49. Exponential Enhancement of Oncolytic VSV Potency by Vector-Mediated Suppression of Anti-Viral Inflammatory Responses In Vivo

peutic drugs. Effective genetic modification of primary murine 'I' cells was achieved by transductionwith eeotropieoneoretroviruses. Transductionefficiencies ofJO-60% were routinelyachieved. These FasL-expressingT lymphocytes induced apoptotic death of RM-I cells in in vitro "Cr-release assays, with up to 30% killing after 6 hrs. This killing was shown to be Fas-mediated,as blocking of the perforin/granzyme pathway by addition of EGTA to cultures had no effect on killing. To determine whether such an approach might apply to the clinical situation, a more pertinentmodel using primary human prostate cells was developed for further studies. Epithelial cells, derived from II tumors obtained from radical prostatectomy, were determined to express high levels of Fas and no surface FasL. When co-incubated with K562 cells engineered to express a membrane bound, non-cleavableFasL (ncFasL) in 6 hr ~ICr-release assays, between 12-87% of prostate cells were killed (average 34 ± 19%). We found that differences in susceptibility to apoptosis among patientcellscannotbe attributedto overexpression of c-FLIP or members of the lAP family. Inductionofapoptosis was partially inhibited by pre-treating a subset of cell lines for 24 hrs with 10 to IOOOnM docetaxel (up to 2-fold less killing compared to untreated cells), while apoptotic response was increasedby up to 3-fold when cells were pretreated for 48 hrs with 100nM mitoxantrone. This data demonstrates for the first time that primary human prostate eancercells are able to undergo Fas-mediated apoptosis, and that the potential for combined chemo- and Fasl.- gene therapies depends on the schedule of administration. Experiments are underway to investigatethe mechanismofthe varied responseof patientsamples to apoptosis.As well, we are testing this 'I' cell-based Fasl, delivery method on tumor growth in an in vivo mouse model. ONCOLITIC VIRUSES

49. Exponential Enhancement of Oncolytic VSV Potency by Vector-Mediated Suppression of Anti-Viral Inflammatory Responses In Vivo

Lan WU,l Jennifer Altomonte,':' Li Chen,' Marcia Meseck,' Oliver Ebert,' :' Adolfo Garcla-Sastrc.' John T. Fallon.' Savio L. C. Woo.I I Department ofGene and Cell Medicine, Mount Sinai School 0/ Medicine, New York. NY; 2Department cfMicrobiology; Mount Sinai School ofMedicine, New }ork, NY; 'Depanment 0/ Pathology, Mount Sinai School 0/ Medicine. New }ork, N}~' "Znd Medical Department. Klinikum Reclus del' lsar; Technical University 0/ Munich, Munich, Germany.

Oncolytic virotherapy is a promising strategy for treatment of malignancy including hepatocellular carcinoma (I·ICC). Vesicular Stomatitis Virus (VSV) is a particularly attractive oncolytic agent because of its short replication cycle and ability to reach high titers in most rodent and human tumor cells. We have previously reported a three-log elevation in intratumoral VSV titer at oneday after vector administration in rats bearing multi-focal HCC, which was followed by a logarithmic decline that correlated with an accumulation of inflammatory cells such as natural killer (NK) cells at the lesions. Upon depletion ofNK cells by intraperitoneal administration of rabbit anti-asialo GMI in tumor-bearingrats, the intratumoral virustitersandextentsoftumor necrosiswereenhanced by 2-logs and >2-fold, respectively. We then constructed a rVSV vector that expresses equine herpesvirus-I glycoprotein G, which is a broad spectrum viral chcmokine binding protein (rVSV-gG).ln vitro functional characterizationof rVSV-gG showed that this new vector had no significantdifferences in viral life cycle or viralyield, and it is equally effective in killing rat and human Hepatoma cells as a control VSV vector. Moreover,NK cell migration was significantly inhibited by conditioned media from rVSV-gG infected rat hepatoma cell as compared to that of a control-VSV vector. In vivo study showed that hepatic artery infusion ofrVSV-gG in immuneMolecular Therapy Volume 15.Supplement I. ~"r Copyright © The American Soc iety of Gene Therapy

2007

competentratsbearingsyngeneicand multi-focal HCC intheir livers resulted in the suppression ofNK and NKT cell chemotaxis to the tumors and a one-log enhancement of intratumoral virus titer over that of a control VSV vector, which Jed to elevated tumor necrosis andsubstantially prolongedanimalsurvivalwithouttoxicities. These results indicate that rVSV-gG could be developed as an effective and safe oncolytic agent to treat advanced HCC patients in the future. Furthermore, the novel concept that oncolytic potency can be substantially enhanced by vector-mediated suppression of host anti-viral inflammatory responses might be generally applicable in the field of oncolytic virotherapy for cancer.

50. Phase I Clinical Trial Study Ultilizing Oncolytic Adenovirus with HSP Gene Expression (H103) in Treating Patients with Advanced Solid Tumor Hongli Liu,' Yu Wang,' Yong Ben,' Datong Chu,' Fang Hu.'

Medical Affairs Department. Shanghai Sunway Biotech Co.. Ltd.. Shanghai. China; 2Cancer Institute and Hospital. Chinese Academy ofMedical Sciences and Peking Union Medical College. Peiking, China. I

Objective: HI 03 is a type 2-adenovirus with E I B-55kD deleted and human HSP70 (heat shock protein)gene inserted using a CMV promoter in viral genome, aiming to stimulate host's immune responsethroughmaximizingHSP-inducedtumor antigenpresenting. Toevaluatethe safety ofH 103when treatingpatientswith advanced solid tumor, a phase I study was conducted from Sep 2003 to April 2004. Methods: Total 27 patients were recruited.(Male 18 cases, female 9 cases; Median age 58; age 26-68). Tumor types included melanoma (II), renal carcinoma (5), lung cancer (5), colon cancer (I), breast cancer (I) and others (4). HI03 was administrated in an intratumoral mode. Viral dosage escalation is from 2.5x107vp to I.5xlO12vp in singledosageregimen (la, 10patients)and I.5x 1012vp, 3.0x1012vp in multiple dosage (lb, 9 patients). Samples of blood, urine, stool, pharyngeal swabs and injection site swabs were collectedat D1,2,3, 5, 7, 14,21 after injectionfor H I 03 biodistribution using PCR. Blood Samples were collected at D3, 7, 14,21 after injectionfor detectingneutralizingantibody, changeoflymphocytes panel and HSP70 expression level.A tissue sample from one patient was analyzed for HSP expression using immunohistochemistry staining.Results: Dose LimitedToxicity(DLT)was observedat one patient of l.5x I 012vp/patient and one patient on.ox I 012vp/patient, however, MaximumTolerated Dose (MTD) was not reached in this phase I study.The commonadverseevents were fever77.8%(21/27, I-II grades except one patient with III grade), local site pain 22.2% (6/27), leucopeniaand thrombopenia 18.5%(5/27, I-II grade except one patient with IV grade). Overall objective response (CR+PR) rate ofHl03-injceted tumor is 11.1%(3/27) and the caleulatcd CBR (CR+PR+MR+SD) is 77.8% among all patients, And the transient abseopalresponsesof distant tumor were observed in 3 pateintsduring the HI03 treatment.Administration ofI-II 03 trendedto stimulate the host's cell immuneresponseand antibodyresponseby increasing major immunecell (lymphocytes,total 'I' cells, 'I' helpercells, CTLs, even NK cells) and anti Ad2 antibody which is unrelated to anti cancer efficacy. HSPexpressionwas confirmedin tumortissue after HI03 injectionwithouteffectingother sites.QuantitativePCR failed in detecting replicating viral copies in the samples of blood, urine, stool, pharyngeal swabs and injection site swabs at D21 after 1-1103 administration.Conclusions: This phase I study showed that 1-1103 was safe when applied intratumorally to patients with advanced solid tumor,showing promising anti cancer efficacyand increasing host's immune response. Phase II study should be investigated for exploring efficacy and monitoring further safety.

S21