530 Retargeting Oncolytic Adenoviruses to Tumors Expressing c Met Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S203 CANCER ONCOLYTIC VIRU[.]
CANCER-ONCOLYTIC VIRUSES I 527 RdB-VSV-G, a Vesicular Stomatitis Virus Glycoprotein Epitope-Incorporated Oncolytic Adenovirus Shows Marked Enhancement in Oncolysis and Suppression of Tumor Growth A.-Rum Yoon,1 Jaesung Kim,1 Renu Wadhwa,2 Sunil Kaul,2 ChaeOk Yun.1 Severance Biomedical Science Institute (SBSI), College of Medicine, Yonsei University, Seoul, Republic of Korea; 2National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba, Ibaraki, Japan Oncolytic adenoviral (Ad) vectors are widely used as a hopeful therapeutic medicine for human cancer therapy However, full utility of traditional oncolytic Ad has been limited in several reasons One of the key problems is low infectivity of oncolytic Ads due to low expression of coxackies and adenovirus receptor (CAR) in cells Thus current studies are focused to improve its limiting potential by overcoming correlation with cellular CAR expression and Ad infection in cancer gene therapy With the aim of improving the efficiency of infections to cancer cells, we have generated a novel tropism-expanded oncolytic Ad, RdB-VSV-G, that contains the epitope of envelope protein VSV-G at the fiber knob between HI loop with CAR entry facility and an additional entry potential Using immunoblot analysis for detection of Ad fiber, VSV-G epitopeincorporated Ad is capable of forming a fiber trimer, just like RdB with the wild-type fiber The newly tropism-expanded Ad, RdBVSV-G, showed a remarkable improvement in the cytotoxicity to a variety of mammalian cells The supreme enhancement in cytotoxicity was observed in cells that were difficult to infect because of low CAR expression when compared with control Ad that lacked the VSV-G epitope Furthermore, the cellualar entry of RdB-VSV-G is mediated by both CAR and PS (phosphatidyl serine) In line with those results, treatment with RdB-VSV-G significantly enhanced anti-tumor effect in vivo Greater survival advantage was observed for RdB-VSV-G-treated tumor bearing mice In summary, we have developed an oncolytic Ad with significantly improved therapeutic profile for cancer treatment 528 Oncolytic Measles Virus Fully Retargeted to the Melanoma Surface Antigen HMWMAA Johanna K Kaufmann,1 Sascha Bossow,2 Christian Grardt,2 Stefanie Sawall,2 Jưrg Kupsch,3 Guy Ungerechts,2 Dirk M Nettelbeck.1 Helmholtz University Group Oncolytic Adenoviruses, German Cancer Research Center & Heidelberg University Hospital, Heidelberg, Germany; 2Department of Translational Oncology, National Center for Tumor Diseases, German Cancer Research Center, Heidelberg, Germany; 3RAFT Institute for Plastic Surgery, Mount Vernon Hospital, Northwood, United Kingdom Advanced melanoma is a substantial clinical challenge with rising incidence and poor survival rates No curative therapy is currently available Therefore, novel treatment methods are urgently needed Oncolytic measles viruses (MV) based on the Edmonston vaccine strain have been shown to be a promising tool for the treatment of various cancer types We generated a fully retargeted melanomaspecific oncolytic MV that enters cells specifically through the High Molecular Weight Melanoma-Associated Antigen (HMWMAA), a molecule widely expressed on the surface of malignant melanoma cells To this end, an optimized single-chain antibody against HMWMAA (RAFT3) was fused to the C-terminus of a mutated attachment protein hemagglutinin (H) unable to recognize its natural receptors Specificity and biological function were demonstrated via fusion assay on HMWMAA-expressing cells after transient plasmid-mediated co-expression of retargeted H glycoprotein (HαHMWMAA) and the fusion protein F Incorporation of Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy HαHMWMAA into virus particles was as efficient as for other retargeted control viruses using the producer cell line Vero-αHis In line with the fusion assays, infection experiments revealed specific entry of MV-HαHMWMAA into antigen-expressing cell lines Viral spread could be detected as broad syncytia formation and infection efficacy correlated with HMWMAA surface expression levels of all cell lines tested Virtually no off-target infection was detected Cytotoxicity assays showed significant cell killing of melanoma cells after infection with the retargeted virus We further evaluated the efficacy and specificity of MV-HαHMWMAA by one-step growth curves After infection of melanoma cells, the retargeted virus showed only a slight growth retardation as maximal titers were 2-fold lower than those of an unmodified control virus In contrast, replication of MV-HαHMWMAA was strongly attenuated by three orders of magnitude on non-target cells With this, selectivity was significantly higher than that of an established control MV We are currently arming MV-HαHMWMAA with an additional payload to further enhance the oncolytic efficacy This highly melanoma-specific virus may be a promising tool for virotherapy of advanced melanoma 529 Modification of the Early Gene EnhancerPromoter Improves the Oncolytic Potency of Adenovirus 11 Han Hsi Wong,1 Guozhong Jiang,1 Rathi Gangeswaran,1 Ming Yuan,1 Hexiao Wang,1 Vipul Bhakta,1 Heike Muller,1 Nick Lemoine,1 Yaohe Wang.1 Molecular Oncology and Imaging, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom Replicating oncolytic adenoviruses based on serotype (Ad5) have several shortcomings, including the downregulation of its receptor in cancer cells, high prevalence of neutralizing antibodies and hepatotoxicity Another adenoviral serotype, Ad11, could overcome these obstacles Here, we show that human cancer cell lines express higher levels of the Ad11 receptor CD46, resulting in much better infectivity than Ad5 Surprisingly, only 36% (9/25) of the cell lines were more sensitive to Ad11- than to Ad5-mediated cytotoxicity Investigations revealed that it was the transcription of Ad11 E1A, not CD46 expression or virus infectivity, which determined the cell’s sensitivity to Ad11 killing Ad11 E1A mRNA levels have an effect on viral DNA replication, structural protein synthesis and infectious particle production To test the hypothesis that increased E1A transcription would lead to improved Ad11 replication in Ad5sensitive (but Ad11-less sensitive) cells, two Ad11 mutants (Ad11Ad5-P and Ad11-Ad5-EP) were constructed where either the E1A promoter or enhancer-promoter, respectively, was replaced by that of Ad5 Ad11-Ad5-EP demonstrated increased E1A mRNA levels and replication, together with enhanced oncolytic potency in vitro and in vivo This effect was found in both the Ad5-sensitive and Ad11sensitive cancer cells, broadening the range of tumors that could be effectively killed by Ad11-Ad5-EP These results imply that the novel Ad11 mutant developed in this study with the Ad5 E1A (instead of wild-type Ad11 E1A) enhancer-promoter is a promising backbone for the future development of more potent and tumor-selective oncolytic viruses 530 Retargeting Oncolytic Adenoviruses to Tumors Expressing c-Met Hany I Sakr,1 Shilpa Bhatia,1 J Michael Mathis.1 Cellular Biology and Anatomy, LSU Health Sciences Center, Shreveport, LA The hepatocyte growth factor (HGF) receptor, or c-Met, is a unique heterodimer with tyrosine kinase activity The physiologic actions mediated by c-Met are widespread, include altering cell proliferation, adhesion, and cell motility during tissue development, and repair S203 CANCER-TARGETED GENE & CELL THERAPY II The c-Met receptor can interact with multiple intracellular signaling pathways, and is up regulated in a majority of malignancies This makes c-Met a relevant target for novel therapeutic interventions Virotherapy employing oncolytic adenoviruses represents a promising biological intervention applicable to a wide array of neoplastic diseases Using oncolytic adenoviruses, designed to divide in and specifically kill tumor cells, is a promising virotherapy approach for cancer treatment Ideally, cancer-specific replication of oncolytic adenoviruses results in viral-mediated replication and lysis of infected tumor tissues Release of virus progeny results in further propagation in surrounding tumor cells but not in those of normal tissues that would be refractory to virus replication To date, achieving replicative specificity of oncolytic adenovirus agents has been accomplished primarily by incorporating tumor selective promoters To achieve a novel alternative specificity of oncolytic adenoviruses, we used a high affinity c-Met ligand to retarget viral infectivity to metastatic disease over expressing the receptor Specifically, we incorporated NK2, a competitive antagonist of the HGF - c-Met association, into the adenovirus fiber In this approach, the NK2 ligand was genetically incorporated by ablating the adenovirus fiber (protein pIV) gene, and replacing it with a recombinant gene consisting of the tail domain from the fiber and a trimerization domain from T4 fibritin fused to NK2 (fiber-fibritin-NK2) This study demonstrates the ability to rescue viable adenoviral particles that display functional fiber-fibritin-NK2 as a component of their capsid surface Importantly, this construct in the context of an oncolytic adenovirus, specifically infected cancer cell lines that over express c-Met, and induced cell lysis These results suggest that a c-Met retargeted virus could be used to counteract tumor mitogenesis, motogenesis, and morphogenesis driven by this receptor If successful, this approach could open new avenues for using adenoviral vectors in cancer gene therapy and hasten the shift from preclinical to clinical trials Cancer-Targeted Gene & Cell Therapy II 531 Stanniocalcin-1 Derived from Multi Potent Stromal Cell Changes Cell Metabolism Dramatically, Decreases Reactive Oxygen Species Stress and Promotes Survival of Cancer Cells with Uncoupling Protein Upregulation Shinya Ohkouchi,1 Gregory J Block,2 Darwin J Prockop,3 Toshihiro Nukiwa.1 Department of Respiratory Medicine, Graduate School of Medicine, Tohoku University, Sendai, Miyagi, Japan; 2Institute of Sten Cell and Regenerative Medicine, University of Washington, Seattle, WA; 3Institute for Regenerative Medicine at Scott and White Hospital, Texas A&M Health Science Center, Temple, TX Previous studies have demonstrated that multipotent stromal cells (MSCs) enhance cell survival through upregulation and secretion of stanniocalcin-1 (STC1) in lactic acidosis and hypoxia These situation induce lots of intracellular ROS, therefore we hypothesized MSCs derived STC1 decreases ROS induced cell death This study shows that MSC derived STC1 promotes survival of lung cancer A549 cells by uncoupling oxidative phosphorylation, reducing intracellular reactive oxygen species (ROS), and shifting metabolism towards a more uncoupling metabolic profile MSC derived STC1 upregulated uncoupling protein (UCP2) in injured A549 cells in an STC1 dependent manner Knockdown of UCP2 reduced the ability of MSCs and recombinant STC1 (rSTC1) to reduce cell death in the A549 population rSTC1 treated A549 cells displayed decreased levels of reactive oxygen species, mitochondrial membrane potential, increased lactate production and increased oxygen consumption, all of which were dependent on the upregulation of UCP2 Our data suggest that S204 MSCs promote cell survival by regulating mitochondrial respiration via STC1 Furthermore, STC1 may provide promising avenues for treatment of reactive oxygen species and metabolic disorders 532 Neuropilin-1 Expressing Mononuclear Cells (NEMs), a Novel Population of Bone Marrow Cells Recruited by AAV2-Sema3A, Contributes to Vessel Normalization and Inhibits Tumor Growth Serena Zacchigna,1 Alessandro Carrer,1 Silvia Moimas,1 Lorena Zentilin,1 Mauro Giacca.1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology - ICGEB, Trieste, Italy Over the last several years, we have extensively exploited the properties of viral vectors based on the Adeno-Associated Virus (AAV) for the in vivo delivery of a variety of genes involved in blood vessel formation in various small and large animal models AAV vectors have the capacity to transduce non-replicating cells with high efficiency, in the absence of inflammation and immune response, and thus promote expression of their transgenes in vivo for indefinite periods of time In particular, we observed that the prolonged expression of VEGF-A165 and Sema3A, two Neuropilin-1 (Nrp-1) ligands, determined a massive infiltration of the expressing tissues by a population of bone-marrow derived mononuclear cells expressing Nrp-1 These Nrp-1 Expressing Mononuclear cells (NEMs) were found to be involved in vessel maturation through a paracrine effect ensuing in the activation and proliferation of tissue-resident mural cells (Zacchigna et al J Clin Invest 2008, 118, 2062) Consistent with the observation that NEMs quench angiogenic burden and promote vessel maturation, we observed that both AAV-Sema3A and NEMs exerted strong anti-tumoral effect In particular, we exploited AAV2-mediated gene transfer to overexpress Sema3A at the site of inoculation of either B16.F10 melanoma or T24.1 fibrosarcoma cells in immunocompetent mice We found that Sema3A markedly reduced tumor growth, without directly affecting tumor cell proliferation Similarly, the direct injection of NEMs, isolated from either the bone marrow or AAV-Sema3A-injected muscles, and administered to B16-F10 tumor-bearing animals, markedly impaired tumor growth Analysis of the vasculature of AAV2-Sema3A- or NEMtreated tumors revealed a more mature vascular network, associated to a higher degree of a-SMA+/NG2+ mural cell coverage, reduced vessel tortuosity and decreased vascular leakiness, indicating a role for NEMs in triggering vessel normalization Tumors treated with Sema3A or NEMs were smaller, better perfused, less hypoxic and with a reduced level of activation of HIF-1a Phenotypic characterization of purified NEMs revealed that they constitute a unique population of cells, having a CD11b+/Nrp1+/Gr1-/Tie2- phenotype These cells constitute ∼1.0% of total bone marrow cells and are characterized by a gene expression profile reminiscent of M1 polarized monocyte/ macrophages 533 Examining the Role of CD44 in the Tumor Tropism of Multipotent Mesenchymal Stem Cell (MSC) for Cancer Microenvironments Erika L Spaeth,1 Brian Toole,2 Ann Klopp,1 Michael Andreeff,1 Frank C Marini.1 Leukemia, MD Anderson Cancer Center, Houston, TX; 2Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC Multipotent mesenchymal stem/stromal cells (MSC) make excellent gene delivery vehicles because they are readily propagated in vitro, can be genetically modified, have high metabolic activity to support the production of therapeutic agents and have tumor tropic properties We have shown the propensity of MSC to migrate to and incorporate into the tumor stroma Once engrafted in the tumor, MSC are capable Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... specificity of oncolytic adenoviruses, we used a high affinity c- Met ligand to retarget viral infectivity to metastatic disease over expressing the receptor Specifically, we incorporated NK2, a competitive... particles that display functional fiber-fibritin-NK2 as a component of their capsid surface Importantly, this construct in the context of an oncolytic adenovirus, specifically infected cancer cell... adenoviruses, designed to divide in and specifically kill tumor cells, is a promising virotherapy approach for cancer treatment Ideally, cancer-specific replication of oncolytic adenoviruses results