Triple-Mutated HSV-1 Expressing Soluble B7-1 Plus CTLA-4 Blockade Suppresses Lymph Node Metastasis in Tongue Cancer

Cervical lymph node metastasis is a major predictor of poor prognosis in patients with oral squamous cell carcinoma (OSCC), underscoring the need for therapies that effectively target metastatic spread. We previously reported that G47Δ, a third-generation oncolytic herpes simplex virus type 1 (HSV-1), rapidly traffics from tongue tumors to cervical lymph nodes and suppresses metastasis in murine models. Based on this lymphotropic property and the immunological significance of CD28-B7-1 interactions for T-cell activation, we pursue the potential of T-mB7-1, a new G47Δ-derived oncolytic HSV-1 expressing soluble murine B7-1. T-mB7-1 secreted soluble B7-1 in vitro without compromising cytopathic activity. In vivo, intratumoral T-mB7-1 suppressed tumor growth in the resistant KLN205-MUC1 subcutaneous model and prolonged survival in SCCVII orthotopic tongue tumors. In the KLN205-MUC1 orthotopic tongue cancer that exhibits rapid lymphatic metastasis, a single dose of T-mB7-1, but not the control virus T-01, significantly reduced cervical lymph node metastasis and improved survival. Repeated low-dose T-mB7-1 further caused near-complete suppression of lymph node metastasis and markedly extended survival. Flow cytometry showed both T-01 and T-mB7-1 induced CD69⁺ T-cell activation in cervical lymph nodes at 18 h, which was reduced with T-mB7-1 at 24 h. Importantly, combining T-mB7-1 with systemic CTLA-4 blockade markedly prolonged survival of mice harboring orthotopic KLN205-MUC1 tumors, with all treated mice surviving beyond 80 days in the otherwise fatal model. These findings demonstrate that soluble B7-1-expressing oncolytic HSV-1 combined with CTLA-4 blockade durably suppresses lymph node metastasis, providing a promising and minimally invasive strategy for regionally metastatic OSCC.