Oncolytic vaccinia viruses are currently in clinical development. However, the safety and the tumor selectivity of these oncolytic viruses must be improved. We previously constructed a first-generation oncolytic vaccinia virus by expressing the suicide gene FCU1 inserted in the J2R locus that encodes thymidine kinase. We demonstrated that the combination of this thymidine-kinase-deleted vaccinia virus and the FCU1/5-fluocytosine system is a potent vector for cancer therapy. Here, we developed a second generation of vaccinia virus, named TG6002, expressing FCU1 and with targeted deletions of the J2R gene and the I4L gene, which encodes the large subunit of the ribonucleotide reductase. Compared to the previously used single thymidine-kinase-deleted vaccinia virus, TG6002 is highly attenuated in normal cells, yet it displays tumor-selective replication and tumor cell killing. TG6002 replication is highly dependent on cellular ribonucleotide reductase levels and is less pathogenic than the single-deleted vaccinia virus. Tumor-selective viral replication, prolonged therapeutic levels of 5-fluorouracil in tumors, and significant antitumor effects were observed in multiple human xenograft tumor models after systemic injection of TG6002 and 5-fluorocytosine. TG6002 displays a convincing safety profile and is a promising candidate for treatment of cancer in humans.

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.

Introduction: Oncolytic virotherapy with tumor selective viruses, such as Vaccinia viruses (VV), offers a promising treatment modality for cancer. TG6002 is a recombinant oncolytic VV deleted in two viral genes (thymidine kinase and ribonucleotide reductase) and armed with the suicide gene FCU1 that encodes a bifunctional chimeric protein which efficiently catalyses the direct conversion of the nontoxic 5-fluorocytosine (5-FC) into the toxic metabolite 5-fluorouracil (5-FU). Canine tumors are relevant predictive preclinical surrogates for human oncology. The first objective was to evaluate the susceptibility, the replication rate and the oncolytic potency of VV in canine tumor cell lines. The second objective was to evaluate oncolytic potency of TG6002 in xenograft model and fresh canine tumor biopsies. The third objective was to assess safety and viral shedding of TG6002 in healthy dogs. Materials and Methods: Transduction efficiency, replication and oncolytic potency of TG6002 were evaluated in vitro in a variety of different canine cancer cell lines. In vivo anti-tumor effect of TG6002 was examined in a canine tumor xenograft model. TG6002 was injected intratumorally or intravenously with or without 5-FC. Three canine mammary adenocarcinoma explants were infected with TG6002 in presence of 5-FC during 6 days. Oncolytic potency was assessed by histological exams. Concentrations of 5-FC and 5-FU were monitored. TG6002 was administered intramuscularly for 7 healthy dogs and intravenously for 4 healthy dogs. Clinical exams, complete blood count and biochemistry analysis were performed. Blood, saliva, urine, feces were collected for virus detection by qPCR and plaque assay. Results: Canine cell lines were highly susceptible to VV infection. A replication factor of 106 to 107 was determined 4 days after infection and a significant reduction of cell viability was noticed 5 days after infection. In xenograft model, intratumoral or intravenous injections of TG6002 with oral 5-FC induced a significant inhibition of tumor growth compared to control groups. In canine mammary adenocarcinoma biopsies, a lysis of 90% of tubular cells was observed on histological exams. Conversion of more than 50% of 5-FC to 5-FU was noticed. In healthy dogs, a good tolerance of intramuscular and intravenous injections of TG6002 without viral shedding was assessed. Conclusion: This study demonstrates that TG6002 is able to infect and replicate in canine tumor cell lines and is oncolytic in both cell lines, xenograft model and canine mammary adenocarcinoma samples. This study also confirms that TG6002 can be safely administered in dogs. These promising results support the use of TG6002 in a clinical trial for both human and canine species. This study emphasizes the importance of a One Health approach in oncology. Citation Format: Jérémy Béguin, Johann Foloppe, Eve Laloy, Virginie Nourtier, Isabelle Farine, Murielle Gantzer, Christelle Pichon, Sandrine Cochin, Pascale Cordier, Dominique Tierny, Jean Marc Balloul, Eric Quémeneur, Christelle Maurey, Bernard Klonjkowski, Philippe Erbs. Characterization, evaluation and safety studies of the oncolytic Vaccinia virus TG6002 for canine cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1446.

Oncolytic virus therapy has recently been recognized as a promising new therapeutic approach for cancer treatment. In this study, we are proposing for the first time to evaluate the in vitro and in vivo oncolytic capacities of the Cowpox virus (CPXV). To improve the tumor selectivity and oncolytic activity, we developed a thymidine kinase (TK)-deleted CPXV expressing the suicide gene FCU1, which converts the non-toxic prodrug 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) and 5-fluorouridine-5′-monophosphate (5-FUMP). This TK-deleted virus replicated efficiently in human tumor cell lines; however, it was notably attenuated in normal primary cells, thus displaying a good therapeutic index. Furthermore, this new recombinant poxvirus rendered cells sensitive to 5-FC. In vivo, after systemic injection in mice, the TK-deleted variant caused significantly less mortality than the wild-type strain. A biodistribution study demonstrated high tumor selectivity and low accumulation in normal tissues. In human xenograft models of solid tumors, the recombinant CPXV also displayed high replication, inducing relevant tumor growth inhibition. This anti-tumor effect was improved by 5-FC co-administration. These results demonstrated that CPXV is a promising oncolytic vector capable of expressing functional therapeutic transgenes.

Vaccinia virus, a member of the Poxviridae family, has been extensively used as an oncolytic agent and has entered late stage clinical development. In this study, we evaluated the potential oncolytic properties of other members of the Poxviridae family. Numerous tumor cell lines were infected with ten non-vaccinia poxviruses to identify which virus displayed the most potential as an oncolytic agent. Cell viability indicated that tumor cell lines were differentially susceptible to each virus. Raccoonpox virus was the most potent of the tested poxviruses and was highly effective in controlling cell growth in all tumor cell lines. To investigate further the oncolytic capacity of the Raccoonpox virus, we have generated a thymidine kinase (TK)-deleted recombinant Raccoonpox virus expressing the suicide gene FCU1. This TK-deleted Raccoonpox virus was notably attenuated in normal primary cells but replicated efficiently in numerous tumor cell lines. In human colon cancer xenograft model, a single intratumoral inoculation of the recombinant Raccoonpox virus, in combination with 5-fluorocytosine administration, produced relevant tumor growth control. The results demonstrated significant antitumoral activity of this new modified Raccoonpox virus armed with FCU1 and this virus could be considered to be included into the growing armamentarium of oncolytic virotherapy for cancer.