840. Markedly Enhanced Cytolysis by E1B-19kD-Deleted Oncolytic Adenovirus in Combination with Cisplatin

ADENOVIRAL VECTORS: CLINICAL APPLICATIONS conjugates can enhance the transduction of colonic tissue by AAV in a mouse colitis model. We are now asking if intra-colonical administration of AAV carrying the mouse IL-10 gene in the form of virus-microbead conjugates could raise local IL-10 levels for the amelioration of colitis by efficient transduction of the inflamed colon. These results suggest the potential for this gene transfer technology to serve as an effective gene therapy strategy for IBD. A clear advantage of this technology over other virus-mediated gene transfer strategies for the therapy of IBD, including those involving systemic delivery of viral vectors, is that the delivery of viral vectors and subsequent production of IL-10 (or other therapeutic proteins) is focused at inflamed lesions. In addition, since all viral particles are anchored stably to microbeads, uncontrolled delivery of viral vectors to and subsequent transduction of other organs should be minimized. These characteristics should not only maximize the therapeutic effectiveness but also enhance the safety of the use of viral vectors.

838. 10 Year Follow-Up of 146 Advanced Stage NSCLC Patients Who Received Adenoviral Based Therapy John Nemunaitis,1 Been Pappen,1 Rosemarie Arzaga,1 Casey Cunningham,1 Neil Senzer.1 1 Mary Crowley Medical Research Center, Mary Crowley Medical Research Center, Dallas, TX. We report the first long-term follow-up of adenoviral vector based therapeutics patients who previously received anticancer therapy. Over the period of 10/26/95 to 8/15/05 146 patients with stage IIIB/ IV NSCLC were treated as out patients. Patients participated in 1 of 8 adenoviral based therapeutic trials involving 6 different agents. All were evaluable for long term unexpected toxic effect and survival. The study agent and number of patients is as follows: Bystander GVAX, 35; GVAX, 53; Adenoviral p53, 51; Onyx 015, 5; TNFeride, 1; MDA7, 1. Early safety and activity results have previously been reported in the literature(see below). Male/female ratio was 55%/ 45%, and median age was 60 years (19-85 years). No long term unexpected toxicity was reported. Overall mean survival was 334 days (95% CI: 269 to 400), median survival was 199 (95% CI: 168 to 320) days. No difference in survival was demonstrated by gender (Log Rank, p = 0.187) or age (Log Rank, p = 0.392). Survival at 1 year and at 2 years for patients <60 years and > 60 years respectively was 32% and 16% vs. 27% and 5%. These results are consistent with expected survival described in the literature of similar advanced stage NSCLC patients over the same time period and suggest no evidence of long term adverse effect related to adenoviral vector therapy. Four patients remain alive more than 3 years after treatment. 1.Nemunaitis J, Jahan T, Ross H, et al. Phase 1/2 trial of autologous tumor mixed with an allogeneic GVAX® vaccine in advanced-stage non-small-cell lung cancer. Cancer Gene Therapy 2006 Jan 6; [epub ahead of print]. 2.Cunningham CC, Chada S, Merritt J, et al: Clinical and Local Biological Effects of an Intratumoral Injection of mda-7 (INGN 241) in Patients with Advanced Carcinoma; a Phase I Study. Molecular Therapy 2005 Jan; 11(1):149-59. 3.Nemunaitis J, Sternman D, Juhn K, et al. Phase I/II Study of GMCSF GeneModified Autologous Tumor Vaccine (GVAX) in Early and Advanced Stage Non Small Cell Lung Cancer. Journal of the National Cancer Institute 2004; 96: 326-331. 4.Senzer N, Mani S, Rosemurgy A, et al. TNFerade Biologic, an Adenovector with a radiationinducible promoter, carrying the Human Tumor Necrosis Factor Alpha Gene: A phase I study in Patients with Solid Tumors. Journal of Clinical Oncology 2004 Feb; 22(4):592-601. 5.Nemunaitis J, Cunningham C, Buchanan A, et al: Intravenous infusion of a replication-selective adenovirus (ONYX-015) in cancer patients: Safety, feasibility and biological activity. Gene Therapy 8(10):74659, 2001 May. 6.Nemunaitis J, Swisher G, Timmons T, et al: Adenovirus-mediated p53 gene transfer in sequence with Cisplatin S324

to tumors of patients with non-small cell lung cancer. Journal of Clinical Oncology 18(3):609-622, 2000. 7.Swisher SG, Roth JA, Nemunaitis J, et al. Adenoviral-mediated p53 gene transfer in advanced non-small cell lung cancer. J Natl Cancer Inst 91(9):763771, 1999. May

839. Long-Term Knockdown of Phospholamban in Cultured Neonatal Rat Cardiomyocytes by a Viral Vector Expressing PLB-shRNAs Lennart Suckau,1 Jens Kurreck,2 Roland Vetter,3 Stefan Weger,4 Jos Lamers,5 Wolfgang Poller,1 Henry Fechner.1 1 Cardiology & Pulmology, Charite - Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany; 2Institute for Chemistry (Biochemistry), Freie Universität Berlin, Berlin, Germany; 3Institute of Clinical Pharmacology and Toxicology, Charite - Universitaetsmedizin Berlin, Berlin, Germany; 4Institute of Infectious Diseases, Department of Virology, Charite Universitaetsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany; 5Department of Biochemistry, Cardiovascular Research School COEUR, Erasmus University, Rotterdam, Netherlands. Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum (SR) Ca2+ pump (SERCA2a) is crucial for contractile dysfunction in heart failure. Recently, downregulation of PLB by a synthetic small interfering (si) RNA was found to be linked to up-regulation of the SR Ca2+ pump activity of neonatal rat cardiomyocytes. However, the application of latter approach under in vivo conditions may be limited due to the low transfer efficacy and the poor stability of siRNA. Therefore, we constructed an adenoviral vector expressing a PLB-shRNA from a murine polymerase III U6 promoter for knocking down PLB expression. The efficacy of this vector to suppress PLB expression and to modulate SERCA2acatalysed reticular Ca2+ transport was examined. Transduction of cultured neonatal rat cardiomyocytes with the vector resulted in a marked and sustained decrease of PLB gene expression. At day 2 after transduction, the PLB-mRNA signal was suppressed by more than 95% and stayed at this low level until the end of the experiment at day 13. At the protein level, maximum PLB downregulation was achieved at day 7 (-90% vs. control). SERCA2a and Na/Ca exchanger expression were not altered. PLB knockdown was associated with a marked increase of the oxalate-supported Ca2+ uptake rate that was measured in cell homogenates at 0.34 µM free Ca2+. Thus, the use of adenoviral PLB-shRNA is a powerful approach for a long-term increase of the SERCA2a-catalysed Ca2+ transport into the SR of cultured neonatal rat cardiomyocytes.

840. Markedly Enhanced Cytolysis by E1B19kD-Deleted Oncolytic Adenovirus in Combination with Cisplatin A-Rum Yoon,1,2 Young-Sook Lee,1,2 Ji-Young Yoo,1,2 Hoguen Kim,1,3 Jinsun Kim,1,2 Joong Bae Ahn,2 Joo-Hang Kim,1,2 ChaeOk Yun.1,2 1 Brain Korea 21 Project for Medical Sciences, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea; 2Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea; 3 Department of Pathology, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea. Oncolytic adenoviruses are currently being developed as novel antitumor therapeutics. To enhance their therapeutic potential, adeonoviruses are being administered in combination with standard chemotherapy. Adenoviral vectors used in these clinical trials, however, can be destructive as they encode intact E1B 19-kDa protein, which can block the apoptotic pathway induced by a variety Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

ADENOVIRAL VECTORS: CLINICAL APPLICATIONS of chemotherapeutic agents. Previously, we have shown that oncolytic adenovirus Ad-∆E1B19/55, deleted for sequence encoding E1B 19-kDa and E1B 55-kDa proteins, exhibits marked enhancement in cytolytic and apototic activity (Kim et al., 2002). In the current study, we assessed the therapeutic value of Ad-∆E1B55 and AdE1B19/55 in combination with cisplatin. A marked increase in cytotoxicity was observed for both Ad-∆E1B55 and Ad-∆E1B19/ 55 when combined with cisplatin. Relative to each other in all cell lines examined, the combination of the double-deleted adenovirus, Ad-∆E1B19/55, plus cisplatin exhibited greater cell-killing effect than did the single-deleted adenovirus, Ad-∆E1B55, plus cisplatin. Propidiun iodide staining and TUNEL assay analysis also revealed that combination of cisplatin with Ad-∆E1B19/55 caused greater induction of apoptosis than that with Ad-∆E1B55. Similarly in vivo, the combination of Ad-∆E1B55 or Ad-∆E1B19/55 with cisplatin also induced greater antitumor effect in a human cervical xenograft model. TUNEL staining showed that the apoptotic level was significantly higher in tumor tissue treated with Ad-∆E1B19/55 plus cisplatin than with any other treatment. In addition, viral presence was confirmed by the immunohistological staining with increased numbers of adenoviral particle detected in wider areas of tumors treated with Ad-∆E1B19/55 oncolytic adenovirus plus cisplatin. Taken together, these findings demonstrate that cisplatin in combination with E1B- 19kD-deleted oncolytic adenovirus may enhance therapeutic efficacy (via active induction of apoptosis), eliciting greater efficacy profile than that with E1B-19kD-expressing oncolytic adenovirus.

841. Anti-Tumor Effect of Tumor Specific Replicating Adenovirus Expressing IL-12 and IL-18 Jing-Hua Huang, Il-Kyu Choi, Sung-Mi Jung, Minjung Kim, JooHyuk Sohn, Chae-Ok Yun, Joo-Hang Kim. 1 Brain Korea 21 Project for Medical Science, Institute for Cancer Research, Yonsei Cancer Reaserch Center, Yonsei University College Of Medicine, Seoul, Republic of Korea. Cancer immunotherapy denotes a strategy for activating a host’s immune response for the treatment of malignancy. Although cancer cells are less immunogenic, immune system is capable of recognizing and eliminating cancer cells. Oncolytic adenoviral vectors are currently being developed as biologic anti-tumor agents, and coupling the lytic function of an oncolytic adenovirus with its ability as a transgene delivery system represents a powerful extension of this methodology. IL-12 is reported to exhibit potent anti-tumor effect by promoting NK cell and cytotoxic T cell activities. IL-18 is also have shown that augments cytotoxicity of NK cells and proliferation of T cells. It stimulates Th1 cells to produce IL-2 and IFN-γ. This effect augmented in a synergistic manner IL-12. To increase the potential anti-tumor effect of the oncolytic adenovirus, we have generated an E1B deleted and E1A mutated oncolytic adenoviruses, Ad-∆B7-IL12, Ad-∆B7-IL18, or Ad-∆B7/IL12-IL18 that expresses IL-12, IL-18, or IL-12 plus IL-18, respectively. The therapeutic efficacy of these oncolytic adenoviruses was then evaluated in immunocompetent C57BL/6 mice bearing murine melanoma B16F10 tumor. The results showed significant inhibition of tumor growth following Ad-∆B7/IL12-IL18 treatment compared to Ad-∆B7, Ad∆B7-IL18, or Ad-∆B7-IL12 treated tumors. Moreover, the oncolytic adenovirus expressing both IL-12 and IL-18 demonstrated enhanced anti-tumor effect and higher incidences of complete tumor regression compared to Ad-∆B7, Ad-∆B7-IL18, or Ad-∆B7-IL12. To establish that the observed anti-tumor effect is associated with the generation of a tumor-specific immune response, we examined the cytolytic activity by IFN-γ ELISpot assay and CTL assay. We observed that Ad-∆B7/IL12-IL18 induced significantly higher T cell-mediated antitumor effect than its cognate controls, Ad-∆B7, Ad-∆B7-IL12, and Ad-∆B7-IL18. Furthermore, immunohistochemical studies Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

demonstrated robust CD4+ and CD8+ T-cell infiltration in these mice compared to the Ad-∆B7, Ad-∆B7-IL18, or Ad-∆B7-IL12treated subjects. These data indicate that oncolytic adenovirusmediated IL-12 and IL-18 gene transfer provides a potential therapeutic strategy for the management of neoplasia.

842. Novel Oncolytic Adenoviruses for Cancer Gene Therapy Devanand Sarkar,1 Zao-zhong Su,1 Paul B. Fisher.1 Urology, Columbia University Medical Center, New York, NY.

1

Conditionally replication competent adenoviruses (CRCA) are being stringently evaluated for their ability to replicate selectively in cancer cells and induce oncolysis. However, recent clinical trials in different cancer indications demonstrate that oncolytic adenoviruses alone are not sufficient to significantly deter the precipitous course of the disease suggesting that additional approaches are necessary to bolster oncolysis by these agents. For replication competent adenoviruses to be successful for cancer treatment, they must replicate only in cancer cells with minimal toxicity to normal tissues. The promoter region of Progression Elevated Gene-3 (PEG-Prom) has the unique ability to function selectively in cancer cells with minimum to no activity in normal cells. We have created several CRCA in which the expression of the E1A gene of Ad, necessary for replication, is under the control of PEG-Prom and which simultaneously express a cancer-specific tumor suppressor/immunomodulatory gene in the E3 region of the Ad. We have demonstrated that these CRCA display cancer cell specific replication, transgene expression and induction of apoptosis and necrosis without harming normal cells. Intratumoral injection of these CRCA completely eradicates both primary and distant (representative of metastasis) tumors in established human cancer xenografts in athymic nude mice. These novel CRCA might thus be effective tools to induce complete remission of cancers and need to be stringently evaluated in immunocompetent animal models of cancer and Phase I/II clinical trials for their effective translation for the actual treatment of cancer patients.

843. MicroPET Imaging of HSV-TK Activity To Assess Cancer-Specific Gene Expression Targeted at the Level of Protein Translation Initiation Don A. Sibley,1 Shayne Barlow,1 Ricky DeBenedetti,1 David T. Curiel,2 J. Michael Mathis.1 1 Gene Therapy Program, LSU Health Sciences Center, Shreveport, LA; 2Center for Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL. Current cancer gene therapies are hindered by poor gene delivery and lack of tumor specificity. In order to enhance tumor specificity, the therapeutic suicide gene HSV-1 thymidine kinase (TK) sequence was modified with a 5’ upstream-untranslated region (5’-UTR) from the rat bFGF mRNA. This modification restricts protein translation of the suicide gene and cytotoxicity to cancer cells previously shown to express high levels of the translation initiation factor eIF4E. MicroPET scans utilizing a MicroPET rodent four-ring system (modelR4) from CTI Concord Microsystems, LLC (Knoxville, TN) imaging the radiolabled HSV1-TK substrate 18F-penciclovir were used to test suicide gene delivery and selective expression in tumor cells with in vitro cell cultures and in vivo animal models. A nonlytic adenovirus vector containing the HSV1-TK gene (Ad-TK) or the HSV1-TK gene modified with a 5’-UTR (Ad-UTK) was used to deliver the suicide gene in both model systems. In vitro model studies compared TK expression from the two vectors in human breast epithelial cells MCF-10A, and MCF-10A cells stably transfected to express high levels of eIF4E (MCF-10A-4E). Administration of Ad-TK and Ad-UTK to non-tumor bearing nu/nu S325