42. Resistance of Primary Ovarian Cancer Cells to Viral Oncolysis

CANCER-APOPTOSIS AND SUICIDE 42. Resistance of Primary Ovarian Cancer Cells to Viral Oncolysis Robert Strauss,1 Pavel Sova,2 Ying Liu,1 Zong Yi Li,1 Nicole Urban,3 Charles Drescher,3 Nancy Kiviat,2 Andre Lieber.1,2 1 Division of Medical Genetics, University of Washington, Seattle, WA; 2Department of Pathology, University of Washington, Seattle, WA; 3Fred Hutchinson Cancer Research Center, Seattle, WA. We investigated the mechanisms which confer resistance to viral oncolysis using a capsid-modified, conditionally replicating oncolytic adenovirus (Ad5/35.IR.E1A/TRAIL) and primary ovarian cancer cells. This vector targets tumor cells by binding through the adenovirus serotype 35 fiber to CD46, which is upregulated on most tumor cells in situ. Tumor-specific viral replication and TRAIL gene expression is achieved upon homologous recombination between inverted repeats (IR) in adenovirus genomes (Ad.IR). Ad5/35.IRE1A/TRAIL was efficient in lysing a variety of established human tumor cell lines in vitro and in eliminating liver metastases in mouse models after intravenous injection. However, in experiments on primary ovarian cancer cell cultures derived from biopsies (N>30), we discovered that in a number of cultures, individual cells remained alive after infection with Ad5/35.IR.E1A/TRAIL, even when high doses of virus were applied. To enrich cultures for cells resistant to viral oncolysis we established a total of 120 cultures derived from single cell clones of a biopsy from a grade IV ovarian cancer patient. Resulting clonal cell cultures were tested for their level of infectibility, their ability to support viral replication and their susceptibility to viral oncolysis by Ad5/35.IR-E1A/TRAIL. We found that about 50% of clonal cultures contained cells resistant to cell lysis, whereby the mechanisms that cause resistance were complex and appeared to include reduced infectivity, reduced ability to support viral DNA replication, and inability to execute apoptosis. Using methylationspecific PCR we found the promoter of a number of cancer-associated genes methylated in resistant cells but not in susceptible cells. Based on this, we tested whether demethylation agents would increase the sensitivity to viral oncolysis. We demonstrated that enhanced viral oncolysis could be detected when Ad5/35.IR.E1A/TRAIL was used in combination with deacetylase inhibitor TSA (trichostatin A) and methyltransferase inhibitor DAC (5-aza-2’-deoxycytidine). We then focused on clones that could be efficiently infected with Ad5/35 vectors and performed genome-wide expression profiling of 15 clones with >90% resistant cells and 15 clones that were 100% susceptible to lysis by Ad5/35.IR-E1A/TRAIL using cDNA arrays. Examples of genes that were upregulated (in array and qRT-PCR analyses) in resistant cells were clusterin, lipocalin2, netrin4, epiregulin, claudin7, defensin beta1, inhibin betaB. We are currently using siRNA specific to these genes to test whether their down-regulation can overcome resistance to oncolysis and results will be reported. Furthermore, we are processing the array data by functional clustering to identify pathways that confer resistance. Our final goal is to “arm” our oncolytic vector with cDNA or siRNA expression cassettes that can sensitize tumor cells to virus and TRAIL mediated cell killing.

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43. Long-Term T Cell Activation Following Combined In Situ Gene Therapy and IntensityModulated Radiotherapy Compared to Gene Therapy as Mono-Therapy in Prostate Cancer Patients Tetsuo Fujita,1 Bin S. Teh,2,5,6 Terry L. Timme,1,5 Wei-Yuan Mai,2 Takefumi Satoh,1 Nobuyuki Kusaka,1 Koji Naruishi,1 Elmoataz Abdelfattah,1 Estuardo Aguilar-Cordova,4 Brian J. Miles,1 E. Brian Butler,2,5,6 Timothy C. Thompson.1,2,3,5 1 Scott Department of Urology, Baylor College of Medicine, Houston, TX; 2Radiology/Section of Radiation Oncology, Baylor College of Medicine, Houston, TX; 3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX; 4Gene Vector Laboratory, Baylor College of Medicine, Houston, TX; 5 Veterans Affairs Medical Center, Houston, TX; 6The Methodist Hospital, Houston, TX. Introduction: A program combining in situ gene therapy and intensity-modulated radiation therapy (IMRT) was implemented for the treatment of prostate cancer because the complementary mechanisms of cytotoxicity may have an enhanced efficacy. The in situ gene therapy comprised of adenoviral vector mediated Herpes Simplex Virus-thymidine kinase (AdHSVtk) + valacyclovir (VCV). We are currently conducting clinical trials using this approach. This study explores long-term immune responses following combined radio-gene therapy compared to gene therapy as mono-therapy. Methods: The study design included two separate clinical trials in the Baylor Prostate Cancer SPORE Program: Trial A gene therapy in combination with IMRT for prostate cancer (31 patients) and Trial B gene therapy as mono-therapy for local recurrent prostate cancer after failing initial radiotherapy (36 patients). Heparinized blood was collected at the time of vector injection and at selected intervals afterward until 12 months. Peripheral blood lymphocytes were analyzed by fluorescent antibody cell sorting (FACS) after the incubation with dual color labeled antibody pairs: CD45/CD14, CD3/CD19, CD3/CD8, CD3/CD4, CD8/HLA-DR, CD4/HLA-DR, CD3/HLA-DR, and CD3/CD56+CD16. Results: The pre-treatment mean percentage of activated CD8+ T cells (DR+CD8+ T cells) was 14.7% and 12.2% (Trials A and B, respectively). Two weeks after the vector injection, this increased to 31.7% and 21.9% (Trials A and B, respectively), and these increases were statistically significant (P < 0.0001 and P = 0.0188, respectively). Only in Trial A were significant increases seen at 4 weeks, 12 weeks, 4 months, 6 months, 8 months and 10 months (P < 0.0001, P = 0.0002, 0.0464, 0.0016, 0.0125 and 0.0354, respectively). In addition, activated CD4+ T cells were noted to increase significantly after the vector injection from 2 weeks till 12 months in Trial A only. (P = 0.0013 and 0.0069, respectively). Conclusions: This is the first report of long-term systemic immune responses following radio-gene therapy compared to gene therapy as mono-therapy. We present evidence showing long-term (up to 12 months) systemic T cell responses to two consecutive AdHSVtk injections during combination in situ gene therapy and IMRT for prostate cancer. These results suggest the potential for sustained activation of cell-mediated immune responses and may have enhanced activities against cancer. This combined approach may maximize tumor control, both local-regional and systemic through radio-gene therapy induced cytotoxicity and anti-tumor immunity.

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