59 Interferon Lambda Suppresses the Growth of Human Esophageal Carcinoma Cells and Enhances the Sensitivity to Anti Cancer Agents Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The Am[.]
CANCER-APOPTOSIS & SUICIDE/CANCER-IMMUNOTHERAPY, ADOPTIVE T CELL THERAPY flank followed by twice-weekly injection of PEI nanoparticles into the tail vein once palpable tumors were observed Control mice had an average tumor volume of 284 mm3 at the time of sacrifice while mice treated with 1.5 mg/kg of PEI:peIF5AK50R:h5A1 had an average tumor volume of only 13 mm3, a 95 % (*p = 0.026) reduction in tumor growth Furthermore, when we attempted to excise the tumors after sacrifice, no residual tumors could be found under the skin of the treated mice Established tumors (>100 mm3) also responded to treatment with 1.5 mg/kg of PEI:peIF5AK50R:h5A1, resulting in the average tumor volume falling from 133 mm3 to 30.5 mm3 after 24 days of treatment TUNEL-labeling revealed evidence of apoptotic cells in the treated tumors indicating that tumor regression occurs through the induction of apoptosis The biodistribution of systemically-delivered PEI nanoparticles was determined using a GFP plasmid and a fluorescently-labeled (DY547) siRNA GFP expression and DY547-siRNA uptake was observed in subcutaneous tumor tissue as well as in cells of the bone marrow The safety of long-term administration of PEI:peIF5AK50R:h5A1was assessed in BALB/c mice Mice receiving twice-weekly administration of PEI nanoparticles continued to gain weight over the nine-week study period and no hematological or liver function changes were observed In summary, our preclinical data indicate that it is efficacious and safe to use systemic administration of PEI:peIF5AK50R:h5A1nanopa rticles in the treatment of multiple myeloma PEI nanoparticles are taken up by cells of the bone marrow following systemic delivery indicating that they may be feasible as a delivery vehicle for gene therapy treatment of multiple myeloma patients 58 Infectivity-Enhanced Oncolytic Adenoviral Vectors Expressing Syngeneic IFNα for Pancreatic Cancer Julia Davydova,1 Eric Brown,1 Selwyn Vickers,1 Masato Yamamoto.1 Surgery, University of Minnesota, Minneapolis, MN Interferon α (IFN) which is commonly used as an antiviral agent exhibits several properties that might be a great use for combination therapy of cancer (e.g direct inhibition of tumor cell growth and indirect antitumor effects through immunomodulation, antiangiogenesis, radio- and chemo-sensitization) Despite encouraging results for survival rates in clinical studies employing IFN chemoradiation therapy, utilization of IFN-based regimen has been impeded by systemic IFN toxicity, unreliable tumor delivery, and its short half-life in blood circulation To reduce IFN toxicity and enhance its efficacy, gene therapy approaches, such oncolytic adenoviral vectors (Ads) expressing human IFN can be used In this work, we apply infectivity-enhanced conditionally replicative Ads (CRAds) expressing syngeneic IFNα in replication-dependent manner to treat pancreatic cancer To increase Ad potency, we enhanced the efficiency of cell lysis through overexpression of adenoviral death protein and equipped the vector with 5/3 and RGD-modified fibers To study antitumor and immunomodulatory effect of replicationdependent IFN expression in syngeneic hamster and immunodeficient mouse models, we designed Ads expressing hamster and human IFNs Comparison of vector functionality in vitro in hamster and human pancreatic cancer cell lines revealed that oncolytic Ads expressing IFN require approximately orders lower titers to successfully kill the cells compare to non-replicative IFN vectors Killing effect and decline in cell viability observed with oncolytic IFN vector were significantly superior compared to that with identical counterpart vectors without IFN The cytotoxicity effect correlated well with IFN production in culture supernatant The IFN concentration increased in a time- and dose-dependent manner after infection with oncolytic Ad, indicating that IFN production depends upon viral replication For instance, infection of MiaPaca2 cells with oncolytic Ad5/3IFN at 0.1 vp/cell resulted in order higher IFN level by day 12 post S24 infection compared to that of non-replicative IFN vector To study direct and indirect antitumor effect of IFN vectors, we established subcutaneous hamster HP1 tumors in immunocompetent Syrian hamsters and human MiaPaca2 xenografts in nude mice In hamsters, the tumors were treated with a single intratumoral injection of RGDmodified vectors expressing hamster IFN Significant antitumor effect was observed with both non-replicative and oncolytic IFN vectors Treatment with non-replicative vector was as effective as that with oncolytic Ad during the first days after injection; however, after day 12 we observed tumor re-growth in the non-replicative vector group and by day 20 oncolytic Ad IFN significantly outperformed the nonreplicative vector Importantly, the hamsters treated with oncolytic IFN vector exhibited significant tumor shrinkage compared to initial tumor size The antitumor effect in MiaPaca2 xenograft in nude mice was less evident compared to those in the HP1 model, suggesting possible indirect antitumor effect of IFNα as an immunomodulator These data indicate that infectivity-enhanced CRAd expressing syngeneic IFNα is a powerful therapeutic modality for pancreatic cancer 59 Interferon-Lambda Suppresses the Growth of Human Esophageal Carcinoma Cells and Enhances the Sensitivity to Anti-Cancer Agents Quanhai Li,1 Kiyoko Kawamura,1 Guangyu Ma,1,2 Nobuo Suzuki,2 Yuki Takei,1,3 Naoto Yamaguchi,3 Fumi Iwata,4 Muneo Numasaki,4 Hideaki Shimada,5 Masatoshi Tagawa.1 Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan; 2Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, Chiba, Japan; 3Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan; 4Department of Nutritional Physiology, Faculty of Pharmaceutical Science, Josai University, Sakado, Japan; Division of Gastroenterological Surgery, Chiba Cancer Center, Chiba, Japan Novel cytokines, interleukin-28A (IL-28A), IL-28B and IL-29, have been identified to belong to a novel interferon (IFN) family, IFN-lambdas All the three cytokines have similar functions and IFN-lambdas are classified as a type III IFN IFN-lambdas could have a distinct activity compared with type I IFN, IFN-alpha and IFN-beta although the precise biological properties of IFN-lambdas remain to be uncharacterized We found that human esophageal carcinoma cell lines expressed the receptor complex of IFNlambdas consisting of IL-10Rbeta and a novel molecule, IL-28R Recombinant IFN-lambda1 up-regulated the expression of level of the class I molecules of the major histocompatibility complexes (MHC) and induced the gene expression of myxovirus resistance A and 2’,5’-oligoadenylate synthetase, which are involved in antiviral protection, in all the esophageal carcinoma cells tested We also found that IFN-lambda1 inhibited the proliferation of esophageal carcinoma cells due to multiple mechanisms including cell cycle arrest or apoptosis The cell cycle arrest was accompanied by upregulated p21 gene expression and increased G0/G1 population Apoptosis was evidenced by increased subG1 population and Annexin-V-positive cells Although all the esophageal carcinoma cells expressed the IFN-lambdas receptor complexes and up-regulated MHC class I expression with IFN-lambda1, inhibited proliferation was not observed in all the cells tested Several human normal cells, positive for IFN-alpha receptor complexes, did not express one of the IFN-lambda receptor complexes, and subsequently did not respond to IFN-lambda1 but were susceptible to IFN-alpha-mediated growth inhibition We demonstrated that combinatory use of 5-FU or cisplatin with IFN-lambdas further enhanced the cytotoxicity to sensitive esophageal carcinoma cells but not to normal cells Adenoviruses with type35 fiber bearing the IL-28A gene induced Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy CANCER-APOPTOSIS & SUICIDE/CANCER-IMMUNOTHERAPY, ADOPTIVE T CELL THERAPY suppressed cell growth of IFN-lambdas-sensitive carcinoma cells These data collectively suggest that IFN-lambdas is a potential anticancer agent for esophageal carcinoma and useful in combination with chemotherapeutic agents 60 Co-Expression of a Chimeric Antigen Receptor Targeting CD19 (CAR19), Optimized Human IL-15, and iCaspase9 To Enhance the Activity and Safety of Cytotoxic T Lymphocytes Valentina Hoyos,1 Barbara Savoldo,1 Juan F Vera,1 Concetta Quintarelli,1 Helen E Heslop,1 Cliona M Rooney,1 Malcolm K Brenner,1 Gianpietro Dotti.1 Pediatrics, Medicine, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX Recurrence of disease remains the largest cause of mortality for many patients transplanted for relapsed or refractory B cell malignancies Modification of primary T cells to express a chimeric antigen receptor (CAR) targeting CD19 represent an attractive strategy to maintain remission after conventional treatment However, their efficacy is limited by poor expansion within the tumor microenvironment The addition of co-stimulatory endodomains, such as CD28, to the CAR may enhance cell expansion in response to the antigen, but cell growth and survival remain suboptimal To further potentiate the expansion and survival of CAR-modified T lymphocytes, we generated a new vector encoding molecules: CAR.19 incorporating the CD28 endodomain, codon optimized hIL15 to enhance cell survival and growth, and an inducible suicide gene based on the expression of Caspase9 (iCasp9) to increase the margin of safety associated with transgenic expression of an autocrine growth factor These three sequences were linked using 2A-like peptide sequences We compared the proliferative capacity, cytotoxic activity and in vivo anti-tumor effects of T lymphocytes expressing either CAR.19-28ζ alone or CAR.19-28ζ, IL15 and the suicide gene T lymphocytes were activated with OKT3/CD28 antibodies and then transduced with retroviral supernatants Phenotypic analysis showed 70±10% and 75±5% transduction efficiency for iCasp9/CAR19-28ζ/ IL15 and CAR19-28ζ T cells, respectively Only the iCasp9/CAR1928ζ /IL15 T cells produced IL15 (>100pg/mL) after stimulation with CD19+ tumor cells T cells kept in culture for weeks stimulated weekly with CD19+ B-CLL cells were co-cultured with CD19+ Daudi cells After 72 hours iCasp9/CAR19-28ζ/IL15 T cells were more efficient eliminating tumor cells compared to CAR19-28ζ T cells (0.7% residual tumor cells vs 14% respectively) Furthermore, labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that the proliferation of the iCasp9/CAR19-28ζ/IL15+ T cells in response to CD19+ tumor cells was greater than that of CAR.19-28ζ control+ T cells Finally, the activation of the suicide gene iCasp9 with a small-molecule dimerizer (CID 50ng/mL) rapidly induced >90% apoptosis of T cells expressing iCasp9/CAR19-28ζ/IL15 Hence all three transgenes were functional To assess the antitumor effects of the modified cells in vivo, we used a xenograft SCID mouse model and an in vivo bioluminescence system CD19+ Daudi cells (1x106) expressing firefly Luciferase (FL) were injected i.p., and on day 4, mice received i.p iCasp9/CAR19-28ζ/IL15+ or CAR1928ζ+ or control T cells (10x106) By day 30 the tumor signal was significantly reduced in mice receiving iCasp9/CAR19-28ζ/IL15+ T cells (ROI2.0x109) In conclusion, our data indicate that a tricistronic vector can effectively be expressed in tumor-redirected human T cells, improving their survival and allowing their destruction should unwanted effects occur Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 61 Effects of Chimeric Antigen Receptor (CAR) Expression on Regulatory T Cells Ibrahim Akalin,1 Serena K Perna,1 Biagio De Angelis,1 Fatma V Okur,1 Cliona M Rooney,1 Helen Heslop,1 Malcolm K Brenner,1 Barbara Savoldo,1 Gianpietro Dotti.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX Adoptive immunotherapies with genetically modified T lymphocytes are endowed with a means for effectively treating malignant and infectious disorders Transduction of T cells with chimeric antigen receptors (CARs) can redirect the cellular immune response to almost any surface target antigen The function of CAR+ T cells in vivo may, however, be impaired by naturally occurring regulatory T cells (Tregs) Moreover, Tregs themselves might be the inadvertent targets of CAR transfer, resulting in an undesired increase in their presence at the site of activity of CAR-expressing effector T cells To discover the importance of this effect and to devise means to avoid this, we isolated CD4+CD25bright Treg cells from peripheral blood of healthy donors and assessed their inhibitory function using carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay We then transduced peripheral blood polymorphonuclear cells (PBMCs) and Tregs with a chimeric antigen receptor targeting CD19 (CAR-CD19-CD28ζ), since this antigen is present on a high proportion of human B cell malignancies, and is the target for several current trials of T cell immunotherapy using such CAR-CD19 Our results showed that both activated PBMCs and Tregs are transduced and express CAR-CD19 (from 20% to 80%) While CAR-CD19+ T cells showed cytotoxic activity against CD19+ target cells as assessed by 51Cr release assay (71%±34% at E:T ratio 20:1), CAR-CD19+ Tregs lacked effector function (6%±0.1% at E:T ratio 20:1) (p=0.01), but retained inhibitory activity in an MLR [% of proliferation were 68%±18% and 28%±19% when activated T cells were incubated with CD4+CD25- control cells and CAR-CD19+ Tregs, respectively (p=0.001)] More importantly, when CAR-CD19+ Tregs were mixed with CAR-CD19+ T cells (ratio 5:1) they inhibited the anti-tumor effects of these cells (% of remaining CD19+ tumor cells were 49%±21% and 2%±4% when tumor cells were co-cultured with CAR-CD19+ T cells with or without the addition of CAR-CD19+ Tregs) (p=0.02) To discover whether the inhibitory effect on CARCD19+ T cells by CAR-CD19+ Tregs was due to competition for the same epitopes on the CD19 target antigen, we redirected Tregs and T cells with two different CARs (CAR-CD19 and CAR-CD30) We found that inhibition mediated by Tregs was maintained, suggesting that specific regulation rather than epitope/antigen competition was responsible for the observed inhibition Hence, transduction of unseparated peripheral blood T cells transduces both effector T cells and Tregs, and the latter are detrimental to the function of the former Future efforts to generate CAR-modified T cells for human use may need to separate effector T cells from Tregs before transduction or before infusion 62 Conditional Activation of T Cells to Specifically Target c-Met under Hypoxia Sonny O Ang,1 Simon Olivares,1 Drew C Deniger,1 Dean A Lee,1 Richard E Champlin,1 Laurence J Cooper.1 University of Texas MD Anderson Cancer Center, Houston, TX Malignancies localized within chronic and intermittent hypoxic niches, is a negative prognosticator for many aggressive, chemoand radio-resistant solid tumors The decreased oxygen tension, depleted nutrient levels, and low extracellular pH in this tumor microenvironment can limit T-cell viability, cytotoxicity, and thus immunotherapeutic efficacy This makes T-cell mediated elimination of bulky solid tumors difficult However, this also represents a microenvironment that can be exploited by genetic engineering to activate T cells for redirected killing We developed a new approach that S25 ... survival and growth, and an inducible suicide gene based on the expression of Caspase9 (iCasp9) to increase the margin of safety associated with transgenic expression of an autocrine growth factor These.. .CANCER- APOPTOSIS & SUICIDE /CANCER- IMMUNOTHERAPY, ADOPTIVE T CELL THERAPY suppressed cell growth of IFN-lambdas-sensitive carcinoma cells These data collectively suggest that IFN-lambdas... effector T cells To discover the importance of this effect and to devise means to avoid this, we isolated CD4+CD25bright Treg cells from peripheral blood of healthy donors and assessed their