632. Improving Cancer Gene Therapy for Advanced Head and Neck Cancer with a Multi-Modal Therapy Approach

tive Adflt-Luc and AdCMV-Lucvectors on a panel of human breast cancer and endothelial cells. The metastasiccapability ofthese cells was determined via stimulation oftumor cell growth and migration using recombinantVEGF.The cytotoxicityofcancerand endothelial cells by Ad infection was analyzed using the crystal violet inclusion assay. CRAd progeny production was determined using real time PCRand a standard50% tissuecultureinfectiousdose assay.A tumor xenograft model was used to determinate the therapeuticefficacy of oncolytic virotherapy to treat human breast tumors. Higher levels of nt- I-driven Luc expression were observed in 2LMP,LCC6, and DY36T2 aggressive metastatic clones in comparison with parental MDA-MB-23I , MDA-MB-435S, and MDA-MB-361 cell lines, respectively. VEGF significantly increased flt-I positive breast cancer cell growth and migration.A CRAd using the flt-I promoter element for specific EI II gene expression was developed, encoding the CDCGRDCFC-containing peptide in the HI loop of the fiber knob domain (CRAdRGDnt). The cancer cells demonstrated different levels of cytotoxicity after CRAdRGDl1t infection which correlated with I1t-1 promoter activity.There was a significantdecrease in viability of LCC6 and 2LMP cells infected with CRAdRGDflt. In vivo therapy studies demonstrated that intratumoral injection of CRAdRGDfitproducedsignificantinhibitionof subcutaneous2LMP breast tumor xenograft growth in athymic nude mice compared to PBS treated control group. These results suggest that selective targeting of oncolytic virotherapy would be a superior strategy for treatment of breast cancer. This work was supported in part by the USAMRMC under W8IXWH-04-1-0663.

631. Therapeutic Efficacy of Midkine PromoterBased Conditionally Replicative Adenovirus Vector for Targeting the Midkine-Expressing Human Bladder Cancer Model

Shuji Tcrao,' Toshiro Shirakawa," Shuji Kubo,' Katsuyuki Hamada,' Masatoshi Tagawa,' Atsushi Takenaka,' Masato Fujisawa ,' Akinobu Gotoh.2 /Division ofUrology, Department ofOrgans Therapeutics, Kobe University Graduate School ofMedicine. Kobe, Japan: lln_ ternational Center for Medical Research and Treatment, Kobe University School ofMedicine. Kobe. Japan; JLaborato/J' of Cell and Gene Therapy Institute for advanced Medical Sciences, Hyogo College ofMedicine, Nishinomiya, Japan; ~ Department of Obstetrics and Gynecology, Ehime University School ofMedicine, Shizukawa, Shigenobu-cho, Onsen-gun, Japan: SDivision ofPathology, Chiba Cancer Center Research Institute, Chiba, Japan. Objectives. Weattempted to develop a novel therapeutic strategy against human bladder cancer usingAd-MK-E Ia-a midkine(MK) promoter-regulated conditionally replicating adenovirus (CRAD). Methods. Wetested several human cancer cell lines in vitro, including those of bladder cancer (KK47, 5637, and T24), lung cancer (A549), and head and neck cancer (H891). In each cell line, we examined MK mRNA expression by TaqMan real-time quantitative PCR, MK promoter activity, post plasmid transfection, by a luciferasc assay, and the transduction efficiency by cotransfection with the cytomegalovirus (CMV)-13-gal plasmid. In the above cells, we assessed the cell type-specific replication ofAd-MK-Ela virus by measuring the EIa DNAcopy number by real-time PCR and the cell growth inhibition due to this virus by the Alamar blue assay. In animal studies, nude mice were subcutaneously inoculated with KK47 cells and later intratumorally injected with PBS or Ad5CMV-LacZ or Ad-MK-Ela. Results. The MK mRNA expression level and MK promoter-driven lucifcrasc activity were relatively higher and markedly increased,respectively, in the 5637,A549, and KK47 cells than in theT24 and H891 cells. FoliowingAd-MK-Ela infection, the Ela DNA copy number increased more significantly S242

in the KK47, 5637, and A549 cells than in the 1'24 and H891 cells. At a multiplicity of infection (MOl) of 0.01, Ad-MK-Ela significantly inhibited KK47 and 5637 cell growth. In vivo, Ad-MK-Ela injection markedly inhibited KK47 tumor growth. Conclusions. We demonstrated the antitumor effect of Ad-MK-Ela in a human bladder cancer model overexpressing MK mRNA.

632. Improving Cancer Gene Therapy for Advanced Head and Neck Cancer with a MultiModal Therapy Approach

Joao D. Dias,1.2 Minna Eriksson.U Dung-TsaChen,' Kilian Guse.l-' Mikko Tenhunen.' Reidar Grenrnan,' Mikko Savontaus,' Akseli Hemminki.'-' J Haartman Inst & Malec. Cane. Bio Prog, Helsinki U; Helsinki, Finland; lDep. ofOnc., HUCH. Helsinki. Finland: 'Btostatistics Division, Dep. ofInterdisciplinary Onc., Moffit! Cancer Center & Research Inst., Tampa; 'Dept. ofOtorhinolaryngology-Head and Neck Surge/Yo Turku 0. & TUCU, Turku, Finland; "Iurku Centre for Biotechnology Med. Dep, Turku 0. , Turku, Finland.

Each year, >500,000 new head and neck cancers (HNC) arc diagnosed worldwide. Approximately 90% ofHNC is squamous-cell carcinoma(HNSCC). Currently, standardcare ofHNSCC combines surgery, radiation, and chemotherapy. Mortality associated with disease and morbidityassociated with its treatments has encouraged the pursuit of alternate therapeutic strategies. Over the past decade interruption of epidermal growth factor and its receptor pathway has been exploited in treatment of various solid tumors. Cetuximab, a monoclonal antibody that blocks this pathway, recently received full FDAapproval for treatment of patients with metastatic HNSCC or locoregionally advanced HNSCC in combination with radiation. Even thoughthere is clinicaldata that has validatedthe inherentsafety and therapeutic potential ofAd vectors, the main conclusion from most cancer trials is that tumor transduction has often been too low for significanttherapeutic antitumor effect. Therefore, for advanced disease, tumor penetration is the key to efficacy. Thus, oncolytic adenovirus have been explored for enhanced tumor transduction and amplificationof effect.Wc investigated which adenoviralcapsid modificationsallow the best gene transfer to HNSCC primarytumor lines (transductional targeting). Determination of transductional efficiency of the modified viruses was performed using replication deficientAds, which express luciferase.To evaluate tumor cell killing effect of capsid modified viruses, cell lines were infected with oncolytic Ad and cell killing assays (MTS) were performed. After determining HNSCC primary cell line oncolytic Ad sensitivity we studied a multimodal approach, more specifically thc combination effect of oncolyticAds with HNSCC standard therapies. Sensitivity of cell lines to 5-FU and/or to cisplatin (chemotherapy); radiation (radiotherapy) and cetuximab (monoclonal antibody therapy) was establishedbefore measuringcombinedtherapy effect. Combination of the two most oncolytic Ads with 5-FU and cisplatin, cetuximab or with radiation was determined using MTS assay. For in vivo experiments, we are setting up animal models in nude mice. We have established cultures of primary HNSCC specimens to ensure good correlation to the clinical situation. Results and conclusions Capsid modified virus showed increased transduction and oncolysis of HNSCCsubstratesin comparisonto Ad5 basedagents.Ad5 chimeric with theAd3 knob fiber, RGDor the polylysine modifications(pK7) resulted in significantly higher antitumor effect than wild type Ad. Ad5/3and pK7 allowedthe bestgene transfer and cell killingeffects. The combination of chemotherapy, radiotherapy and monoclonal antibody therapy with CRAD therapy results in an increased cell killing effect in vitro. In vivo data will be presented.

Molecular Therapy Volume15.Supplement t, .\by 2007 Copyright © '111C American Society of Gene Therapy