CANCER-ONCOLYTIC VIRUSES II irradiation prolonged the survival of mice. However, the mechanisms further sought various applications of IL-12-expressing oncolytic involving in the effect of irradiation on enhanced therapeutic outcome HSV-1 for treating bladder cancer. A lung metastases model was are still unclear and are under investigation. generated by injecting MB-49 cells intravenously in C57BL/6 mice, and viruses were repeatedly administered into the tail vein. Both 6 795. Treatment of Cancer with Oncolytic T-01 and T-mfIL12 (5 x 10 pfu) signifi cantly prolonged the survival Adenovirus Combined with Temozolomide, an compared with mock, with T-mfIL12 being more effi cacious than T-01 (Fig. 1). In an orthotopic bladder cancer model, intravesical Autophagy Inducer inoculations with T-mfIL12 (1 x 106 pfu) prolonged the survival of 1 1 1 Ilkka Liikanen, Laura Ahtiainen, Vincenzo Cerullo, Petri C57BL/6 mice with MB-49 bladder cancer compared with T-01. 1 1 1 1 Nokisalmi, Sophie Escutenaire, Iulia Diaconu, Sari Pesonen, Further, using the orthotopic model, cancer-bearing mice were treated 1 2 2 Anniina Koski, Lotta Kangasniemi, Elina Haavisto, Kaarina with intravesical administration of T-mfIL12 (1 x 106 pfu) or mock 3 3 1 1 Partanen, Timo Joensuu, Anna Kanerva, Akseli Hemminki. in combination with BCG (1.35mg) or PBS. The combination of 1 Cancer Group, Molecular Cancer Biology T-mfIL12 and BCG prolonged the survival of the mice with MB-49 Program, Transplantation Laboratory, Haartman Institute and bladder cancer compared with T-mfIL12 alone or BCG alone. When Finnish Institute for Molecular Medicine, University of Helsinki, athymic mice bearing subcutaneous human RT-4 bladder cancer 2 Helsinki, Finland; Oncos Therapeutics, Inc., Helsinki, Finland; were treated with intraneoplastic inoculation of G47∆ (4 x 104 pfu) 3 International Comprehensive Cancer Center Docrates, Docrates or mock in combination with intraperitoneal injection of Gefi tinib, an Clinic, Helsinki, Finland. epidermal growth factor receptor inhibitor, or PBS, the combination Purpose: Oncolytic viruses can induce autophagic cancer death therapy was signifi cantly more effi cacious than G47∆ alone or and preclinical data suggests that autophagy modulating drugs can Gefi tinib alone (Fig.2). Results indicate that the G47∆-derived enhance their effi cacy. This, however, has not been previously studied oncolytic HSV-1’s are useful for treating bladder cancer in a variety in humans. Experimental design: We assessed the combinations of ways. The effi cacy of G47∆ may be greatly enhanced by arming of , autophagy inducing temozolomide and the virus with IL-12. immunomodulatory cyclophosphamide in a prostate tumor model in mice. 17 patients with solid tumors refractory to conventional treatment modalities were treated in a personalized advanced therapy access program with oncolytic adenovirus together with a low-dose pulse of temozolomide (100mg/d). Concurrent low-dose metronomic cyclophophamide was administered to reduce regulatory T-cells. The primary end-point was safety. In addition, immunological, virological and effi cacy parameters were studied. Results: In mice, the best tumor growth inhibition was observed with combination of oncolytic adenovirus, temozolomide and cyclophosphamide. Electron microscopy revealed increased autophagy in combination treated tumors compared to controls. Combination treatments in humans were safe with grade 1–2 fl u-like symptoms, fever, and fatigue and nausea in most patients. Non-hematological grade 3–5 side effects were seen in one patient with grade 3 ileus and one patient with grade 3 abdominal pain. Overall, objective evidence of anti-tumor activity was seen in 12/17 evaluable treatments. Conclusions: Low-dose pulse temozolomide and low-dose cyclophosphamide in combination with oncolytic adenovirus showed increased effi cacy in mice and were well tolerated in humans. Further studies are needed to assess if temozolomide increases the effi cacy of oncolytic in humans.

796. Therapy for Bladder Cancer Using a Third-Generation HSV-1 Armed with Interleukin 12 Jiangang Hou,1 Hiroshi Fukuhara,1 Yuzuri Tsurumaki,1 Yukio Homma,1 Yasushi Ino,2,3 Tomoki Todo.2,3 797. Oncolytic Adenovirus Induces Autophagic 1Urology, The University of Tokyo, Tokyo, Japan; 2Neurosurgery, Cell Death through an E2F1-microRNA-7- The University of Tokyo, Tokyo, Japan; 3Translational Research Epidermal Growth Factor Receptor Axis in Human Center, The University of Tokyo Hospital, Tokyo, Japan. Cancer Cells 1,2 2 2 Oncolytic herpes simplex viruses type 1 (HSV-1) that replicate Hiroshi Tazawa, Shuya Yano, Ryosuke Yoshida, Yuuri Hashimoto,2 Yasuo Urata,3 Toshiyoshi Fujiwara.2 selectively in cancer cells are promising therapeutic agents for 1 cancer. G47∆ is a third-generation oncolytic HSV-1 that has triple Center for Gene and Cell Therapy, Okayama University Hospital, γ α Okayama, Japan; 2Department of Gastroenterological Surgery, in the 34.5, 47 and ICP6 genes, and the fi rst recombinant 3 oncolytic virus to be used in a clinical trial in Japan. Using the G47∆ Okayama University Graduate School, Okayama, Japan; Oncolys backbone, we newly constructed T-mfIL12, an HSV-1 armed with BioPharma, Inc., Tokyo, Japan. fusion-type murine interleukin 12 (IL-12). We have previously shown Background: Oncolytic virotherapy is a promising anticancer that, when inoculated intraneoplastically at 1 x 106 pfu, T-mfIL12 treatment to induce oncolytic cell death that is distinct from was signifi cantly better at inhibiting the growth of subcutaneous caused by conventional chemoradiotherapy in human cancer cells. MB-49 tumors (murine bladder cancer) in syngeneic C57BL/6 mice Recent accumulating evidences have suggested the implication of compared with T-01, a control virus. From a clinical standpoint, we autophagy on the oncolytic adenovirus-mediated cell death. However, S304 Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy CANCER-ONCOLYTIC VIRUSES II molecular mechanism underlying oncolytic adenovirus-mediated prodrug convertase cytosine deaminase (CD) which converts the non- autophagic cell death remains unclear. We previously generated a toxic prodrug 5-fl uorocytosine (5-FC) to the potent chemotherapeutic genetically engineered oncolytic adenovirus (Telomelysin®: OBP- 5-fl uorouracil (5-FU). Since EGFR is frequently overexpressed in 301) that replicates and induces autophagic cell death against human HNSCC, we fused a single-chain (scFv) specifi c for the cancer cells in a telomerase-dependent manner. As microRNA extracellular domain of EGFR as a C-terminal extension to the MV (miRNA), a post-transcriptional regulator of gene expression, has attachment H which was blinded for the natural MV receptors been recently shown to regulate diverse cell fates including autophagy, CD46/SLAM. Specifi city of by the EGFR-retargeted MV we hypothesized that OBP-301 infection induces autophagic cell was compared to the parental MV-EGFP with unmodifi ed tropism death through miRNA modulation in human cancer cells. In this and to a control virus which binds to CD20, a B cell specifi c . study, we attempted the role of miRNA on the OBP-301-mediated It was demonstrated in vitro that EGFR-retargeted MV can infect autophagic cell death in human cancer cells. Methods: We used 3 HNSCC specifi cally and kill target cells very effi ciently. Combined human lines (H1299, A549 and T.Tn), that showed different treatment of MV-CD-antiEGFR infected cells with 5-FC substantially sensitivities to OBP-301. In vitro cytopathic effect of OBP-301 in enhanced the cytoreductive impact on infected and bystander cells. each cell line was confi rmed by XTT assay. Induction of autophagy In a subcutaneous tumor xenograft model of HNSCC intratumoral was analyzed by western blot for autophagy-related (LC3, administration of MV-EGFP-antiEGFR resulted in a statistically Atg5 and p62). To investigate whether OBP-301 infection modulates signifi cant tumor growth delay and prolongation of survival in contrast miRNA expression, the expression levels of 757 human miRNAs to mock treated animals. Importantly, combined chemovirotherapy were analyzed in OBP-301-infected H1299 cells using miRCURY using MV-CD-antiEGFR together with subsequent prodrug miRNA microarray (EXIQON). Modulation of miRNA expression administration yielded signifi cantly consolidated tumor regression as was further validated by quantitative real-time RT-PCR (qRT-PCR). well as extended survival compared to mice treated with virus only. Induction of autophagy by miRNA was determined using artifi cial This EGFR-retargeted and armed virus may be a promising tool for miRNA-transfected cells by fl owcytometric and western blot analysis. chemovirotherapy of advanced HNSCC. Expression of miRNA’s target gene was confi rmed by western blot analysis. Results: XTT assay revealed that H1299 and A549 cells 799. Generation of Fiber-Modifi ed, were sensitive, whereas T.Tn cells were resistant to cytopathic effect Conditionally-Replicating Adenovirus Vectors of OBP-301. In OBP-301-sensitive H1299 and A549 cells, autophagy, which includes the conversion of LC3-I to LC3-II, upregulation of Expressing the Sodium Iodide Symporter for Atg5 and downregulation of p62, was induced by OBP-301 infection. Therapy However, no autophagy was induced in OBP-301-resistant T.Tn cells. Michael J. Oneal,1 Miguel A. Trujillo,2 Julia Davydova,3 Masato miRNA microarray and qRT-PCR revealed that OBP-301 infection Yamamoto,3 John C. Morris.2 upregulated miR-7 expression, which was signifi cantly correlated with 1Department of Molecular Medicine, Mayo Clinic, Rochester, OBP-301-mediated cytopathic activity and autophagy induction. We MN; 2Department of Endocrinology, Mayo Clinic, Rochester, MN; also revealed that OBP-301-mediated miR-7 upregulation depended 3Department of Surgery, University of Minnesota, Minneapolis, on enhanced E2F1 expression. Furthermore, ectopic expression MN. of miR-7 in human cancer cells suppressed cell viability and As the second leading cause of cancer death in men, and as a subsequently induced autophagy. Flowcytometric and western blot cancer for which few curative treatments exist, there is an urgent analysis revealed that downregulation of epidermal growth factor need for novel therapies for prostate cancer. Replicating oncolytic receptor (EGFR) was involved in the induction of autophagy by viruses expressing the sodium iodide symporter (hNIS) can readily miR-7. Conclusions: Our results suggest that oncolytic adenovirus transduce target cells and effect functional NIS expression and OBP-301 induces autophagic cell death through an E2F1-miR-7- uptake of radioactive iodine. This vector would extend radioiodine EGFR pathway in human cancer cells, providing a novel insight into therapy, a well-established thyroid cancer treatment, to other the molecular mechanism of an oncolytic virotherapy. cancers. Previous work in our lab has elucidated the potential of radiovirotherapy using a conditionally replicating vector, Ad5PB- 798. EGFR-Targeted and Cytosine Deaminase- RSV-NIS, where E1a expression is regulated by the prostate-specifi c Armed Measles Virus in Combination with probasin promoter. Transduction of tumors with Ad5PB-RSV-NIS 5-Fluorocytosine as a Novel Chemovirotherapy for followed by administration of radioactive iodide showed signifi cant Head and Neck Squamous Cell Carcinoma improvement over vector alone. This approach utilizes the bystander Karim Zaoui,1,2 Sascha Bossow,1 Christian Großardt,1 Mathias effects of replication and radiation to compensate for low in vivo F. Leber,1 Christoph Springfeld,3 Christof von Kalle,1 Peter K. transduction effi ciency, a problem frequently observed in virotherapy Plinkert,2 Guy Ungerechts.1 trials. We have modifi ed the adenovirus vector structure to further 1Department of Translational Oncology, National Center for improve efficacy. Specifically, we modified our vectors with a Tumor Diseases, German Cancer Research Center, Heidelberg, hybrid fi ber protein containing the knob domain of Ad serotype 3, Germany; 2Department of Otorhinolaryngology and Head and which binds to CD46 rather than the less-available CAR. Also, the Neck Surgery, University Hospital Heidelberg, Heidelberg, Adenovirus death protein (ADP), an E3 splice product absent in our Germany; 3Department of Internal Medicine IV, University previous vector, hastens the release of viral particles after assembly. Hospital Heidelberg, Heidelberg, Germany. Increasing the speed of viral spread by the introduction of cytolytic proteins or transductional modifi cation may increase the number For locally advanced or metastatic head and neck squamous cell of infected tumor cells able to take up radioiodide, and the dose of carcinoma (HNSCC) no curative therapy is currently available. radiation absorbed by the tumor as a consequence. To determine Therefore, novel treatment methods are urgently needed. Measles the effect of these changes on effi cacy, we generated two vectors, viruses (MV) derived from vaccine strain have shown considerable Ad5/3PB-ADP-hNIS, and Ad5/3PB-hNIS, and assessed their ability oncolytic activity against lymphomas and a wide variety of solid to improve outcome. Infection and NIS-expression in vitro was adenocarcinomas from different entities. In this study, we investigated characterized by MTS assay, staining of infected cells with crystal for the fi rst time the susceptibility of HNSCC cell lines to MV violet, and radioisotope uptake assays. These assays demonstrated infection using MV expressing enhanced green fl uorescent protein that ADP-expressing vector lysed cells tenfold more quickly than (MV-EGFP). Next, we generated targeted and armed MV encoding the their ADP-lacking counterpart, but induced about a third of the

Molecular Therapy Volume 19, Supplement 1, May 2011 S305 Copyright © The American Society of Gene & Cell Therapy