Oncolytic virotherapy with intratumoral injection of vaccinia virus TG6002 and 5-fluorocytosine administration in dogs with malignant tumors.

TG6002 is an oncolytic vaccinia virus expressing FCU1 protein, which converts 5-fluorocytosine into 5-fluorouracil. The study objectives were to assess tolerance, viral replication, 5-fluorouracil synthesis, and tumor microenvironment modifications to treatment in dogs with spontaneous malignant tumors. Thirteen dogs received one to three weekly intratumoral injections of TG6002 and 5-fluorocytosine. The viral genome was assessed in blood and tumor biopsies by qPCR. 5-Fluorouracil concentrations were measured in serum and tumor biopsies by liquid chromatography or high-resolution mass spectrometry. Histological and immunohistochemical analyses were performed. The viral genome was detected in blood (7/13) and tumor biopsies (4/11). Viral replication was suspected in 6/13 dogs. The median intratumoral concentration of 5-fluorouracil was 314 pg/mg. 5-Fluorouracil was not detected in the blood. An increase in necrosis (6/9) and a downregulation of intratumoral regulatory T lymphocytes (6/6) were observed. Viral replication, 5-fluorouracil synthesis, and tumor microenvironment changes were more frequently observed with higher TG6002 doses. This study confirmed the replicative properties, targeted chemotherapy synthesis, and reversion of the immunosuppressive tumor microenvironment in dogs with spontaneous malignant tumors treated with TG6002 and 5-fluorocytosine.

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.

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.

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.

Cowpox Virus: A New and Armed Oncolytic Poxvirus.

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.