Johann Foloppe

Targeted chemotherapy for head and neck cancer with a chimeric oncolytic adenovirus coding for bifunctional suicide protein FCU1.

Purpose: Transfer of prodrug activation systems into tumors by using replication-deficient viruses has been suggested to be an effective method for achieving high local and low systemic anticancer drug concentrations. However, most current suicide gene therapy strategies are still hindered by poor efficiency of in vivo gene transfer, inefficient tumor penetration, limited bystander cell killing effect, and need of large prodrug doses. We hypothesized that local amplification provided by a replication competent platform would help overcome these limitations. Experimental Design: We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Δ24FCU1 expressing the fusion suicide gene FCU1. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Results: We examined the efficacy of Ad5/3-Δ24FCU1 and the replication-defective control Ad5/3-FCU1 with and without 5-FC. FCU1 expression was confirmed by Western blot, whereas enzymatic conversion levels in vitro and in vivo were determined by high-performance liquid chromatography separation. Significant antitumor effect was observed in vitro and in vivo in a murine model of head and neck squamous cell carcinoma. Although we observed a decrease in viral DNA copy number in vitro and in tumors treated with Ad5/3-Δ24FCU1 and 5-FC, suggesting an effect on virus replication, the highest antitumor effect was observed for this combination. Conclusions: It seems feasible and efficacious to combine adenovirus replication to the FCU1 prodrug activation system. Clin Cancer Res; 16(9); 2540–9. ©2010 AACR.

Oncolytic virotherapy with an armed vaccinia virus in an orthotopic model of renal carcinoma is associated with modification of the tumor microenvironment

ABSTRACT Oncolytic virotherapy is an emergent promising therapeutic approach for the treatment of cancer. We have constructed a vaccinia virus (WR strain) deleted for thymidine kinase (TK) and ribonucleotide reductase (RR) genes that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. We evaluated this construct (VV-FCU1) in the orthotopic model of renal carcinoma (RenCa). Systemic administration of VV-FCU1 resulted in orthotopic tumor growth inhibition, despite temporary expression of viral proteins. VV-FCU1 treatment was associated with an infiltration of tumors by CD8+ T lymphocytes and a decrease in the proportion of infiltrating Tregs, thus modifying the ratio of CD8+/CD4+ Treg in favor of CD8+cytotoxic T cells. We demonstrated that VV-FCU1 treatment prolonged survival of animals implanted with RenCa cells in kidney. Depletion of CD8+ T cells abolished the therapeutic effect of VV-FCU1 while depletion of CD4+ T cells enhanced its protective activity. Administration of the prodrug 5-fluorocytosine (5-FC) resulted in a sustained control of tumor growth but did not extend survival. This study shows the importance of CD4+ and CD8+ T cells in vaccinia virus-mediated oncolytic virotherapy and suggests that this approach may be evaluated for the treatment of human renal cell carcinoma.

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.

Ultrasound-mediated cavitation does not decrease the activity of small molecule, antibody or viral-based medicines

The treatment of cancer using nanomedicines is limited by the poor penetration of these potentially powerful agents into and throughout solid tumors. Externally controlled mechanical stimuli, such as the generation of cavitation-induced microstreaming using ultrasound (US), can provide a means of improving nanomedicine delivery. Notably, it has been demonstrated that by focusing, monitoring and controlling the US exposure, delivery can be achieved without damage to surrounding tissue or vasculature. However, there is a risk that such stimuli may disrupt the structure and thereby diminish the activity of the delivered drugs, especially complex antibody and viral-based nanomedicines. In this study, we characterize the impact of cavitation on four different agents, doxorubicin (Dox), cetuximab, adenovirus (Ad) and vaccinia virus (VV), representing a scale of sophistication from a simple small-molecule drug to complex biological agents. To achieve tight regulation of the level and duration of cavitation exposure, a “cavitation test rig” was designed and built. The activity of each agent was assessed with and without exposure to a defined cavitation regime which has previously been shown to provide effective and safe delivery of agents to tumors in preclinical studies. The fluorescence profile of Dox remained unchanged after exposure to cavitation, and the efficacy of this drug in killing a cancer cell line remained the same. Similarly, the ability of cetuximab to bind its epidermal growth factor receptor target was not diminished following exposure to cavitation. The encoding of the reporter gene luciferase within the Ad and VV constructs tested here allowed the infectivity of these viruses to be easily quantified. Exposure to cavitation did not impact on the activity of either virus. These data provide compelling evidence that the US parameters used to safely and successfully delivery nanomedicines to tumors in preclinical models do not detrimentally impact on the structure or activity of these nanomedicines.

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.

Abstract LB-287: Pseudocowpox: A next generation viral vector for cancer immunotherapy

Engineered viral vectors are effective approaches to stimulate anti-tumor immunity, and change the tumor immune environment. Several viruses and strains have been developed to express tumor antigens and cytokines, and corresponding products are in advanced clinical trials. However, novel viral strains with improved immunogenic properties are sought. In this perspective, we screened a variety of poxviridae potentially usable in humans: Cowpox (CPX), Pseudocowpox (PCPV), Parapoxvirus Ovis (ORF), Myxoma virus (MYX), Swinepox (SWP), Yaba-like disease virus (YLDV), Raccoonpox (RCN), Cotia virus (CTV), and compared them to the well-established vaccine strain Modified Virus Ankara (MVA), and oncolytic Vaccinia Virus, strain Copenhagen (VV). Both in vitro with human primary immune cells, and in vivo with syngeneic mouse tumor models, PCPV proved to be a very promising vector for immunotherapy. Compared with MVA, PCPV induced a 1000-fold higher expression of IFN-alpha in human PBMCs, whereas SWPV and ORF displayed a lower 10 to 100-fold induction. Other viruses (i.e. VV, RCN, CTV, or MYX) did not raise the IFN-alpha level. PCPV was also shown to increase the level of GM-CSF, and to be safe for PBMCs, in contrast to ORF which displayed some cytotoxic effects. When tested for its capacity to trigger the expression of CD86, a co-stimulatory factor for T-cell activation, PCPV was shown to be superior than MVA in primary moDCs. Furthermore, PCPV treatment increased CD86 expression in human CD163+CD206+ “M2”-type macrophages derived from CD14+ monocytes, suggesting a shift to an antigen-presenting phenotype. In these cells, PCPV increased significantly the secretion of IL-18, IL-6 and IP-10, signing a conversion towards a less suppressive macrophage phenotype. Last, incubation of PCPV in a co-culture model overcame the immunosuppressive effect of MDSCs on human autologous CD8+ T. A GFP-encoding recombinant PCPV vector was generated, and we could demonstrate that PCPV was capable of infecting human primary immune cells, comparably to recombinant MVA vectors, except for activated T cells. A recombinant PCPV encoding for the HPV E7 protein was generated to assess the anti-tumor activity and immunogenicity in a syngeneic murine tumor model. Like MVA-E7, PCPV-E7 induced a strong cellular response (ELISPOT on splenocytes, and frequency of antigen-specific short-lived effector cells), but PCPV-E7 displayed a different cytokine/chemokine profile at the site of injection, with increased levels of pro-immune cytokines including IP-10, IFN-gamma, GM-CSF, IL-18, MIP-1 alpha, MIP-1 beta, IL-12 and IL-6. When injected intratumorally into fast growing MC-38 tumors, PCPV led to tumor control. Analysis of tumors infiltrates showed that PCPV treatment led to higher levels of neutrophils and decreased the frequency of MHCIIlo TAMs. Our data demonstrate that PCPV might display better properties than current viral vectors, in terms of local response and priming activity, of ability to induce effector T cells and to reshape the tumor infiltration profiles. It has the same capacity as other poxviruses to encode and deliver large genetic payload, which will be useful for designing advanced anti-tumor vaccines. Citation Format: Karola Rittner, Marine Ricordel, Caroline Tosch, Christine Thioudellet, Christelle Remy-Ziller, Marie-Christine Claudepierre, Chantal Hoffmann, Doris Schmitt, Benoit Sansas, Johann Foloppe, Philippe Erbs, Nathalie Silvestre, Kaidre Bendjama, Eric Quemeneur. Pseudocowpox: A next generation viral vector for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-287.