Purpose: Early clinical trials are under way exploring the direct oncolytic potential of reovirus.This study addresses whether tumor infection by reovirus is also able to generate bystander, adaptive antitumor immunity. Experimental Design: Reovirus was delivered intravenously to C57BL/6 mice bearing lymph node metastases from the murine melanoma, B16-tk, with assessment of nodal metastatic clearance, priming of antitumor immunity against the tumor-associated antigen tyrosinase-related protein-2, and cytokine responses. In an in vitro human system, the effect of reovirus infection on the ability of Mel888 melanoma cells to activate and load dendritic cells for cytotoxic lymphocyte (CTL) priming was investigated. Results: In the murine model, a single intravenous dose of reovirus reduced metastatic lymph node burden and induced antitumor immunity (splenocyte response to tyrosinase-related protein-2 and interleukin-12 production in disaggregated lymph nodes). In vitro human assays revealed that uninfected Mel888 cells failed to induce dendritic cell maturation or support priming of an anti-Mel888 CTL response. In contrast, reovirus-infected Mel888 cells (reo-Mel) matured dendritic cells in a reovirus dose-dependent manner. When cultured with autologous peripheral blood lymphocytes, dendritic cells loaded with reo-Mel induced lymphocyte expansion, IFN-g production, specific anti-Mel888 cell cytotoxicity, and cross-primed CD8 + Tcells specific against the human tumor-associated antigen MART-1. Conclusion: Reovirus infection of tumor cells reduces metastatic disease burden and primes antitumor immunity. Future clinical trials should be designed to explore both direct cytotoxic and immunotherapeutic effects of reovirus.

Oncolytic viruses, which preferentially lyse cancer cells and stimulate an antitumor immune response, represent a promising approach to the treatment of cancer. However, how they evade the antiviral immune response and their selective delivery to, and replication in, tumor over normal tissue has not been investigated in humans. Here,we treated patients with a single cycle of intravenous reovirus before planned surgery to resect colorectal cancer metastases in the liver. Tracking the viral genome in the circulation showed that reovirus could be detected in plasma and blood mononuclear, granulocyte, and platelet cell compartments after infusion. Despite the presence of neutralizing antibodies before viral infusion in all patients, replication-competent reovirus that retained cytotoxicity was recovered from blood cells but not plasma, suggesting that transport by cells could protect virus for potential delivery to tumors. Analysis of surgical specimens demonstrated greater, preferential expression of reovirus protein in malignant cells compared to either tumor stroma or surrounding normal liver tissue. There was evidence of viral factories within tumor, and recovery of replicating virus from tumor (but not normal liver)was achieved in all four patients from whom fresh tissue was available. Hence, reovirus could be protected from neutralizing antibodies after systemic administration by immune cell carriage, which delivered reovirus to tumor.These findings suggest new preclinical and clinical scheduling and treatment combination strategies to enhance in vivo immune evasion and effective intravenous delivery of oncolytic viruses to patients in vivo.

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