410 T Cell Engager Armed Oncolytic Vaccinia Virus Significantly Enhances Antitumor Therapy Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S[.]
CANCER - ONCOLYTIC VIRUSES I 408 Systemic Administration of Fiber Redesigned Infectivity-Selective Oncolytic Adenovirus for Cancer Treatment Yoshiaki Miura,1 Joohee Han,1 Julia Davydova,1 Masato Yamamoto.1 Department of Surgery, University of Minnesota, Minneapolis, MN Adenovirus (Ad) has been used as a backbone of oncolytic viral agents, while lack of selectivity to cancer cells at the stage of infection has greatly hampered in vivo efficacy Unlike loco-regional therapy, systemic application of cancer gene therapy mandates different level of selectivity of gene delivery The controlled vector distribution and cancer selective transduction would overcome the obstacles for systemic delivery and enable efficient systemic treatment of cancer We have recently developed a new class of oncolytic virus system, Infectivity-Selective Oncolytic Adenovirus (ISOAd) via direct screening of a high diversity (109-level) targeting ligand library in replicative Ad format The AB-loop region of the fiber knob has been applied to our library system The Ad library has been screened against a target molecule, mesothelin (MSLN) which is highly over-expressed in pancreatic cancer, not in normal tissue, as well as against an unknown target on PC3 cells which rarely express MSLN and primary Ad receptor Ads with redesigned AB-loop, AdML-VTIN targeting MSLN and AdML-VRLL targeting PC3 cells, were successfully isolated from the high-throughput screening of same Ad library by infectivity The in vitro binding of AdML-VTIN corresponded to the level of MSLN-expression and the suppression of MSLN with siRNA or antibody significantly reduced the binding to MSLN-expressing cells, which indicates that mesothelin is a receptor moiety for the isolated targeting ligand In PC3, the binding of AdML-VRLL was significant higher than that of wild-type Ad5 and AdML-VTIN The viral burst corresponded with the binding ability of each virus These data indicate that ISOAd system can identify transductionallytargeted Ad against both particular target molecules and unknown targets Next, the in vivo selectivity and therapeutic effect of the MSLN-retargeted Ad was examined The intra-tumor injection of AdML-VTIN showed significant antitumor effect against Panc-1 tumors (MSLN-positive), and about half of the tumor disappeared Contrarily, the same virus showed no antitumor effect in MiaPaCa-2 (MSLN-negative) Viral DNA quantitation also supported the selective viral replication in Panc-1 Upon systemic administration in the nude mice with Panc-1 subcutaneous xenografts, AdMLVTIN showed significantly lower liver sequestration compared to the wild-type Ad5, and the copy number of this retargeted virus was significantly (1000-fold) higher in the tumor These data exhibits that AB-loop redesigned Ad-vector enables efficient systemic delivery and treatment of target-molecule expressing cancer and enables reduction of vector distribution to liver In this study, our AB-loop redesigned Ad vectors exhibited selective infectivity to a known target (MSLN) and an unknown target expressed on cancer cells Moreover, MSLN-targeted Ad showed selective and potent antitumor effect against MSLN-positive pancreatic cancer cells in vivo, dramatically augmented delivery to the tumors, and significantly reduced liver sequestration upon systemic administration This new genetically modified Ad, which is transductionally retargeted to cancer, may embody a next generation of systemic therapy for cancer 409 Therapeutic Efficacy of Oncolytic Herpes Virus and Chemotherapy for Treatment of LateStage Ovarian Cancer Chelsea M Bolyard,1 Balveen Kaur.1 Neurosurgery, The Ohio State University, Columbus, OH Ovarian cancer is the 5th most deadly cancer in women, with twothirds of women presenting with disease that has spread to abdominal Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy organs or distant sites We utilize a murine xenograft model of ovarian cancer In this model, we inject female nude mice with ovarian cancer cells intraperitonally, which yields progressive disease that includes abdominal dissemination, tumor nodular formation through the IP cavity and bloody ascites With this study, we examine the use of oncolytic HSV-1(oHSV) with the conventional chemotherapeutic drug doxorubicin for treatment of the murine xenograft model of ovarian cancer We utilize two engineered oHSV, both of which are oncolytic due to a truncated UL39, which ablates the viral ribonucleotide reductase 34.5-Expresed by Nestin promoter and Vasculostatin-120 Expressing (34.5ENVE) also expresses immunomodulatory protein 34.5 under the nestin promoter, and the extracellular fragment of anti-angiogenic molecule Brain Angiogenesis Inhibitor-1 (BAI1), Vasculostatin-120 under viral Immediate Early 4/5 promoter The second virus, revertant ENVE, was generated by removing the transgene containing the 34.5 and VStat120 genes from 34.5ENVE Ovarian cancer(OvCa) cell lines displayed at least a 2-fold increase in sensitivity to 34.5ENVE-mediated killing as compared to revertant, with some cell lines showing over a 10-fold increase in sensitivity Further, when SKOV3.ip1 cells were grown in vivo, and tumor ascites were isolated from the mice (ascites-1 ascites-2), the cells from ascites had an increase in Nestin expression over the parental adherent cell line, of 10.4- and 11.1-fold These ex vivo ascites tumor cells also had increase sensitivity to 34.5ENVE, as compared to cells grown in tissue culture The EC50 for ascites-1 and -2 were an MOI of 0.0026 and 0.0085, respectively; EC50 for SKOV3.ip1 was 0.052 OvCa cell lines infected with 34.5ENVE secreted functional Vstat120 that was capable of inhibiting HDMEC migration by 26.4%, as assessed by migration assay (p=0.0074) An in vivo survival study showed a significant increase in survival of mice with human ovarian cancer when treated with oHSV, with a greater increase in median survival time in 34.5ENVE-treated cohort as compared to revertant-treated Median survival of PBS treated mice was 37 days, revertant was 49 days, and 34.5ENVE extended survival to 63 days Furthermore, 34.5ENVE treatment reduced the number of mice dying due to bloody ascities from 100% in PBS cohort (8/8), to 20% in ENVE-treated cohort (2/8) We also examined whether conventional chemotherapeutic doxorubicin could augment cytotoxicity mediated by oHSV We found that in the cell lines we tested, doxorubicin was able to synergize with 34.5ENVE, as assessed by Chou-Talalay analysys (using CompuSyn program) Doxorubicin pre-treatment increased viral transduction and transgene expression in both SKOV3 and A2780-CR When OvCa cells are pretreated with doxorubicin, and then infected with ENVE, there is a more robust increase in apoptotic cells, as assessed by Annexin V/7AAD staining Animal studies testing the ability of doxorubicin and ENVE to increase survival in vivo are on-going 410 T-Cell Engager-Armed Oncolytic Vaccinia Virus Significantly Enhances Antitumor Therapy Feng Yu,1 Stephen M Gottschalk,1 Xiao-Tong Song.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX Oncolytic vaccinia virus (VV) therapy has shown promise in preclinical models and in clinical studies Complete responses, however, have rarely been observed The limitations presented by VV therapy typically have been suboptimal virus spread through the tumor and the limited induction of antigen-specific T-cell responses Here we describe a novel strategy to overcome these limitations We have constructed T-cell Engager Armed Vaccinia Viruses (TEA-VVs) encoding secretory bi-specific T-cell engager (single chain variable fragment, scFv) that binds both to CD3 and a tumor cell surface antigen EphA2 (EphA2-TEA-VV) The expression of EphA2-TE was demonstrated in cell culture Also, the replication and oncolysis efficiency of EphA2-TEA-VV in cell culture were similar to that of S157 CANCER - ONCOLYTIC VIRUSES I the parental virus However, the EphA2-TEA-VV killed EphA2positive tumor cells A549 more effectively in the presence of human PBMC as determined by target cell lysis, compared to unmodified VV In addition, EphA2-TEA-VV induced bystander killing of tumor cells that are not infected with virus as determined by in vitro assay Importantly, EphA2-TEA-VV resulted in a significant decrease in tumor growth in an established A549 lung cancer model Thus, TEAVV represents a unique and effective strategy to improve oncolytic virus therapy by engaging T cells for cancer therapy 411 Improving the Oncolytic Activity of Measles Virus by Enhancing the Neutrophil-Mediated Response to Infected Tumor Cells Aditi Dey,1 Yu Zhang,1 Anna Z Castleton,1 Bella Patel.1 Haematology, UCL, London, United Kingdom We have previously shown that neutrophils play a role in the oncolytic activity of attenuated measles virus (MV) both in-vivo and in-vitro To investigate further, we carried out neutrophil depletion experiments using the Ly6G antibody (or isotype control) in two subcutaneous SCID mouse xenograft models of B cells malignancy; Raji - Burkitt’s lymphoma and Nalm-6 - acute lymphoblastic leukaemia (ALL) Both tumors responded to MV therapy as compared to UV-inactivated control, with the Nalm-6 tumors regressing more quickly and completely than Raji tumors Depletion of neutrophils in the Nalm-6 model did not abrogate the therapeutic effect of MV By contrast, depletion of neutrophils in the Raji model abrogated the therapeutic effect of MV whereas the isotype control antibody did not affect anti-tumor response In order to investigate potential mechanisms underlying the differential anti-tumor effect of neutrophils in the two models, we examined neutrophil-mediated cytotoxicity in-vitro, using chromium release assays MV-infected (or uninfected) targets (Jurkat, Raji and Nalm-6 cells) were incubated with neutrophils from a number of different healthy donors We used two different vaccine strains of MV; MV-NSe and MV-Moraten (MV-Mor) MV-Mor-enhanced neutrophil cytotoxicity was seen against all three cell lines tested whereas MV-NSe only significantly enhanced neutrophil cytotoxicity against Jurkat cells To investigate whether antibody dependent cellular cytotoxicity was involved, the experiment was repeated with high-titre anti-MV anti-serum, in the Jurkat model Surprisingly, there was a different response to the antiserum between the two strains of MV with significant enhancement in neutrophil mediated killing with MV-Mor infection (p=0.038) but a significant decrease with MV-NSe infection (p=0.0005) Various explanations for this are being investigated Since neutrophils clearly play a role in MV-mediated cytotoxicity both in-vivo and in-vitro, with a possible role for ADCC, we cloned and rescued MVs expressing the human cytokine granulocyte colony stimulating factor (MVhGCSF), which also has significant activity at the murine GCSF receptor MVhGCSF showed similar growth characteristics to unmodified MV in-vitro and could infect Raji, Nalm-6 and Jurkat cells, resulting in production of hGCSF at concentrations 200ng-600ng/ml Infection of neutrophils from healthy donor resulted in hGCSF production of 2-3ng/ml In-vitro evaluation of neutrophil mediated cytotoxicity by MVhGSCF is ongoing Meanwhile the response of Raji xenografts to MVhGCSF was evaluated in SCID mice MVhGCSF resulted in a better anti-tumor response than the non-modified MV In conclusion, neutrophils play a role in the anti-tumor response to MV both in-vivo and in-vitro although there are clearly several parameters permitting response which remain to be more closely defined Enhancement of neutrophil function may have utility in enhancing the anti-tumor response to oncolytic MV S158 412 Directed Evolution of Oncolytic HSV To Overcome Resistance to Virotherapy Due to Low Entry Receptor Expression Pin-Yi Wang,1 Pearlly Yan,3 Ralf Bundschuh,4 Hok-Hei Tam,5 Timothy P Cripe.1,2 Center for Childhood Cancer and Blood Diseases, The Research Institute and the Division of Hematology/Oncology/BMT, Nationwide Children’s Hospital, Columbus, OH; 2Department of Pediatrics, The Ohio State University, Columbus, OH; Comprehensive Cancer Center, The Ohio State University, Columbus, OH; 4Department of Physics and Deaprtment of Chemistry & Biochemistry, The Ohio State University, Columbus, OH; 5Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA Oncolytic Herpes simplex virus (oHSV) has shown promising antitumor efficacy in many preclinical models oHSV exhibits a wild range of cell tropism, in which at least four cell surface molecules are recognized as redundant major HSV entry receptors We identified a primary neuroblastoma patient-derived EBV-infected lymphocyte culture that expresses very low level of known HSV entry receptors making it relatively resistant to oHSV infection To overcome the low receptor expression barrier, we adapted a “directed evolution” strategy to select out recombinants via serial passage of oHSV in this non-permissive culture To encourage genetic diversity, we used two different mixed pairs in the initial infection comprised of: (i) the wild types KOS plus 17syn+ or (ii) the strain KOS ICP6- mutant rRp450 plus the strain 17 ICP34.5 mutant 17termA, followed by 19 additional passages in the non-permissive line Compared with the “input” parent viruses, significant cytotoxicity was seen even as early as passage and was more pronounced by passages 10 and 15 Two potent viruses that effectively target the low receptor expression line were selected and plaque-purified The mutant pair clearly demonstrated a giant syncytial plaque phenotype while the wild-type pair plaque remained normal size Whole genome sequence analysis revealed that the mutant pair acquired the intact ICP34.5 from rRp450 and ICP6 from 17termA except for two nucleotide differences, suggesting a wild-type virus-like genotype In addition, at least gene loci contain unique alterations compared with either parent virus that lead to amino acid changes in the recombinant virus Since the newly selected mutants were even more potent than the parent or corresponding wild-type viruses, our results suggest those unique genomic alterations may play a significant role in bypassing the low receptor barrier The most interesting mutant involved Us8, which encodes glycoprotein E that mediates cell-cell fusion Further study will focus on unveiling the underlying mechanism and translating this knowledge to design a therapeutic agent that better targets low receptor-expressing tumors 413 VSV Pseudotyped with Maraba Virus G Glycoprotein Escapes Serum Neutralization Mulu Z Tesfay,1 Arun Ammayappan,1 Mark J Federspiel,1 Glen N Barber,2 David F Stojdl,3 Kah-Whye Peng,1 Stephen J Russell.1 Department of Molecular Medicine, Mayo Clinic, Rochester, MN; Department of Medicine and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL; Children’s Hospital, Eastern Ontario Research Institute, Ottawa, ON, Canada To enhance the utility of oncolytic vesicular stomatitis virus (VSV) for systemic treatment of multiple myeloma (MM), an incurable, disseminated malignancy of antibody secreting plasma cells, we are developing strategies to enhance its systemic delivery One of the presumed advantages of using VSV as an oncolytic virus is that human infections are rare and preexisting anti-VSV immunity is therefore lacking, which is very important for clinical success However, our studies show that non-immune human serum rapidly Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy ... neutrophil cytotoxicity against Jurkat cells To investigate whether antibody dependent cellular cytotoxicity was involved, the experiment was repeated with high-titre anti-MV anti-serum, in the... not affect anti-tumor response In order to investigate potential mechanisms underlying the differential anti-tumor effect of neutrophils in the two models, we examined neutrophil-mediated cytotoxicity... in the Nalm-6 model did not abrogate the therapeutic effect of MV By contrast, depletion of neutrophils in the Raji model abrogated the therapeutic effect of MV whereas the isotype control antibody