509 Dose Escalation of Therapeutic High Capacity, Gutless Adenoviral Vectors in the Naïve Rat Brain Pre Clinical Safety and Toxicity Evaluation as a Prelude to a Phase I Clinical Trial for Glioma Mol[.]
CANCER - IMMUNOTHERAPY II Apoptosis was induced by upregulation of caspases 3, and Secondly, we compared the therapeutic efficacy of anthocyanins with a leukemia specific DNA vaccine to treat mice with minimal residual disease (MRD) of syngeneic Ph+ ALL Non-viral minimalistic immunogenically defined gene expression vectors (MIDGE) encoding a BCR-ABL fusion specific peptide, GM-CSF and IL12 were used for in vivo transfection of murine skin As TLR-9 agonist we used a DNAbased double stem-loop immunomodulator (dSLIM), containing six CpG-motifs Following a lethal leukemia challenge mice received either daily oral applications of berry extract or intraperitoneal applications of cyanidin from day – 21 or were immunized with the DNA vaccine on days and In addition, two groups received a combination of the DNA vaccine and berry extract or cyanidin In contrast to the anti-leukemia activity in vitro the anthocyanins were not efficient to treat mice with Ph+ ALL in vivo However, mice which received the DNA vaccine showed a significant longer tumor-free and overall survival compared to the control and a survival rate of 56% Intriguingly, the combined treatment with DNA vaccine and berry extract but not with cyanidin further improved the outcome and lead to a significant longer tumor-free and overall survival compared to the DNA vaccine alone and a survival rate of 90% In conclusion, we provide data that a leukemia-specific DNA vaccine and berry extract act synergistically in the treatment of mice with MRD This approach may have implications to optimize maintenance therapy in patients with Ph+ ALL 508 IL-21 Has an Anti-Apoptotic Effect on NK Cells during Activation Vladimir Senyukov,1 Cecele J Denman,1 Laurence J N Cooper,1 Dean A Lee.1 Pediatrics, UT MD Anderson Cancer Center, Houston, TX Natural killer (NK) cells, as a key component of innate immunty, have recently shown clinical potential for adoptive immunotherapy against broad spectrum of oncological diseases One of the major obstacles for adoptive NK cell immunotherapy is obtaining sufficient numbers of NK cells for effective therapy To address this hurdle, we developed an artificial antigen presenting cells (aAPC) to expand primary NK cells in vitro using K562 cells gene-modified to express membrane-bound cytokine, and showed that mIL21 promoted stronger NK cells expansion than mIL15 To investigate this phenomenon we compared NK cell proliferation kinetics (by SFCE dilution) in parallel with daily assessment of apoptosis (by annexin V biding) in response to mIL15 and mIL21 We find that in the presence of mIL21 NK cells proliferated much faster than in response to mIL15 (fig.1a) Moreover, in the presence of mIL15 as much as 90% of NK cells (versus 15% in presence of mIL21) became able to bind annexin V by the 3rd day after aAPC stimulation (fig1b) Furthermore, we have found that mIL21 up-regulated and supported CD160 expression by NK cells during expansion time (42% in presence of mIL21 vs 6% in response to mIL15 on day and 83% vs 16% on day 7) CD160 is a GPI-anchored protein and its expression is down-modulated by GPI-specific phospholipase D and synthesis of which, in turn, is induced in NK cells in response to IL15 and IL2 It has been shown that CD160 signaling mediates PI3K-dependent survival and growth in CLL cells that was associated with up-regulation of Bcl-2, Bcl-xL, and Mcl-1 In addition to the herpes virus entry mediator that is a high affinity ligand for CD160 it has been shown that classical and nonclassical MHC class I molecules bind to CD160 with low affinity The K562 cell line used to generate the aAPC is characterized as HLA-Cw3+/Cw5+,with low-level constitutive expression that can be augmented by IFN-γ To verify if CD160 engagement with MHC class I is important for NK cell survival in this system we applied w6/32 antibodies to block HLA-C recognition We stained fresh purified NK cells with CFSE and blocked Fc-receptors with human IgG to prevent CD16 involvement in NK cell activation In parallel, irradiated S196 aAPC-mIL21 were incubated with w6/32 antibodies or with isotypematched control antibodies and after washing cells were co-cultured with NK cells We found that blocking of HLA class I recognition did not affect NK cell proliferation in response to aAPC-mIL21 (fig.2a) but increased apoptosis (fig.2b) This data suggests that IL21might prevent activation-induced NK cell death through up-regulation of anti-apoptotic factors through a CD160–mediated mechanism Future experiments will focus on identifying the mechanisms of suppressing apoptosis in this system 509 Dose Escalation of Therapeutic HighCapacity, Gutless Adenoviral Vectors in the Naïve Rat Brain: Pre-Clinical Safety and Toxicity Evaluation as a Prelude to a Phase I Clinical Trial for Glioma A K M G Muhammad,1 Mariana Puntel,1 Weidong Xiong,1 Kyle Kelson,1 Alireza Salem,1 Kurt M Kroeger,1 Chunyan Liu,1 Catherine Farrokhi,1 Liliana Lacayo,1 Robert N Pechnick,1 Donna Palmer,2 Philip Ng,2 Pedro R Lowenstein,1 Maria G Castro.1 Cedars-Sinai Medical Center/UCLA, Los Angeles, CA; 2Baylor College of Medicine, Houston, TX Glioblastoma multiforme (GBM) carries a dismal prognosis with a median survival of 18-21 months post-diagnosis We have recently demonstrated the therapeutic efficacy and high safety profile of intratumoral delivery of a novel, combined gene therapy consisting of first generation, or high-capacity adenoviral vectors (HC-Ads) encoding either the conditionally cytotoxic gene herpes simplex type 1-thymidine kinase (TK) or the immunostimulatory gene fms-like tyrosine kinase ligand (Flt3L) transgenes in a syngeneic, orthotopic rat model of GBM In anticipation of an upcoming dose finding, Phase I clinical trial for primary GBM, we herein performed a thorough assessment of the safety and toxicity of HC-Ads encoding TK and Flt3L upon administration into the naïve rat brain Lewis rats were injected intracranially with escalating doses of HC-Ad-TK and HC-Ad-TetON-Flt3L (1x10^8, 1x10^9, or 1x10^10 vp of each) followed by systemic administration of gancyclovir and doxycycline Rats were evaluated at days, month, months and 12 months for biodistribution of HC-Ad vector genomes, HC-Ad vector induced behavioral deficiencies, neurotoxicity, peripheral blood cell counts and serum biochemistry, circulating levels of Flt3L, anti-adenovirus neutralizing antibodies, and anti-TK antibodies Even at the highest dose tested, real-time quantitative PCR did not detect evidence of biodistribution of HC-Ad genomes to peripheral organs and a comprehensive panel of behavioral testing did not reveal any vector mediated abnormalities Peripheral blood cell counts and serum biochemistry were within normal ranges during all time points, at all doses tested Flt3L expression was tightly regulated, with Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy CANCER - IMMUNOTHERAPY II expression dynamics that coincided with the administration of the inducer doxycycline However, a comprehensive neuropathological panel revealed evidence of neurotoxicity at the highest dose tested (1x10^10 vp of each), i.e loss of brain tissue, and high levels of inflammation We did not detect any evidence of neurotoxicity or long-term inflammation at either lower dose tested Taken together, these data indicate that 1x10^9 vp of each HC-Ad is the maximally tolerated dose (MTD) that can be safely administered into the brains of naïve Lewis rats These well controlled, non-clinical safety and toxicity studies are required by the FDA in order to file of an IND and constitute a major milestone in the path towards implementation of the first ever Phase I clinical trial for GBM using gutless, HC-Ad vectors 510 Role of STAT3 Signaling in TK/Flt3L Gene Therapy Mediated Brain Tumor Regression Hikmat H Assi,1,2 James Curtin,1 Chunyan Liu,1 Maria G Castro,1,2 Pedro R Lowenstein.1,2 Gene Therapy Research Institute, Cedars Sinai Medical Center, Los Angeles, CA; 2Molecular and Medical Pharmacology, UCLA, Los Angeles, CA STAT3 signaling is constitutively active in high-grade tumors and its transcriptional activity is required by malignant cells for their proliferation well as promotion of an immunosuppressive tumor microenvironment Recently pre-clinical models have investigated whether STAT3 antagonists can be used in combination with the immunotherapy to induce tumor cell death and enhance immune responses against GBM antigens Owing to the multiple roles of STAT3 in both tumor cells and immune cells, we wished to investigate whether STAT3 signaling is involved in Ad-TK/Ad-Flt3L gene therapy-mediated T cell dependent brain tumor regression We used adenovirus expressing STAT3 shRNA as well small molecule inhibitors to silence STAT3 and assessed its role in tumor cell proliferation as well as dendritic cell (DC) activity We report that expression of STAT3 is up regulated in various syngeneic glioma mouse models Treating Gl26 or SMA560 glioma cells with the novel small molecule inhibitor CPA-7 blocks STAT3 in vitro and leads to cell death through a loss of Cyclin D, Bcl-xl and survivin expression We also show that STAT3 mediates the activation and maturation of DCs Using bone marrow cells with GM-CSF we generated immature mouse DCs and examined the effects on DC maturation elicited by the TLR ligand CpG and the STAT3 inhibitor CPA7 Treatment of DCs with CPA-7 induced their maturation evidenced by increased expression of co-stimulatory molecules CD80, CD86 and CD40 as well as decreased antigen uptake indicative of a more mature phenotype CPA-7 treatment enhanced the maturation activity of CpG DCs treated with CPA7 in combination with CpG were able to better stimulate the proliferation of T cells in an allogeneic MLR when compared to DCs treated with CpG alone We have previously demonstrated that intra-tumoral delivery of Flt3L and TK using adenoviral vectors drives T cell dependent tumor regression in intracranial syngeneic glioma (GBM) models in rodents (Ali et al, 2005; Curtin et al 2009; Candolfi et al, 2009) Intra-tumoral delivery of STAT3 shRNA, however, completely blocks Flt3L and TK mediated tumor regression compared with a scrambled non-specific shRNA This was confirmed using the small molecule inhibitor CPA-7 Our data suggest that STAT3 signaling is involved in tumor cells’ survival and DCs’ functions, thus constituting an attractive therapeutic target for brain cancer Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy 511 An Enhanced Non Viral Vector (EEV) Giving Improved Responses in Immunogene Cancer Treatment over Standard Plasmid Vectors Patrick F Forde,1 Lindsay J Hall,1 Marcel de Kruijf,1 Gerald C O’Sullivan,1 Declan M Soden.1 Cork Cancer Research Centre, University College Cork, Cork, Ireland We compared the efficacy of an enhanced expression vector (EEV) with CMV standard plasmid vector for non viral immunogenetherapy of solid tumours The EEV contains its own RNA replicase and transcribes mRNA cytoplasmically The transgenes LacZ and GMCSF/B71 were under the control of the CMV promoter Comparison of the EEV with the standard vector in normal and growing tumours used quantitative RT-PCR, histological tools and in vivo murine tumour growth and survival curves In all normal tissues (liver, spleen, and colon and oesophageal epithelium) there were significant increased RNA/DNA ratios for the EEV over the standard plasmid The EEV gives superior results than standard vector in non viral immunogene therapy of solid growing tumours The EEV was curative in two tumour models and has potential for clinical development Antitumor responses of the EEV vector were both from immune cell recruitment to the tumour environment and the unrestrained RNA production in the cytoplasm 512 A Novel Adjuvant Ling Zhi-8 Enhances the Efficacy of DNA Cancer Vaccine by Activating Dendritic Cells Chi-Chen Lin,1 Chia-Chiao Shih,1 Ching-Liang Chu.2 Institute of Biomedical Sciences, National Chung Hsin University, Taichung, Taiwan; 2Yeastern Biotechnology Inc, Taipei, Taiwan DNA vaccine has been applied in cancer therapy, but the efficacy remains to be improved The immunostimulatory effect of a fungal immunomodulatory protein Ling Zhi-8 (LZ-8) isolated from Ganoderma lucidum has been reported on human dendritic cells (DCs) In this study, we tested the adjuvanticity of LZ-8 for HER-2/ neu DNA vaccine against p185neu expressing tumor MBT-2 in mice We found that recombinant LZ-8 activated mouse bone marrowderived DCs via TLR4 and this stimulation was not due to any microbe contaminant In addition, LZ-8 enhanced the ability of DCs to induce antigen-specific T cell activation in vitro and in a subunit vaccine model in vivo Surprisingly, LZ-8 co-treatment strongly improved the therapeutic effect of DNA vaccine against MBT-2 tumor in mice This increase of anti-tumor activity was attributed to the enhancement of vaccine-induced Th1 and CTL responses Consistent with the results from DCs, the promoting effect of LZ-8 on DNA vaccine was diminished when the MBT-2 tumor cells were grown in TLR4 mutant mice Thus, we concluded that LZ-8 may be a promising adjuvant to enhance the efficacy of DNA vaccine by activating DCs 513 Prostate Cancer Immunotherapy Using BiSpecific Tumor-Reactive T Cells Robert C Chan,1 Ceidy Sanchez,1 Anna Worth,1 Ann M Leen,1 Malcolm K Brenner,1 Ganesh Palapattu,2 Juan F Vera.1 Baylor College of Medicine, Texas Children’s Hospital, The Methodist Hospital, Houston; 2Department of Urology, The Methodist Hospital, Houston Although localized prostate cancer (PC) can often be cured by surgery and/or radiation therapy, the therapeutic options for castrateresistant disease (CRPC) are largely palliative, underscoring the need for alternative therapies Like many other tumors, CRPC expresses tumor-associated antigens (TAAs), which are potential targets for immune destruction However, tumors use multiple mechanisms of immune evasion including downregulation of target antigen S197 ... non -clinical safety and toxicity studies are required by the FDA in order to file of an IND and constitute a major milestone in the path towards implementation of the first ever Phase I clinical. .. stimulation was not due to any microbe contaminant In addition, LZ-8 enhanced the ability of DCs to induce antigen-specific T cell activation in vitro and in a subunit vaccine model in vivo Surprisingly,... Sciences, National Chung Hsin University, Taichung, Taiwan; 2Yeastern Biotechnology Inc, Taipei, Taiwan DNA vaccine has been applied in cancer therapy, but the efficacy remains to be improved The