812. Bacterial Vector Mediated Vaccination for Prostate Cancer

CANCER - IMMUNOTHERAPY III profoundly suppress antitumor immunity and inhibit vaccine efcacy, resulting in insufcient reduction of tumor burdens. To overcome these obstacles and enhance the efciency of DC vaccination, we selected both interleukin (IL)-12 and granulocyte-macrophage colony stimulating factor (GM-CSF) as suitable cytokines to eliminate immune suppression and promote DC function. By treating tumors with an IL-12- and GM-CSF-coexpressing oncolytic adenovirus (Ad-∆B7/IL12/GMCSF) prior to DC vaccination, DCs elicited greater antitumor effects than in response to either treatment alone. In addition, combination therapy using high doses of Ad-∆B7/IL12/ GMCSF induced more effective antitumor effects than the lowdose combination therapy. DC migration to draining lymph nodes (DLNs) dramatically increased in mice treated with the combination therapy. This result was associated with upregulation of CCL21+ lymphatics in tumors treated with Ad-∆B7/IL12/GMCSF. Moreover, the proportion of CD4+CD25+ T cells decreased in DLNs of mice treated with the combination therapy. VEGF expression was also downregulated and angiogenesis was inhibited in tumor tissues treated with the combination therapy. Furthermore, combination therapy using immature DCs also showed effective antitumor effects when combined with Ad-∆B7/IL12/GMCSF. The combination therapy had a remarkable therapeutic efcacy on large tumors. Taken together, oncolytic Ad coexpressing IL-12 and GM-CSF in combination with DC vaccination has synergistic antitumor effects and can act as a potent adjuvant for promoting and optimizing DC vaccination.

812. Bacterial Vector Mediated Vaccination for Prostate Cancer

Garrett Casey, Sarfraz Ahmad, Michelle Cronin, Simon Rajendran, Mark Tangney, Gerald C. O’Sullivan. Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laboratory, University College Cork, Cork, Ireland. Induction of systemic immune responses directed against antigenic targets over-expressed by cancer cells represents a powerful therapeutic strategy to target metastatic cancers. Bacterial vectormediated mucosal delivery of tumour-associated antigen expressing plasmids to antigen presenting cells has potential to induce effective, durable, systemic immune responses. We aimed to generate specic anti-tumor immune responses in a murine model of prostate cancer, by oral administration of an attenuated replication-incompetent strain of Salmonella typhimurium containing a plasmid coding for murine Prostate Stem Cell Antigen (mPSCA). Trafcking of Salmonella typhimurium in the initial 10 hours post-gavage feeding was determined with the aid of bacterial lux transformed strain. Clearance of the bacterial strain from the animal was shown to be complete 9 hours post-feeding. Meanwhile, delivery of a Salmonella typhimurium strain transformed with a eukaryotic luciferase reporter plasmid, resulted in maximal gene expression in splenocytes at 48 hours post-feeding. For vaccination trials, male C57 BL/6 mice were gavage fed transformed Salmonella typhimurium (SL7207/pmPSCA) or controls, twice at fortnightly interval. One week after last feed, mice were challenged subcutaneously with tumourigenic doses of TRAMPC1 cells. Tumor dynamics and animal survival were recorded. Induction of tumor protective immunity was achieved by oral feeding of the antigen plasmid bearing bacteria, with > 50% immunised mice remaining tumour free. No significant toxicity was observed. Induction of cell mediated immune responses was demonstrated by the demonstration of IFN γ levels by splenocytes, of vaccinated mice, exposed to the tumor cells. Also splenocytes derived from vaccinated mice, when adoptively transferred to naive animals, prevented tumor growth in 66% of the challenged animals while control cell transfer had no inuence. Endogenous prostate cancer antigen gene delivery S314

using a bacterial vector resulted in a breaking of an immune tolerance to this antigen (mPSCA) and signicant growth retardation of the prostate cancer.

813. Evaluation of Long-Term Transgene Expression in piggyBac-Modied Human T Lymphocytes

Yozo Nakazawa,3 Sunandan Saha,2 Daniel Galvan,2 Cliona M. Rooney,3 Matthew H. Wilson.1,2 1 Michael E. DeBakey VA Medical Center, Houston, TX; 2Medicine and Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 3Pediatrics and Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX.

The nonviral piggyBac (PB) transposon system enables efcient and stable modication of human T lymphocytes with reporter genes. We undertook the current study to further evaluate the regulation and stability of transgene expression in human T cells after nucleofection with PB transposons. Peripheral blood mononuclear cells were nucleofected with a transposon containing eGFP and transposase followed by single cell sorting for eGFP expression, stimulation and expansion. Eight separate T cell clones exhibited complete and homogeneous eGFP expression with mean uorescent intensities ranging from 43 to 479 units. All 8 clones exhibited stable eGFP expression for >24 weeks in culture. CD3/CD28 stimulation of the T cell receptor (TCR) resulted in approximately tenfold transient and reversible increase in eGFP expression. Immunomodulatory cytokines including interferon gamma, interleukin 12, interleukin 4, and transforming growth factor beta, did not alter transgene expression in actively dividing, activated, or resting T cells. 5 azacytidine treatment of T cell clones resulted in increased transgene expression indicating that PB-mediated transgene expression may be regulated by methylation. Trichostatin-A treatment of T cell clones indicated that transgene expression may also be regulated by histone deacetylation. Current studies are underway to quantitate the number of PB integrations achieved in T cell clones. We conclude that PB can mediate very stable expression of transgenes in human T cells beyond 6 months in cell culture. TCR stimulation results in transient increase in PB-mediated gene expression which would be advantageous in immunotherapy applications. Cytokines, known to be in T cell environments, did not appear to alter PB-mediated transgene expression. Although PB can mediate long-term stable gene expression, at least some of PB-mediated transgene expression appears to be regulated by epigenetic mechanisms. Our results provide important insight into the ability of PB to achieve stable genetic modication of T cells for immunotherapy applications and how transgene expression might be regulated by TCR activation, cytokines, and epigenetic mechanisms.

814. Engineered Drug Resistant Immunocompetent Cell Therapy for Neuroblastoma

Anindya Dasgupta,1 David McCarty,1 H. Trent Spencer.1 Department of Pediatrics, Aac Cancer Center and Blood Disorders Service, Emory University School of Medicine, Atlanta, GA.

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The knowledge of the molecular events that result in drug resistance has allowed us and others to test the feasibility of gene transfer strategies to confer drug resistance to anti-cancer cells, and clinical trials are being conducted with an aim of decreasing the myelosuppressive side effects of chemotherapy. We previously showed that protection of the hematopoietic system from chemotherapy-induced toxicity can also be useful in providing the foundation on which to administer a combination of immunotherapy and chemotherapy. It was hypothesized that chemo-protection of immunocompetent cells would Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy