644. A Dual-Action Armed Replicating Adenovirus for the Treatment of Bone Metastases of Breast Cancer

CANCER-TARGETED GENE THERAPY: ADENOVIRUS AND HERPES VIRUS 643. Human Matrix Metalloproteinase-8 Gene Delivery Enhances the Oncolytic Activity of a Replicating Adenovirus Jin Cheng,1 Harald Sauthoff,1,2 YaoQi Huang,1 David I. Kutler,2,3 Sheila Heitner,1 William N. Rom,1 John G. Hay.1,2 1 Medicine, NYU School of Medicine, New York, NY; 2Medicine, Veterans Administration, New York, NY; 3Otolaryngology, NYU School of Medicine, New York, NY. The success of replicating adenoviruses in cancer therapy is limited by inefficient delivery. The suboptimal transduction of cancer cells is compounded by poor distribution of the virus within the tumor mass. The hypothesis that is addressed in this study is that matrix within the tumor hinders the free cell-to-cell spread of replicating adenoviral vectors. Collagen I, collagen IV, fibronectin and laminin matrix components were obtained commercially pre-coated on membrane inserts. The membrane inserts were placed into the chambers of 24-well plates that had been pre-seeded with 293 cells growing as a monolayer on the base. Adβ-gal virus was added to the upper chamber, and in order to infect the 293 cells plated on the base of the wells, the virus had to spread through the matrix precoated insert. The collagen I insert blocked the spread of Adβ-gal virus so that only a few 293 cells stained blue after the addition of X-gal. In contrast, control, collagen IV, fibronectin and laminin inserts allowed the passage of virus with subsequent intense blue staining of the 293 indicator cells. A significant reduction of viral passage through the Collagen 1 matrix was confirmed with assays of β-gal acivity. Based on reports of the effective collagen I-degrading activity of MMP-8, we chose to construct an non-replicating adenovirus to express the MMP-8 transgene. Established human A549 cell lung cancer xenografts were injected with a wild-type replicating adenovirus Adwt (5 x 108 PFU) together with the non-replicating AdMMP-8 virus (5 x 10 8 PFU). Histochemical analysis demonstrated reduced amounts of collagen within necrotic areas of Adwt plus AdMMP-8 injected tumors compared to controls. The addition of AdMMP-8 to the wild type virus also significantly reduced the growth of tumors compared with control tumors injected with the wild type virus in combination with the non-replicating control virus. AdMMP-8 injection alone did not affect the growth of the lung A549 tumor xenografts. These results suggest that intratumoral AdMMP-8 and collagen disruption is a possible strategy for improving viral spread and improving the oncolytic activity of replicating adenovirus.

644. A Dual-Action Armed Replicating Adenovirus for the Treatment of Bone Metastases of Breast Cancer 1

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James J. Cody, Joanne T. Douglas. Department of Pathology, University of Alabama at Birmingham, Birmingham, AL; 2Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL.

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The majority of patients with advanced breast cancer develop osteolytic metastases in the skeleton, which lead to such complications as pain, fractures, hypercalcemia and a general decrease in the quality of life. Current therapies for bone metastases are not curative in nature. Thus, new treatments are needed for osteolytic bone metastasis of breast cancer. Conditionally replicating adenoviruses (CRADs) are a new class of anti-cancer agents that offer the potential ability to infect and lyse all the cells of a tumor. In clinical trials, replicating adenoviruses have exhibited safety but not the desired level of efficacy, demonstrating a need for improvement. One approach to enhance the efficacy of a CRAD is to arm it with a therapeutic transgene. Osteoprotegerin (OPG), the S248

recently described inhibitor of osteoclastic bone resorption, represents a promising candidate with which to arm a replicating adenovirus designed to treat osteolytic bone metastasis. We hypothesize that a replicating adenovirus armed with a gene for OPG will be able to inhibit breast cancer bone metastasis and reduce the tumor burden in the bone through two actions: direct lysis of the tumor cells by replication of the virus and inhibition of bone resorption by the production of OPG. Previously, we have shown the feasibility of this hypothesis by employing a two-component system in which cells are coinfected with a non-replicating OPG expressing adenovirus and wild-type adenovirus. It was shown that the production of OPG neither inhibits viral replication nor reduces the oncolytic potency of a replicating adenovirus. We have now constructed a conditionally replicating adenovirus in which E3B has been replaced by an OPG-Fc fusion gene (Ad-∆24-OPG-Fc-RGD). The placement of the transgene within the genome is intended to result in high levels of expression, late in the infection cycle. Selectivity at the level of replication is conferred by the deletion of 24 base pairs within E1A (∆24), which prevents the binding of E1A and cellular Rb and limits replication to cells with disregulated cell cycles. Transductional selectivity is conferred by the incorporation of an RGD peptide sequence within the fiber knob, which directs infection towards cells expressing αv integrins. In order to evaluate the various modifications of this virus, a panel of control viruses has been constructed as well. These viruses are being evaluated in vitro for selectivity of replication in the target breast cancer cells as well as transgene expression levels and kinetics. These viruses will also be evaluated to ensure that the expression of OPG-Fc does not reduce the oncolytic potency of a CRAd. This armed, replicating adenovirus will then be evaluated in vivo in a murine model of breast cancer bone metastasis. This CRAd represents a new agent with which to treat breast cancer bone metastasis.

645. Combination of Oncolytic Adenovirotherapy and Bax Gene Therapy Does Not Augments Antitumor Efficacy Masayoshi Hioki,1 Shunsuke Kagawa,1,2 Toshiya Fujiwara,1 Yoshihiro Ikeda,1 Toru Kojima,1 Ryo Sakai,1 Futoshi Uno,1 Fuminori Teraishi,1 Yoshiko Shirakiya,3 Yuichi Watanabe,3 Yuuri Hashimoto,3 Yasuo Urata.3 1 Department of Surgery, Okayama University Graduate School, Okayama, Japan; 2Center for Gene and Cell Therapy, Okayama University Hospital, Okayama, Japan; 3R&D Planning, Oncolys BioPharma, Inc., Tokyo, Japan. Cancer gene therapy and oncolytic virotherapy have been studied extensively. However, the clinical application is hampered by its weak anticancer activity. We constructed a replicating adenovirus (OBP-301, Telomelysin), in which the human telomerase reverse transcriptase (hTERT) promoter drives expression of the adenoviral E1 genes, could replicate in and causes selective lysis of human cancer cells. We hypothesized that combination adenoviral therapy containing OBP-301 and a non-replicating E1-deleted adenovirus expressing the proapoptotic Bax gene (Ad/Bax) could overcome the weakness and augment anticancer efficacy of each modality. The human cancer cell line A549, SW620, DU145, MKN28, MKN45 were treated with OBP-301 with Ad/Bax, and cell viability, transgene expression, viral replication were assessed in vitro. To test the antitumor effects of combination treatment in vivo, subcutaneous tumors and pleural dissemination of A549 cells were generated in nude mice. Each mouse underwent intratumoral or intrapleural administration of viruses and the growth of implanted tumors were observed after trearment. Combination treatment resulted in marked Bax protein expression, and enhanced efficay in in vitro cell viability assay, as compared Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy