844. A Single Vector-Dependent, Doxycycline Regulatable, Lung Cancer Specific Oncolytic Adenovirus

ADENOVIRAL VECTORS: CLINICAL APPLICATIONS mice and nu/nu mice bearing human breast cancer tumors was used to compare in vivo expression of TK in non-tumor and tumor tissues, respectively. Analysis of emission data from these studies showed that Ad-TK administration resulted in broad expression of TK activity in normal and tumor tissues, whereas expression of suicide gene activity from Ad-UTK vector administration was restricted to the human breast epithelial cells expressing high levels of eIF4E (MCF10A-4E) and the human breast cancer tumor tissues.

844. A Single Vector-Dependent, DoxycyclineRegulatable, Lung Cancer Specific Oncolytic Adenovirus Isaac Sipo,1 Almudena Hurtado Pico,1 Judith Wildner,1 Lennart Suckau,1 Sandra Pinkert,1 Stefan Weger,2 Wolfgang Poller,1 Henry Fechner.1 1 Cardiology & Pulmology, Charite - Universitaetsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany; 2Institute of Infectious Diseases, Department of Virology, Charite Universitaetsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany. Tight regulation of oncolytic adenoviruses (oAdV) represents an important requirement for their safe application. We have developed a new doxycycline (Dox)-dependent oAdV with a bidirectional expression cassette, which drives the expression of the reverse tetracycline-controlled transactivator (rtTAs-M2) from a lung tumorspecific promoter and, in the opposite direction, the expression of the adenoviral E1A gene from a second generation tetO7 sequence linked to an isolated TATA box. In H441 lung cancer cells, this oAdV showed a strictly Dox-dependent E1A expression, adenoviral replication, cell killing activity and a 450-fold induction of progeny virus production. Furthermore, the virus could be shut off again by withdrawal of Dox and, in contrast to an oAdV expressing E1A directly from the SP-B promoter, did not replicate in non-target HeLa cells. In contrast, when the isolated TATA box in the E1A expression cassette was replaced by a CMV minimal promoter, the corresponding oAdV already expressed significant amounts of E1A in the absence of the inducer. These were sufficient to prevent any Dox-dependent regulation of adenoviral replication. These results underline the importance of non-leaky E1A expression for preventing undesirable oAdV replication and demonstrate, for the first time, Dox-dependent oAdV replication from a single adenoviral vector genome.

845. The Loss Expression of Coxsackie and Adenovirus Receptor Enhances the Feature of Aggressive Bladder Cancer Kazumasa Matsumoto,1 Sharokh F. Shariat,2 Akira Irie,1 Tetsuo Fujita,1 Hiroshi Okusa,1 Takefumi Satoh,1 Masatsugu Iwamura,1 Shiro Baba,1 Seth P. Lerner.1 1 Department of Urology, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan; 2Scott Department of Urology, Baylor College of Medicine, Houston, TX. Objectives. Adenovirus is a useful vector for cancer gene delivery because of its high gene transfer efficacy, high titer production, and safety. The initial binding of adenoviruses except for serotype B to cell surface is mediated via a specific 46 kDa coxsackie and adenovirus receptor (CAR). In addition, recent evidence suggested that CD46, a complement inhibitory protein, plays a role as a serotype B Ad receptor. Low levels of CAR are associated with decreased efficiency of adenovirus-mediated gene transduction of bladder cancer. In present study, we evaluated the expression of CAR and CD46 in urologic cancer cell lines, and then examined whether CAR expression is associated with bladder cancer characteristics and clinical outcomes. S326

Methods. CAR and CD46 expression in urologic cancer cell lines were determined by flow cytometry. Immunohistochemical staining for CAR was carried out on surgical specimens from 62 patients who underwent radical cystectomy. Immunoreactivity was categorized on a 0-3+ semiquantitation system for both intensities of staining and the percentage of positive cells (labeling frequency %). We also stained 30 tumor section specimens from normal sections for microvessel density, E-cadherin, pRB, p16, p21, p53, cyclooxygenase-2, and transforming growth factor-beta1 and its receptors type I and II. Results. Low expression of CAR and high expression of CD46 were determined in urologic cancer cell lines. In immunohistochemical staining, CAR expression was lost in 17/62 (27%) tumors. Loss of CAR expression was associated with metastases to regional lymph nodes (p=0.049), muscle-invasive disease (p=0.025), high grade disease (p=0.038), altered p53 status (p=0.041), and loss of Ecadherin expression (p=0.042). With a median follow-up of 60 months, loss of CAR expression was associated with decreased bladder cancer-specific survival (p=0.029) but not disease progression. When adjusted for the effects of clinicopathologic findings, only lymph node metastasis was associated with bladder cancer progression and mortality. Conclusions. Loss of CAR expression is associated with established markers of biologically aggressive bladder cancer. The CAR dependence of the adenovirus vector may result in sequestration of recombinant virions by biologically less aggressive yet high-CARexpressing bladder cancer cells, whereas the targeted aggressive bladder cancer cells, low in CAR, will be poorly transduced. The association of CAR with E-cadherin and p53 suggests a potential role for CAR in the regulation of urothelium integrity and cell cycle.

846. Enhanced Antitumor Effect of Oncolytic Adenovirus Expressing Interleukin-12 and B7.1 in an Immuneocompetenet Mouse Model Ji Young Yoo, Young-Sook Lee, Jaesung Kim, Hyejin Choi, JooHang Kim, Chae-Ok Yun. 1 Brain Korea 21 Project for Medical Science, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Republic of Korea. Interleukin (IL)-12 is reported to exhibit potent anti-tumor effect by promoting NK cell and cytotoxic T cell activities. The B7 family has been shown to take an important role in stimulating anti-tumor response, and synergy between IL-12 and B7-1 for cancer immunotherapy has previously been demonstrated. To increase the potential antitumor activity of the oncolytic adenovirus, we have constructed an E1B 55kDa deleted oncolytic adenoviral vector, YKLIL12/B, which expresses murine IL-12 and B7.1. The therapeutic efficacy of YKL-IL12 and YKL-IL12/B was evaluated in an immunocompetent mouse bearing murine melanoma B16F10 tumor. The results showed significant inhibition of tumor growth following YKL-IL12/B treatment compared to PBS treated tumors. Moreover, YKL-IL12/B adenoviral vector elicited enhanced antitumor activity and a higher incidence of complete tumor regression compared to tumors treated with the analogous vector (YKL-1) that lacks IL-12 and B7.1. Survival was also significantly prolonged in YKL-IL12/ B-treated mice, and immunohistochemistry analysis demonstrated robust CD4+ and CD8+ T-cell infiltration in these mice compared to the YKL-1 subjects. In addition, we observed that YKL-IL12/B induced significantly higher CTL activity than YKL-1, demonstrating that anti-tumor effect is associated with the generation of tumorspecific immune response. In summary, these data indicate that local expression of IL-12 and B7.1 in tumor bed may be an attractive alternative treatment against metastatic carcinoma.

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy