIM3) or human (hNbTIM3) TIM-3. The therapeutic efficacy of OVV-mNbTIM3 was evaluated across multiple murine cancer models, demonstrating that localized VHHTIM3 delivery via OVV-mNbTIM3 promotes systemic anti-tumor immunity in established cancers. Mechanistically, OVV-mNbTIM3 treatment enhances DCs maturation and tumor-specific CD8+ T cells activation. Notably, intratumoral expression of hNbTIM3 also conferred therapeutic benefit in humanized mice bearing patient-derived cancer xenografts. These findings provide mechanistic insights for enhancing OVV combination therapies and inform the rational design of next-generation oncolytic viruses with engineered immunomodulatory properties.