590. Antitumor Effect of Intravenous Administration of Oncolytic Herpes Simplex Virus Expressing Interleukin 12 on Systemic Metastases

event to express a CAR transgene is mediated by clinical-grade DNA plasmid or viral vectors requiring prolonged ex vivo cell culturing and/or integration of vectors. To avoid the expense of viral vectors for early phase clinical trials, we use non-viral gene transfer and routinely observed that eleetrotransfer of DNA plasmid resulted in rapid and high levels of transgene expression in human T and NK cells. Thus, in addition to developing DNA plasmid vectors for stable (integrated) CAR expression and long-term disease eradication. we are using a non-viral non-integrating gene transfer approach to introduce vector-free in vitro transcribed CAR mRNA into T and NK cells for disease control. Compared with DNA transfection or viral transduction the benefits of mRNA-mediated transfection include low potential for permanently altering the genome as expression is not dependent on nuclear entry and translation occurs in the cytosol such that patient safety is protected as there is no need for transcriptional promoters, DNA nanking sequences for integration , and immunogenic antibiotic resistance genes. We demonstrate using the Nucleofector device, that codon-optimized mRNA coding for CD 19-specific CAR, which can activate cells through chimeric CD28 and/or CD3-zeta, can be expressed in T and NK cells to redirect specificity for CD 19' tumor targets . When scaled-up, the introduction of one or more mRNA species into T and/or NK cells has the advantage of (i) minimal manipulation ofT and NK cells with availability of genetically modified cells within a day ofapheresis, (ii) efficiency, (iii) high levels of transgene expression, (iv) being inexpensive, and (iv) safety (no insertional mutagenesis). To overcome the loss of transgene expression we propose a clinical trial in which patients receive recursive infusions of CAR' T and/or NK cells following repetitive c1cctroporation ofmRNA using clinically meaningful numbers of lymphocytes. This application oftransient transfection to adoptive immunotherapy can be used to treat patients with rapidly growing tumors while T and NK cells expressing CAR from integrated DNA plasm ids are being prepared.

588. Anticancer Activity of Oncolytic Adenovirus Vector Armed with IFN-alpha and ADP Is Enhanced by Pharmacologically-Controlled Expression of TRAIL Elena V. Shashkova,' Mohan N. Kuppuswamy.- William S. M. Wold.' Konstantin Doronin. ? 'Preclinical development, VirRx, Inc., Saint Louis, "'10; lMolecular Microbiology and Immunology, Saint Louis University School ofMedicine, Saint Louis, MO. Replicative adenoviruses (Ad) represent a novel class of cancer therapeutic agents. Expression of therapeutic genes in the context of Ad replication allows for enhanced therapeutic protein production and for augmented oncolysis. We have previously described the oncolytic Ad vectors KD3 and KD3-IFN that were rendered cancer-specific by mutations in the EIA region ofAd; these mutations abolish binding ofEIA proteins to pRB and p300/CBP. The antitumor activity of the vectors was increased by overexpression of the adenovirus death protein (ADP, E3-11.6K) and by replication -linked expression of IFN-alpha. We hypothesized that the anticancer efficacy ofthe KD3-IFN vector could be further improved by expression ofTNF-related apoptosis-inducing ligand (TRAIL). First-generation E I-dcleted Ad vectors carrying reporter genes for EGFP or SEAP and a therapeutic gene for TRAIL under control of the TetON system were constructed. Expression of EGFP, SEAP, and TRAIL was increased in the presence ofa helper virus and the inducer doxycycline such that up to 23 I-fold activation of expression for the TctON-SEAP vector was obtained. Coinfcction of the TetOn-TRAIL vector with KD3 or KD3-IFN resulted in increased anticancer activity in cancer cell cultures. Induction of TRAIL expression at various stage s of virus replication did not reduce the yield of progeny virus. Treatment of established hepatocellular S226

carcinoma xenografts with the combination of TetON-TRAIL and KD3-IFN was more efficient than treatment with either vector alone; this result demonstrates the efficacy of a four-pronged cancer gene therapy approach, which includes Ad oncolysis, ADP overexpression, IFN-alpha-mcdiated immunotherapy, and pharmacologically controlled TRAIL activity.

589. Generation of a New Type of YeastSecreted Biotinylated Recombinant Antibodies Directed Against Mesothelin and Able To Prevent CA125/Mesothelin-Dependent Cell Attachment Nathalie Scholler,' Barbara Garvik ,' Lindsay Bergan,' Barry Nevin, I Jennifer Gross, I Nicole Urban.' 'Molecular Diagnostics Program, Public Healtlz Sciences, Fred Hutchinson Cancer Research Center; Seattle, IfA. Recombinant antibodies arc emerging as an attractive alternative to mouse antibodies for diagnostic tests as well as in vivo imaging and therapy. We developed a novel and original system of yeast expression enabling the secretion of recombinant proteins that are biotinylated in C-terminaL Yeast-secreted in vivo biotinylated recombinant antibodies (biobodies) can tetramerize with one single streptavidin, which stabilizes their conformation and increases their affinity binding ofat least 10 fold in average, By combining our biobody platform with a library of yeast-display recombinant proteins (scFv) we can rapidly select antigen-specific scFv. Antigen-specific recognition sequences are first isolated by magnetic and now sorting using yeast-secreted biotinylated recombinant proteins to capture specific seFv, and then changed into biobodies (Bbs) with two simple steps : yeast co-transformation with pTOR2 vector and mating with a biotin-ligase expressing yeast. We generated Bbs that specifically bind to mesothelin , a GPI-anchored protein constitutively expressed by peritoneal cells that is also a ligand for CA 125, an ovarian cancer biomarker. We validated anti-mesothelin Bbs' affinity and specificity by ELISA assays and now eytometry and we tested the function in an in vitro cell adhesion assay combining ovarian tumor cells expressing CA 125 and mesothclin-transfectcd cells. We demonstrated that CAI25/mcsothelin-dependent cell adhesion could be blocked with mesothelin recombinant protein fused to an Ig domain and dimcrized with anti-Ig antibodies, or with anti-CA 125 mouse monoclonal antibodies (mAb), or with anti-mesothelin Bbs derived from high affinity yeast-display scf'v, Blocking CA I25/mesothelin-dependent cell attachment may prevent or delay the peritoneal implantation of ovarian carcinoma cells and thus could prolong ovarian cancer patient remission and survival, Our results suggest that novel therapies based upon affinity agents able to compete with or to block the CA I25/mesothelin interaction could prevent or delay the development of peritoneal metastasis.

590. Antitumor Effect of Intravenous Administration of Oncolytic Herpes Simplex Virus Expressing Interleukin 12 on Systemic Metastases Yi Guan,' Yasushi Ino,' Fukuhara Hiroshi,' Tomoki Todo.'" I Department ofNeurosurgery. Graduate Sclzool ofMedicine, University ofTokyo, TOkyo. Japan; 'Deponmen: cfNeuro-Oncology and Molecular Therapeutics. Graduate School ofMedicine, University ofTokyo, Tokyo, Japan; ' Dep artment ofUrology; Graduate School ofMedicine, University ofTokyo, Tokyo, Japan. Object: Genetically-engineered oncolytic herpes simplex viruses type I (HSV-I) vectors have been proven to be effective therapeutic reagents for cancer when administered intratumorally. However, cancer is a systemic disease in almost all fatal cases, therefore intravenous delivery would broaden the clinical application of oncolytic HSV-I vectors if shown effective, The purpose of this Molecular Therapy Volume 15, Supplemen t I. ,\br 2007 Copyright © 'Ihc Ame rican Society o f Gene Thcr.Lp}'

study was to examine whether expression of interleukin 12 (IL-12) by oncolytic HSY-I vectors improves the antitumor efficacy when delivered intravenously in mice with systemically disseminated tumors. Methods: A third-generation HSY-l (T-Ol) has triple deletions in the g34.5. ICP6 and a47 genes. T-mflL12 was created by inserting the fusion-type murine IL- 12 gene into the deleted ICP6 region ofT-OI. HSY- I-susceptible All mice and syngeneic, poorlyimmunogenic Neuro2a (neuroblastoma) cells were used for in vivo evaluation. Mock, T-OI orT-mllLI2 (5 x I06pfu) was injected into the tail vein three times, either on days 1,3,5 or on days 1,4, 7, in A/I mice or athymic mice bearing intravenously disseminated tumors. The antitumor efficacy was also evaluated in All mice with preexisting anti-HSY-l antibody. Results: In All mice with systemic Neuro2a dissemination, intravenous delivery of T-O I and T-mllL12 both prolonged the survival compared with mock treatment. T-mflL 12 was significantly more efficacious than T-O I with both dosing schedules. In athymic mice, while both T-OI and T-mllL 12prolonged the survival compared with mock, there was no significant difference between T-mllL12- and T-OI-treated groups, indicating that T cells are required for the enhanced efficacy by IL12 expression. In A/J mice with preexisting anti-HSY-I antibody, the therapeutic efficacy of intravenously administered T-mflL12 was significantly decreased compared with that in nalvc mice. We observed no apparent toxicity with the treatment regimen used. Conclusion: Intravenous delivery of oncolytic HSY-I vectors was effective in treating systemic metastases, and the efficacy could be augmented by "arming" the vectors with IL-12. Further optimization oftherapeutic designs may make intravenous delivery of oncolytic HSY- I a useful strategy for treating metastases.

591. Combination Treatment Strategies That Modify Ceramide Metabolism To Increase the Efficacy of FasL Gene Therapy Lorianne S. Turner,' Xiang LU,I Saeed Elojcimy,' Ayman E. M. Mahdy,' Antonio F. Saad,' Jian-Yun Dong,' Terrence Day,' Thomas Keane,' Yusuf Hannun,' James S. Norris.' 'Microbiology and Immunology; Medical University ofSouth Carolina. Charleston. SC; 'Otolaryngology - Head and Neck Surgery; Medical University ofSouth Carolina. Charleston. SC; JUrology. Medical University ofSouth Carolina . Charleston. SC; "Btochemtstry and Molecular Biology, Medical University 0/ South Carolina. Charleston. Sc. Ceramide is a lipid second messenger involved in cellular response to a variety of cancer treatments including chemotherapy and radiation. In addition, ceramide is up-regulated in response to many endogenous cellular stresses, including Fas/FasL interactions , and increased ceramide can initiate signaling pathways leading to apoptosis. Therefore, it follows that cells which are able to metabolize ceramide and keep cellular concentrations down might be resistant to Fas/FasL-induced apoptosis. Our laboratory has documented that the ceramide metabolizing enzyme acid ceramidase (AC) is overexpressed in >65% of prostate cancer (PCa) and head and neck squamous cell carcinoma (HNSCC) samples compared to matched normal tissue controls. These results suggest that increased clearance ofeeramide by overexpression ofAC might allow cancer cells to escape eeramide-indueed apoptosis, and thus highlight a novel target for cancer therapy. Treatment of PCa and HNSCC cell lines with Fas agonists, in an attempt to induce apoptosis, have demonstrated these cells are nearly universally resistant to this form of signaling. However, adenovirus-mediated intracellular expression of Fasl. successfully induced apoptosis in target cells, suggesting internal interactions can overcome the signaling block and highlighting the possibility of FasL gene therapy as a treatment option for solid tumors. Based on this we set out to determine what effects alterations ofceramide signaling would have on the cellular Molecular Therapy Volume 15 ~ Supplement I. May2007 Coprright © Th e AmericanSociety of Gf,.'f11; Th erapy

response to Fas/FasL interactions. Previously we demonstrated that while the HNSCC cell line SCC-I expresses Fas receptor, it is resistant to Fas-mediated apoptosis. In the current work we show that siRNA against AC sensitizes SCC-I cells to the Fas agonist CH I I. Conversely, when SCC-I cells were transfected with an AC expression vector they became resistant to cell death induced by the AdGFPFasL vector. This was followed up by analyzing the effect of LCL204 , a novel inhibitor of AC, on FasL-indueed cell death. Our results demonstrated that AC inhibition by LCL204 sensitizes both PCa and HNSCC cell lines to FasL-mediated cell death. Additional studies on cells that overexpress AC indicate that it plays a role in resistance to chemotherapy or radiation as well. Therefore, we conclude that agents that enhance ceramide accumulation by down-regulating or inhibitingAC function will improve therapy of these cancers. Finally our results show that FasL gene therapy can be a viable treatment option for resistant cell types when used in combination with AC inhibitors. This work is supported by NIHINCI POI CA97132-01AI.

592. In Vivo Monitoring of Efficacy of Human Genetically Modified PSMA-Specific Lymphocytes Konstantin Dobrenkov,' Malgorzata Olszewska,' Yuri Likar,' Jelena Vider,' Larissa Shenker,' Michel Sadelain," Yladimir Ponomarev. I ,Radiology. Memorial-Sloan Kettering Cancer Center; New York. N}~' 2Aledicine. Memorial-Sloan Kettering Cancer Center; New York. Nl: Background: in vivo non-invasive assessment oftrafficking, expansion and survival ofgenetically modified 'I-lymphocytes bearing chimeric antigen receptors (CARs) would provide new insight in tumor-immune system interaction biology. It may also be useful to monitor immunotherapy and predict therapeutic outcomes. Bioluminescent imaging (BLI) represents a powerful tool for the investigation ofrepetitive tumor and immune cell visualization in vivo. Methods: SCIDlBeige immunodeficient mice were challenged with RM I murine prostate carcinoma cells expressing human prostate-specific membrane antigen (hPSMA) and a red fluorescent protein-Renilla lueiferase (RFP/Rlue) reporter gene. Human T-Iymphoeytes were transduced with CARs specific for either hPSMA (Pz l receptor) or carcino-embryonic antigen (hCEA, Cz I control receptor), and a herpes simplex virus type I thymidine kinase (HSYitk), green fluorescent protein (GFP), and Click Beetle luciferase (CBRLuc) triple fusion reporter gene. After ex vivo expansion, 'l-cells were adoptively transferred into tumor bearing mice. 'l-cell distribution was visualized by BLI ofCBRLue expression. Metastatic lung tumors were assessed using BLI ofRluc expression. GFPwas used for assessment ofT-cell transduction efficiency. Results: 85% ofT-celis were CAR- and GFP-positive. Since different luciferases were used, independent BLI of tumor progression and T-cell localization and persistence was repeatedly performed during the course ofthe study without bioluminescent signal interference. BLI showed prolonged 'f-cells persistence at tumor sites in PzI treated group comparing to control Cz l-treated group on days +1 to +6 after 'l-cell injection. Tumor growth delay was observed in PzI treated group but not in control Cz I treated group. Survival advantage in the group , injected with PzI -transduced T-lymphocytes was significantly higher (p