Preclinical Evaluation of the Oncolytic Vaccinia Virus TG6002 by Translational Research on Canine Breast Cancer.

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.

Safety studies and viral shedding of intramuscular administration of oncolytic vaccinia virus TG6002 in healthy beagle dogs.

BACKGROUND: Cancer is a leading cause of mortality for both humans and dogs. As spontaneous canine cancers appear to be relevant models of human cancers, developing new therapeutic approaches could benefit both species. Oncolytic virotherapy is a promising therapeutic approach in cancer treatment. TG6002 is a recombinant oncolytic vaccinia virus deleted in the thymidine kinase and ribonucleotide reductase genes and armed with the suicide gene FCU1 that encodes a protein which catalyses the conversion of the non-toxic 5-fluorocytosine into the toxic metabolite 5-fluorouracil. Previous studies have shown the ability of TG6002 to infect and replicate in canine tumor cell lines, and demonstrated its oncolytic potency in cell lines, xenograft models and canine mammary adenocarcinoma explants. Moreover, 5-fluorouracil synthesis has been confirmed in fresh canine mammary adenocarcinoma explants infected with TG6002 with 5-fluorocytosine. This study aims at assessing the safety profile and viral shedding after unique or repeated intramuscular injections of TG6002 in seven healthy Beagle dogs. RESULTS: Repeated intramuscular administrations of TG6002 at the dose of 5 × 107 PFU/kg resulted in no clinical or biological adverse effects. Residual TG6002 in blood, saliva, urine and feces of treated dogs was not detected by infectious titer assay nor by qPCR, ensuring the safety of the virus in the dogs and their environment. CONCLUSIONS: These results establish the good tolerability of TG6002 in healthy dogs with undetectable viral shedding after multiple injections. This study supports the initiation of further studies in canine cancer patients to evaluate the oncolytic potential of TG6002 and provides critical data for clinical development of TG6002 as a human cancer therapy.

The Enhanced Tumor Specificity of TG6002, an Armed Oncolytic Vaccinia Virus Deleted in Two Genes Involved in Nucleotide Metabolism.

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.

In Vitro Assay for the Evaluation of Cytotoxic Effects Provided by a Combination of Suicide and Killer Genes in a Bicistronic Vector.

When using bicistronic expression constructs the issue arises concerning proper evaluation of the cytotoxic efficiency of a combination of therapeutic genes. For this purpose, an approach can be applied based on the transient transfection of cultured human cells with a specifically designed set of mono- and bicistronic expression constructs and on the comparison of their cytotoxic effects. Here the application of this approach is described using an example of the evaluation of the combined cytotoxic action of bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) and hepatitis A virus 3C protease in a bicistronic plasmid construct.

Oncolytic properties of non-vaccinia poxviruses.

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.

Vaccinia Virus Shuffling: deVV5, a Novel Chimeric Poxvirus with Improved Oncolytic Potency.

Oncolytic virus (OV) therapy has emerged as a promising approach for cancer treatment with the potential to be less toxic and more efficient than classic cancer therapies. Various types of OVs in clinical development, including Vaccinia virus (VACV)-derived OVs, have shown good safety profiles, but limited therapeutic efficacy as monotherapy in some cancer models. Many different methods have been employed to improve the oncolytic potency of OVs. In this study, we used a directed evolution process, pooling different strains of VACV, including Copenhagen, Western Reserve and Wyeth strains and the attenuated modified vaccinia virus Ankara (MVA), to generate a new recombinant poxvirus with increased oncolytic properties. Through selective pressure, a chimeric VACV, deVV5, with increased cancer cell killing capacity and tumor selectivity in vitro was derived. The chimeric viral genome contains sequences of all parental strains. To further improve the tumor selectivity and anti-tumor activity of deVV5, we generated a thymidine kinase (TK)-deleted chimeric virus armed with the suicide gene FCU1. This TK-deleted virus, deVV5-fcu1 replicated efficiently in human tumor cells, and was notably attenuated in normal primary cells. These studies demonstrate the potential of directed evolution as an efficient way to generate recombinant poxviruses with increased oncolytic potency, and with high therapeutic index to improve cancer therapy.