Oncolytic virotherapy is a promising therapeutic approach for the treatment of cancer. TG6002 is a recombinant oncolytic vaccinia virus deleted in the thymidine kinase and ribonucleotide reductase genes and armed with the suicide gene FCU1, which encodes a bifunctional chimeric protein that efficiently catalyzes the direct conversion of the nontoxic 5-fluorocytosine into the toxic metabolite 5-fluorouracil. In translational research, canine tumors and especially mammary cancers are relevant surrogates for human cancers and can be used as preclinical models. Here, we report that TG6002 is able to replicate in canine tumor cell lines and is oncolytic in such cells cultured in 2D or 3D as well as canine mammary tumor explants. Furthermore, intratumoral injections of TG6002 lead to inhibition of the proliferation of canine tumor cells grafted into mice. 5-fluorocytosine treatment of mice significantly improves the anti-tumoral activity of TG6002 infection, a finding that can be correlated with its conversion into 5-fluorouracil within infected fresh canine tumor biopsies. In conclusion, our study suggests that TG6002 associated with 5-fluorocytosine is a promising therapy for human and canine cancers.

As a live biologic agent, oncolytic vaccinia virus has the ability to target and selectively amplify at tumor sites. We have previously reported that deletion of thymidine kinase and ribonucleotide reductase genes in vaccinia virus can increase the safety and efficacy of the virus. Here, to allow direct visualization of the viral genome in living cells, we incorporated the ANCH target sequence and the OR3-Santaka gene in the double-deleted vaccinia virus. Infection of human tumor cells with ANCHOR3-tagged vaccinia virus enables visualization and quantification of viral genome dynamics in living cells. The results show that the ANCHOR technology permits the measurement of the oncolytic potential of the double deleted vaccinia virus. Quantitative analysis of infection kinetics and of viral DNA replication allow rapid and efficient identification of inhibitors and activators of oncolytic activity. Our results highlight the potential application of the ANCHOR technology to track vaccinia virus and virtually any kind of poxvirus in living cells.

Purpose: Effective treatment for patients with metastatic cancer is limited, particularly for those with colorectal cancer with metastatic liver lesions, in which accessibility to numerous tumors is essential for favorable clinical outcomes. Oncolytic viruses (OV) selectively replicate in cancer cells; however, direct targeting of inaccessible lesions is limited when using conventional intravenous or intratumoral administration routes. Patients and Methods: We conducted a multicenter, dose-escalation, phase I study of vaccinia virus, TG6002, via intrahepatic artery (IHA) delivery in combination with the oral prodrug 5-fluorocytosine to 15 patients with metastatic colorectal cancer. Results: Successful IHA delivery of replication-competent TG6002 was achieved, as demonstrated by the virus within tumor biopsies. Functional transcription of the FCU1 transgene indicates viral replication within the tumor, with higher plasma 5-fluorouracil associated with patients receiving the highest dose of TG6002. IHA delivery of TG6002 correlated with a robust systemic peripheral immune response to the virus with activation of peripheral blood mononuclear cells, associated with a proinflammatory cytokine response and release of calreticulin, potentially indicating immunogenic cell death. Gene Ontology analyses of differentially expressed genes reveal a significant immune response at the transcriptional level in response to treatment. Moreover, an increase in the number and frequency of T-cell receptor clones against both cancer antigens and neoantigens, with elevated functional activity, may be associated with improved anticancer activity. Despite these findings, no clinical efficacy was observed. Conclusions: In summary, these data demonstrate the delivery of OV to tumor via IHA administration, associated with viral replication and significant peripheral immune activation. Collectively, the data support the need for future studies using IHA administration of OVs.