609 improving the lytic capacity of a conditionally replicative adenoviruses by incorporating promoter elements responsive to tumor environmental conditons

609 Improving the Lytic Capacity of a Conditionally Replicative Adenoviruses by Incorporating Promoter Elements Responsive to Tumor Environmental Conditons Molecular Therapy Volume 18, Supplement 1, M[.]

CANCER-ONCOLYTIC VIRUSES II with systemic delivery of oncolytic viruses to treat established tumors Since this novel combinatorial approach targets the tumor endothelium, these data have direct, wide-ranging and immediate clinical applicability across a broad range of tumor types 607 The Type III Interferon IL-28 Mediates Anti Tumor Efcacy of Oncolytic VSV in a Fully Immune Competent Model Phonphimon Wongthida,1 Rosa Diaz,1,2 Feorillo Galivo,1 Timothy Kottke,1 Jill Thompson,1 Jose Pulido,3 Kevin Pavelko,2 Larry Pease,2 Alan Melcher,4 Richard Vile.1,2,4 Department of Molecular Medicine, Mayo Clinic, Rochester, MN; 2Department of Immunology, Mayo Clinic, Rochester, MN; 3Department of Ophthalmology and Ocular Oncology, Mayo Clinic, Rochester, MN; 4Institute of Molecular Medicine, University of Leeds, Leeds, United Kingdom Several studies have suggested that activation of innate immune effector mechanisms against oncolytic viruses, in addition to, or even instead of, progressive viral-mediated replication and lytic spread, contribute signicantly to clearance of both infected and uninfected tumor cells in an immunocompetent host Here, we developed an in vitro tumor cell/bone marrow co-culture assay to dissect the innate immune sensor/effector responses to intratumoral VSV We established an assay in which host derived bone marrow cells were cultured with B16ova tumor cells in the presence, or absence, of virus Exposure of B16ova tumor cells to VSV, even at low MOI, induced rapid and extensive cytotoxicity, which was almost completely inhibited by co-culturing with anti-VSV neutralizing serum These data suggested that exposure of bone marrow cells to VSV induces bystander killing of B16ova tumor cells, distinct from direct viral-mediated oncolysis Moreover, ELISA analysis of supernatant from co-culturing showed the induction of the type III interferon, IL28 Antibody mediated blockade of IL-28 signaling in the in vitro bone marrow co-culture assay abrogated VSV-activated bone marrow-mediated killing In addition, prior depletion of the bone marrow cultures of both macrophages and GR1+ cells reproducibly led to signicant reductions in the levels of VSV-activated IL-28 secretion and cell killing Finally, we tested the role of IL-28 in VSV treatment of tumors in vivo B16ova tumor lysates expressed IL-28 following exposure to VSV in vivo When this intratumoral IL-28 was neutralized by antibody blockade, concomitant with treatment with VSV, anti tumor efcacy was signicantly reduced to levels similar to those seen with control treatment Taken together, these studies open new avenues to understand the immuno-virological mechanisms associated with oncolytic virotherapy in immune competent hosts Finally, our data suggest novel approaches by which key immune signals might be manipulated to enhance both viral replication, and anti tumor immune activation, to improve efcacy of oncolytic virotherapies 608 A New Oncolytic Herpes Simplex Virus That Synergizes with Small Molecule Inhibitors of PI3K/Akt To Treat Malignant Glioma Ryuichi Kanai,1 Matthew Gardner,1 Hiroaki Wakimoto,1 Robert L Martuza,1 Samuel D Rabkin.1 Department of Neurosurgery, Massachusetts General Hospital, Boston, MA The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway has been implicated as one of the central pathways contributing to the formation, survival and recurrence of a variety of tumors, including malignant gliomas, and cancer stem cells A number of inhibitors targeting PI3K/Akt have been isolated, and some have successfully entered or completed clinical trials in the US They exhibit anti-tumor activity as single agents and possess anti-angiogenic properties S236 However, targeting only one signaling pathway often results in the emergence of resistant cancer cells, as frequently seen with molecularly targeted drugs The HSV Us3 gene product inhibits apoptosis and therefore Us3 mutants (R7041) have been evaluated as oncolytic herpes simplex virus (oHSV) vectors In addition, HSV infection of cells induces Akt activation, which is limited by Us3 However, for clinical translation there are concerns about singly mutated oHSVs Here, we describe a new ICP6-/Us3- double mutant oHSV, MG18L, that has strong oncolytic activity against not only established glioma cell lines (U87, T98), but also glioma stem cells (GSCs) in vitro In contrast to G207, which is a ICP6-/gamma34.5- double mutant, MG18L replicates well in GSCs Importantly, MG18L did not show pathogenicity after intracerebral injection of HSV sensitive A/J mice, whereas 7041 did We previously reported that R7041 synergizes with small molecule inhibitors of PI3K/Akt to kill cancer cell lines MG18L synergizes with small molecule inhibitors of PI3K/Akt (LY294002 and Triciribine) to kill GSCs and glioma cell lines in vitro, while other oHSV vectors without Us3 mutations (G207, G47∆) only exhibit additive interactions MG18L infection of GSCs induced increased levels of activated Akt compared with G207 or G47∆ infection, and also induced apoptosis more rapidly In vivo, intratumoral administration of MG18L prolonged the survival of mice bearing intracerebral GSC (BT74EF) derived tumors We are currently testing the combination of MG18L with NVP-BEZ235, an orally available PI3K/mTor inhibitor in clinical trial MG18L has sufcient safety and efcacy to be considered for the treatment of malignant gliomas In addition, the combination with PI3K/Akt/mTOR inhibitors provides a new multimodal strategy that may be particularly useful for targeting cancer stem cells 609 Improving the Lytic Capacity of a Conditionally Replicative Adenoviruses by Incorporating Promoter Elements Responsive to Tumor Environmental Conditons Diego L Viale,1 Veronica M Lopez,1 Eduardo G Cafferata,1 David Gould,2 Yuti Chenajovsky,2 Osvaldo L Podhajcer.1 Laboratory of Molecular and Cellular Therapy, Leloir InstituteCONICET, Buenos Aires, Argentina; 2Bone and Joint Research Unit, Queen Mary’s School of Medicine and Dentistry, University of London, London, United Kingdom Human tumors are composed of a heterogeneous mass of malignant cells that also include tumor-associated stromal cells that might become a barrier for successful therapies Conditionally Replicative Adenoviruses (CRAds) are a new modality for cancer therapy We have previously reported that a new CRAd (Ad-F512) where E1A transcription is regulated by a 0.5 kb fragment of the SPARC promoter (SPPr) exhibited strong killing effect over established human melanomas xenografted in nude mice However, its oncolytic effect was strongly reduced on established tumors made of a mix of melanoma cells and human broblasts (Lopez MV et al, PLOS One, 2009) In order to improve the therapeutic efficacy of Ad-F512 we hypothesized that addition of DNA sequences containing responsive elements to different patho-physiological conditions that characterize tumor tissue, such as hypoxia and inammation, would increase its replication and, therefore its oncolytic activity We constructed twelve different chimeric promoters containing SPPr combined with Hypoxia Response Elements (HRE), NFκBresponse elements (NFκB) or both and compared its transcriptional activity in luciferase expressing plasmids Based on their activity/ specicity ratio in different cell types we selected the chimeric promoter HRE-SPPr where three HREs were placed upstream of SPPr and a triple chimeric promoter NFκB-SP(HRE)Pr where twelve NFκB Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy CANCER-ONCOLYTIC VIRUSES II response elements were placed upstream of SPPr and HREs inside SPPr sequence We further evaluated the transcriptional activity of the chimeric promoters in an adenovirus backbone using luciferase as a reporter gene We observed 2-10 fold induction of luciferase activity under hypoxia of both chimeric promoters and a 5-fold induction under TNFα treatment of NFκB-SP(HRE)Pr in malignant cells Based on these results, we constructed two CRAds (Ad-HRE-SPPr and Ad-NFκB-SP(HRE)Pr) where E1A transcription was regulated by the chimeric promoters Both CRAds exhibited to 10 fold increased lytic effect under hypoxia on melanoma cells and broblasts compared to the parental Ad-F512 Finally, nude mice harboring tumors made of a mix of melanoma cells and broblasts that were resistant to three administrations of Ad-F512 (Lopez et al, PlosONE 2009) were treated intratumorally with ve administrations of 1010 vp/mouse with either of the CRAds Ad-F512 treatment induced the elimination of tumors in out of mice, while the other mice showed a slight tumor growth However, in mice treated with Ad-NFκB-SP(HRE)Pr we observed a complete elimination of the tumor in all mice that did not recur even after a follow up period of more than 100 days These results indicate that addition of enhancer elements responsive to tumor-environmental conditions improved the therapeutic effect of a CRAd and might overcome the restriction imposed by the presence of stromal cells 610 Dening the Role of Innate Immune Mechanisms in the Efcacy of Oncolytic VSV Virotherapy in a Fully Immune Competent Model Phonphimon Wongthida,1 Rosa Diaz,1,2 Feorillo Galivo,1 Timothy Kottke,1 Jill Thompson,1 Jose Pulido,3 Alan Melcher,4 Richard Vile.1,2,4 Department of Molecular Medicine, Mayo Clinic, Rochester, MN; 2Department of Immunology, Mayo Clinic, Rochester, MN; 3Department of Ophthalmology and Ocular Oncology, Mayo Clinic, Rochester, MN; 4Institute of Molecular Medicine, University of Leeds, Leeds, United Kingdom We have shown previously that VSV therapy depends on host CD8 and NK cells (Cancer Res 2007 15:2840) and that intra-tumoral injection of VSV induces several pro-inammatory cytokines and chemokines in the B16ova tumor microenvironment (Galivo et al,.Gene Ther 2010 In Press) We have also observed that virus replication was, at best, minimal in the B16ova model and that therapy was as effective with a VSV that could only undergo a single cycle of replication as it was with a fully replication competent virus These data suggest that therapy of VSV in the B16ova model is, at least in part, immune mediated Therefore, we investigated the role of the immune system in more detail in this model MyD88 is a critical adaptor protein for IL-1 and TLR signaling in the innate immune response to viruses and other pathogens VSV-mediated therapy was signicantly decreased in MyD88 knock out mice although levels of inltrating CD8 T cells, anti virus antibody or virus replication were not signicantly affected in these mice Therefore, innate immune signaling through the MyD88 pathway is critical for anti tumor therapy However, VSV therapy in TLR4 or IL-1 receptor knock out mice was not signicantly different from that in C57Bl/6 control mice We are currently testing the effects of other TLR signaling pathways on the efcacy of VSV therapy against B16ova tumors Furthermore, we observed a highly signicant increase in the levels of plasmacytoid DC (CD11b-GR1+), known to be major mediators of IFN-α/β secretion in response to viral infection, reaching the draining LN following VSV injection In this respect, following intratumoral injection with VSV in IFN α/β receptor knock out mice, viral spread was uncontrolled leading to rapid toxicity Therapy was also lost when single cycle virus was administered Taken together, our data show that VSV acts as a potent immune adjuvant in the immune competent setting and that therapeutic efcacy – at least in this model – is immune based Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy Therefore, the balance between enhancing virus replication and reducing benecial immune mediated rejection responses need to be considered when using immunomodulatory strategies to improve the therapeutic efcacy of oncolytic virotherapy 611 Differential Effects of VSV and Cyclophosphamide on NK Cells Leads to Antagonistic Effects on Anti-Tumor Therapy of a Murine Mesothelioma Model Candice L Willmon,1 Jill Thompson,1 Timothy Kottke,1 Stephen Albelda,2 Richard Vile.1 Department of Molecular Medicine, Mayo Clinic, Rochester, MN; 2Pulmonary, Allergy & Critical Care Division, University of Pennsylvania Medical Center, Philadelphia, PA Malignant mesothelioma is an aggressive neoplasm of the lining of the pleura or peritoneum and, to date, conventional treatments for mesothelioma, including chemotherapy, radiation and surgical resection, tend to be more palliative than efcacious However, recent evidence has indicated that mesothelioma is sensitive to immunotherapy This led us to hypothesize that the treatment of mesothelioma with a combination of chemo-immunotherapy and an oncolytic virus may provide both enhanced tumor cell killing and immunotherapeutic benets Using the oncolytic virus, vesicular stomatitis virus (VSV), in conjunction with cyclophosphamide (CPA), we investigated the generation of both anti-viral and anti-tumor responses and how such immune responses can be manipulated to improve on the therapy of a murine mesothelioma model, AE17ova Treatment of AE17ova-bearing mice with CPA, with or without virus, prolongs survival (p