698. IL-23 Transduced Dendritic Cells Produce a Potent Tumor-Specific Immunity Against Intracranial Gliomas

CANCER-IMMUNOTHERAPY: CYTOKINE TRANSGENES AND VACCINATIONS are effective in stimulating an anti-tumor immune response and prolonging survival in mice with an intracerebral tumor upon injection of these cells directly into the tumor.

698. IL-23 Transduced Dendritic Cells Produce a Potent Tumor-Specific Immunity Against Intracranial Gliomas Jinwei Hu,1 Xiangpeng Yuan,1 John M. Ong,1 Christopher J. Wheeler,1 Maria L. Belladonna,2 John S. Yu,1 Keith L. Black.1 1 Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, Los Angeles, CA; 2Department of Experimental Medicine, University of Perugia, Perugia, Italy. IL-23 is a recently discovered cytokine consisting of a heterodimer of the IL-12 p40 subunit and a novel 19-kDa protein, termed p19. IL-23 preferentially induces proliferation of, and IFN-γ production by, memory T cells. In the present study, we transduced freshly cultured dendritic cells (DC) with an adenoviral vector carrying the single-chain IL-23 cDNA. Message RNA and protein expression of IL-23 from the transduced DC were detected by RT-PCR and ELISA respectively. Intratumoral implanting IL-23 expressing DC into intracranial GL-26 glioma bearing C57bl/6 mice resulted in prolonged survival compared with the control mice, which were intracranial bearing GL-26 glioma implanted with empty vector transduced DC. Enhanced CD8+ T cells infiltration within the tumors were detected by immunohistochemistry in brain sections from the mice treated by IL-23 expression DC. Cytotoxic T lymphocyte (CTL) assay of the spleen cells from the IL-23 expressing DC treated mice shown that CTL activities against GL-26 glioma but not irrelevant tumors were augmented. In addition, the mice survived intracranial glioma by IL-23 expressing DC treatment were resistant to intracranial rechallenge with the same GL-26 cell line. This tumor-specific protective immunity was associated with enhanced IFN-γ expression in the brain. Furthermore, the antitumor activities of IL-23 expressing DC was also observed in athymic nude mice but greatly impaired compared with that in C57bl/6 mice. Natural killer cells depletion for the athymic nude mice abolished the antitumor function. Taken together, these data suggest that DC as powerful antigen presenting cells can elicit tumor-specific antitumor immune responses after transduced by IL-23. T cells are crucial for the antitumor activity of the IL-23 expressing DC and natural killer cells are also involved in the process.

699. Evaluation of Potential Toxicity of Electroporation Mediated Deliver of a Plasmid Encoding for IL-12 in a Mouse Melanoma Model Richard Heller,1,2,3 Adil Daud,2 Jeffrey Westover,3 Kathleen Merkler,3 Yolmari Cruz,3 Richard Gilbert,4 Loree Heller.1,3 1 Medical Microbiology and Immunology, University of South Florida, College of Medicine, Tampa, FL; 2Cutaneous Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL; 3Center for Molecular Delivery, University of South Florida, Tampa, FL; 4Chemical Engineering, University of South Florida, Tampa, FL. Previous reports from our laboratory have shown that intra-tumor injection of a plasmid encoding IL-12 (pIL-12) followed by electroporation results in an 80% cure rate of established B16.F10 tumors in mice. Twelve of twelve cured mice were resistant to subsequent challenge with B16.F10 cells. In addition, intra-muscular injection of plasmid encoding IL-12 followed by electroporation significantly reduced the formation of lung colonies following intravenous injection of B16.F10 cells. These pre-clinical studies demonstrated the therapeutic potential of this approach. To translate this to the clinic, the following study was conducted to evaluate the potential toxicity of plasmid delivery with electroporation. Mice Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy

were divided into 5 groups: intratumor injection of saline (Group 1) or injections of plasmid at 0.1 mg/ml (Groups 2, 3) or 1mg/ml (Groups 4, 5) with or without electroporation. The plasmid (50 µl) was injected directly into the tumor and immediately followed by electroporation. Treatments were administered on days 1, 5 and 8. Each mouse was monitored for body weight and general condition on a daily basis beginning the day prior to the first treatment. On days 9, 11, 16, 23 and 30 after treatment, 10 mice from each group (5 male and 5 female) were euthanized. Blood was collected from each animal and used to perform serum chemistries and CBCs. In addition, liver, lung, lymph node, tumor, skin, heart, kidney, spleen and brain samples were collected and histologically analyzed in mice from Groups 1, 4, and 5. All mice appeared to have a generally healthy appearance. Increased weight (10-20%) was noted at the later time points. Mice in Group 5 were found to have the most pronounced reduction in tumor volume including a large percentage of complete regressions. Tumor growth was decreased in Group 3 and the most pronounced increase in tumor size was observed in Groups 1, 2 and 4. With respect to laboratory findings, Group 1 showed the maximum incidence and severity of side effects. The vast majority of ill effects were related to the growth of tumors. For example, Group 1 showed increased AST, increased BUN, decreased lymphocytes, decreased hemoglobin and increased platelets. In comparison to this group, Groups 2 and 4 had reduced magnitude of laboratory abnormalities. Group 5 showed the least laboratory abnormalities. With respect to histopathology, the untreated animals had the most profound abnormalities. Specifically, Group 1 had pronounced tumor involvement in multiple organs. In addition, Group 4 mice showed increased metastatic spread over Group 5 mice. Overall, minimal toxicity and significant tumor regression were observed when pIL-12 is delivered with electroporation. The results of this toxicity study demonstrate that it is safe to test this approach in a clinical study. Authors 1 and 6 have stock and stock options in Gentronics, Inc, an electroporation company developing commercial applications for in vivo electroporation. These two authors also have ownership interest in RMR Technologies, LLC, which is also developing commercial applications of in vivo electroporation.

700. Regression of Squamous Cell Carcinoma by Single Administration of IL-12 and B7.1 Genes Via Electroporation Jiaguo Liu, Marina Torrero, Shulin Li. 1 Comparative Biomedical Sciences, Louisiana State University, Baton Rouge, LA; 2Comparative Biomedical Sciences, Louisiana State University, Baton Rouge, LA; 3Comparative Biomedical Sciences, Louisiana State University, Baton Rouge, LA. Plasmid-based intratumoral injection of IL-12 gene via electroporation is safe, inexpensive and simple, but multiple administrations are required to induce a 40% tumor regression and long-term antitumor memory (Li et al, 2002, JNCI). IL-12-combined B7.1 gene therapy improved the therapeutic efficacy, but the underlying mechanism is less understood, and the delivery of the combined therapeutic genes using electroporation for treating malignant carcinomas is largely unexplored. In this report, electroporation-based IL-12 and B7.1-combined gene therapy was studied in a murine squamous cell carcinoma (SCC) model, SCCVII. A single injection of IL-12 and B7.1 plasmid DNA via electroporation eradicated tumors in 80% of mice, which was superior to IL-12 gene alone in both the inhibition of tumor growth (P = 0.002) and extension of survival time (P = 0.007). The IL-12-combined B7.1 electro-gene therapy-mediated tumor eradication was associated with an increased tumor-specific CTL activity (P = 0.0001), and with an extended duration of B7.1 expression (P <0.05). Neither IL-12 therapy, nor B7.1 therapy alone produced similar results. Importantly, we S271