A Fiber Modified SLPI Promoter Based Conditionally Replicating Adenovirus for Treatment of Ovarian Cancer

TARGETED CANCER THERAPIES III We tested its efficiency and specificity by infection of EC and nonEC. The biodistribution of the vector and transgene was assayed by RT-PCR conducted on organs of systemically injected tumor-bearing mice. Humoral response against the vector and the transgene was evaluated by ELISA test. The anti-tumoral of the vector was assayed on the models of B16 melanoma and Lewis Lung Carcinoma (LLC) lung metastases. Results Infection of EC with Ad-PPE-Fas-c resulted in transgene mRNA transcription and dose-dependent Fas-c protein expression. Infection of EC but not of non- EC induced cell death and sensitized the cells to the pro-apoptotic effect of TNFα In contrast, the vector containing Fas-c gene driven by the non-EC-specific promoter CMV (AdCMV-Fas-c) induced apoptosis both in EC and non- EC. Biodistribution assay demonstrated that following systemic administration, transcriptional control of Fas-c by PPE led to restricted expression in the tumor-bearing lung while no expression was detected in other tissues. In contrast, Ad-CMV-Fas-c injected mice expressed Fas-c mRNA in most tissues tested. Humoral immune response to the transgene was minimal in Ad-PPE-Fas-c injected mice, compared to Ad-CMV-Fas-c injected mice. In the model of B16 melanoma, systemic administration of the vector resulted in growth retardation of tumor starting at day 7 post injection. In LLC model, vector administration led to 56% reduction of lung metastases mass (p< 0.05). Histological analysis, including CD31 staining and TUNEL showed damaged tumor blood vessels and apoptosis of vascular endothelium and adjacent tumor cells. In contrast livers of treated animals were not affected. Conclusions We developed a system for transcriptional targeting of tumor vasculature. This system has several advantages: i) It makes use of a promoter that is expressed efficiently and selectively in angiogenic endothelial cells ii) The vector utilizes a potent apoptosis-inducing gene activated by a “tumor-specific”, TNFα ligand. Hence, a supplementary level of specificity to tumor angiogenesis is provided

970. A Fiber Modified SLPI Promoter Based Conditionally Replicating Adenovirus for Treatment of Ovarian Cancer 1

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Daniel T. Rein, Martina Breidenbach, Minghui Wang, Tyler O. Kirby,2 Gene P. Siegal,3 Gerd J. Bauerschmitz,4 Peter Dall,4 Dirk M. Nettelbeck,1 Akseli Hemminki,5 Ronald D. Alvarez,2 David T. Curiel.1 1 Division of Human Gene Therapy, Departments of Medicine, Surgery, Pathology and the Gene Therapy Center; 2Department of Obstetrics and Gynecology; 3Departments of Pathology, Cell Biology and Surgery, University of Alabama at Birmingham, Birmingham, AL; 4Department of Obstetrics and Gynecology, University of Düsseldorf Medical Center, Düsseldorf, Germany; 5 Rational Drug Design Program, University of Helsinki and Department of Oncology University Central Hospital, Helsinki, Finland. Conditionally replicating adenoviruses (CRADs) are a novel approach for treatment of cancer. The oncolytic potency of CRADs is determined by their capability to infect tumor cells. Most adenoviruses used for gene therapy have been based on serotype 5 (Ad5). Unfortunately, expression of the primary receptor for Ad5 is highly variable on ovarian cancer cells. Genetic fiber pseudotyping is an approach to circumvent this problem by using the alternative Ad serotype 3 receptor for entry into, and killing of ovarian cancer cells. Furthermore, restriction of the CRAD replication to tumor cells minimizes nontumor tissue injury. One way to gain this selectivity is to control replication regulation genes with promoters active only in specific tumor cells. In this study, we constructed a fiber-modified CRAD containing the secretory leukoprotease Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

inhibitor (SLPI) promoter to control viral replication via the E1A gene (Ad5/3SLPI). SLPI has been shown to be over-expressed in various tumors including ovarian cancer but minimally active in normal tissues. Viral replication and oncolysis were assessed in various ovarian cancer cell lines. Replication was also analyzed with quantitative PCR on three-dimensional primary tumor cell sheroids purified from fresh patient samples. Moreover, in a therapeutic orthotopic mouse model of peritoneal carcinomatosis, dramatically enhanced survival was noted with Ad5/3SLPI. To assess the selectivity of Ad5/3SLPI in comparison with Ad5wt, Ad5/3wt and a non replicative control virus Ad5/3luc, we evaluated the ability of the viruses to replicate in non-target liver cells in vivo. Ad5/3∆24, a type 1 CRAD replicates in cancer cells inactive in the Rb/p16 pathway, was included as a control. Serum levels of liver enzymes, liver pathology, viral E1A DNA copy numbers and E1A RNA levels were evaluated 48 hours after i.v. injection of viruses into immunocompetent mice. Little to no hepatotoxicity was seen with the non replicative E1-deleted vector whereas pronounced effects were seen with the replication competent Ad5/3wt vector. Toxicities included submassive hepatic necrosis, high E1A DNA copy number and E1A RNA levels. Ad5/3SLPI demonstrated significantly lower liver pathology and E1A DNA and RNA copy numbers in comparison to Ad5/3wt and Ad5/3∆24. In summary, Ad5/3SLPI is a promising candidate for treating metastatic ovarian cancer and showed robust virus replication, oncolysis, and in vivo therapeutic efficacy. Ad5/3SLPI showed comparatively low liver toxicity and therefore holds great potential for clinical testing.

971. New Generation Conditionally Replicative Adenovirus for Hormone-Refractory Prostate Cancer Tatyana Gavrikova,1 Julia Davydova,1 Long P. Le,1 Hidetaka A. Ono,1 Pedro J. Ramirez,1 Victor Krasnykh,2 David T. Curiel,1 Masato Yamamoto.1 1 Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL; 2Department of Experimental Diagnostic Imaging, University of Texas, Houston, TX. Although anti-androgen therapy is initially effective for advanced prostate cancer, all patients eventually progress to hormonerefractory (HR) prostate cancer (PC) due to biological selection. In this stage, a median survival is only 6-12 months. Therefore, new therapeutic approaches for hormone-refractory prostate carcinoma (HRPC) are needed. Conditionally replicative adenoviruses (CRAds) are attractive anti-cancer agents. These agents are designed to replicate specifically in tumor cells followed by spread of the viral progeny to neighboring cancer cells. We used the cyclooxygenase-2 (COX-2) promoter for transcriptional targeting of tumor, which is active in many cancers but minimally active in normal tissues, including the liver. The COX-2 promoter (-1432/+59: COX-2L) was tested with luciferase (Luc) expression vectors in PC-3, Du145, and LnCap prostate cancer cell lines. Very strong COX-2 promoter activity comparable to the CMV promoter was observed in PC-3 and Du-145 HRPC cell lines. We next examined infectivity enhancement in the PC cells by using fiber modification of Ad5 vectors, including incorporation of an RGD4C motif into the HI loop (RGD) and replacement of the Ad5 knob region with the Ad3 knob (Ad5/3 chimera),. Three CMV promoter driven Luciferase expression vectors with the wild type fiber (Ad5Luc1), RGD insertion (AdRGDLuc1), and Ad5/3 chimera fiber (Ad5/3Luc1) were used. The RGD-modified vector did not show enhanced infectivity in either of these PC cell lines, while the Ad5/3 chimera demonstrated significantly higher levels of infection in PC-3 and Du-145 HRPC cells. To analyze the effect of fiber modification on the cytocidal effect of CRAds we constructed COX-2 CRAds with wild type, RGD-modified, and Ad5/3 chimeric fibers, respectively. All COXS371