503 Safety of Intravenous Ad5 Mda 7, a Potential Anti Metastatic Agent in Gynecologic Cancer Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy[.]
CANCER-ONCOLYTIC VIRUSES II 5-uorocytosine (5FC), cytotoxicity against RCR-yCD infected malignant mesothelioma cells was assessed in vitro by Alamar blue assay, and tumor growth inhibition effects in vivo were assessed in both subcutaneous xenograft tumor and disseminated peritoneal tumor models of malignant mesothelioma Results: Even at multiplicities of infection (MOI) as low as 0.01, RCR vectors successfully infected and efciently replicated in human malignant mesothelioma cell lines, as compared to nonmalignant transformed mesothelial cells In mice with pre-established subcutaneous tumor xenografts, the RCR-GFP showed robust spread throughout entire tumor masses by Day 12 after intratumoral administration of x 104 total infectious units per 100 µl inoculum Notably, no RCR infection was detectable in adjacent normal tissue RCR-yCD showed efcient transmission of the suicide gene associated with replicative spread of the virus, resulting in efcient killing of malignant mesothelioma cells in a 5FC-dose dependent manner in vitro After intratumoral injection of RCR-yCD followed by intraperitoneal administration of 5FC prodrug, RCR vectormediated suicide gene therapy achieved signicant inhibition of subcutaneous tumor growth, and signicantly prolonged survival in the disseminated peritoneal model of malignant mesothelioma Conclusions: These data indicate that RCR vector-mediated suicide gene therapy may represent a highly useful new treatment strategy for malignant mesothelioma 501 Oncolytic Adenovirus with Somatostatin Motif for Selective Infection of Neuroendocrine Tumors Di Yu,1 Justyna Leja,1 Berith Nilsson,1 Magnus Essand.1 Clinical Immunology, Uppsala University, Uppsala, Sweden We have produced an oncolytic serotype adenovirus, Ad[CgAE1A-miR122], where the human chromogranin A (CgA) promoter and miR122 target sequences control E1A gene expression It selectively replicates in and kills neuroendocrine cells, including neuroblastoma and carcinoid cells Neuroendocrine tumors (NETs) contain a high density of homogeneously distributed somatostatin receptors (SSTR) on their surface and somatostatin analogues, such as octreotide, are used for tumor imaging and treatment In order to target SSTRs and increase the infectious capacity for NETs, we have introduced a cyclic loop with four amino acids (FWKT) from octreotide into either the HI-loop of the ber knob or the hexon hypervariable region (HVR) of the capsid We investigate binding properties of GFP-expressing adenoviral vectors with FWKT motif in the ber knob Ad[CMVGFP]HIFWKT and HVR-5 region Ad[CMV-GFP]HVR5FWKT The vectors were evaluated in various NET cell lines as well as in freshly isolated carcinoid cells and normal hepatocytes Both Ad[CMV-GFP] HIFWKT and Ad[CMV-GFP]HVR5FWKT infect SSTR-positive, CAR-negative neuroendocrine tumor cells with greater efcacy than Ad[CMV-GFP] Furthermore, Ad[CMV-GFP]HVR5FWKT can detarget factor X binding and neutralization antibody binding to the virus capsid and hopefully prolong the systematic circulation time We conclude that modication of the capsid with the FWKT motif can be benecial for adenovirus-based NET therapy 502 Silencing a p53 Inhibitor in Cancer Cells Increases Killing Potency of Oncolytic Adenovirus Christie Vermeulen,1 Nikki Tol,1 Winald R Gerritsen,1 Victor W van Beusechem.1 Dept Medical Oncology, VU University Medical Center, Amsterdam, Netherlands Virotherapy of cancer using oncolytic adenoviruses has shown promise in both preclinical and clinical settings The potency of oncolytic adenoviruses depends on their replication efciency in cancer cells and their capacity to destroy these cells by oncolysis S194 Previously, we found that the oncolytic potency of adenoviruses was enhanced by expressing tumor suppressor p53 from the virus genome Not unexpectedly, the effect was minimal in cancer cells expressing high levels of the p53 negative regulator human double minute (HDM2) By abrogating the interaction between HDM2 and p53 the oncolytic adenovirus potency was elevated This suggested that oncolytic adenoviruses could be made more effective by suppressing p53 inhibitor expression in cancer cells We envision that this could be done by incorporating a gene silencing cassette into the oncolytic adenovirus genome To develop such next generation viruses, the p53 inhibitor expression prole in cancer cells should be determined and it should be conrmed that silencing these inhibitors increases oncolytic potency In the present study, we screened derivatives of U2OS and A549 cancer cells carrying a p53 reporter construct for 18 putative p53 inhibitors using siRNA In U2OS cells, several molecules including HDM2 were found to inhibit p53 activity In contrast, in A549 cells considerable p53 activation was observed only upon silencing the SYVN1 gene SYVN1 encodes synoviolin, an ER-resident ubiquitin ligase that is highly expressed in rheumatoid arthritis synoviocytes Interestingly, functional screening for inhibition of adenovirusinduced cell death by the 18 putative p53 inhibitors in A549 cells also revealed only SYVN1 Using three independent cell viability assays (i.e., CellTiter-Blue conversion, BCA protein assay and microscopic cell counting), we found that silencing SYVN1 reproducibly and selectively increased adenovirus killing potency Adenovirus late gene expression, as tested using a virus with endogenous MLP-driven GFP expression, was not signicantly affected Our results establish that SYVN1 silencing empowers adenovirus-mediated killing of A549 cells without affecting early events in the virus life cycle They also show that endogenous p53 reactivation in cancer cells correlates with oncolytic adenovirus potency Therefore, our ndings suggest that knowledge on p53 inhibitor expression in cancer could be exploited to develop a new class of potent oncolytic adenoviruses 503 Safety of Intravenous Ad5-Mda-7, a Potential Anti-Metastatic Agent in Gynecologic Cancer Kenneth H Kim,1 Meredith A Preuss,1 Minghui Wang,1 Anand C Annan,1 Justin A Barnes,1 Devanand Sarkar,2 Steven Grant,2 Paul Dent,2 Paul B Fisher,2 Ronald D Alvarez,1 David T Curiel.1 University of Alabama, Birmingham, AL; 2Virginia Commonwealth University, Richmond, VA Objective: Mda-7 is a cytokine protein with many antitumoral properties that is highly conserved across multiple species; its progressive loss of expression has been shown to correlate with cancer progression Preclinical trials involving its use within an adenoviral viral vector Ad5-mda-7, have shown cancer-cell-selective apoptosis induction, and enhancement of cancer-selective cytotoxicity paired with adjuvant therapy in various solid tumors This study evaluated safety prole after IV injection of AdCMVmda-7, and characterizes its expression and secretion from the liver, in anticipation of ongoing evaluation as an anti-metastasis agent Methods: Two cohorts of mice were treated with doses of 2x10^10 vp/mL of either Ad5-mda-7 (5 mice/cohort) or Ad-CMV.CEA (3 mice/cohort) via tail vein injection, while a third control group (2 mice/cohort) remained uninjected On study days 0, 1, 3, 7, 14, and 21, blood samples were taken from each cohort of mice for chemistry panel analysis Animals were then euthanized and the following organs were removed from each animal for histopathological examination Immunohistochemistry was performed to evaluate mda-7 and CEA tissue expression in harvested organs Results: On immunohistochemistry analysis, mda7 expression within the liver was noted to increase, peaking on day and then returning to normal Similar mda-7 expression patterns were also noted within the kidney, also returning to normal after day There was minimal to no mda-7 expression in other organs At the Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy CANCER-ONCOLYTIC VIRUSES II time of necropsy, no gross abnormalities were noted at any of the time points, and there were no microscopic abnormalities were noted in any of the tissues In the serum analysis, comparison between the mda-7 and the CMV-CEA cohorts, the only clinically signicant difference noted in the levels of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) These values peaked on day 3, and then returned to baseline levels by the end of the study The only other signicant difference was elevated amylase levels in mda-7 versus CMV-CEA (528 vs 428, p=0.04) Of note, there were no signicant differences between any of the groups, indicating normalization over time Conclusions: Ad5-mda7 has an acceptable safety prole with IV administration Ongoing studies involving metastatic challenge are currently underway to evaluate its role in preventing metastasis 504 Characterization of a Novel Dual-Lock Oncolytic Cadvex-Vector That Combines Efcient Prostate-Specic Replication with Cytokine Expression Brian D Hoel,1 Min Luo,2 David A Einfeld,2 Danher Wang,2 John Y Dong.2 Department of Microbiology & Immunology, Medical University of South Carolina, Charleston, SC; 2GenPhar, Inc., Mount Pleasant, SC Prostate cancer remains the second leading cause of cancer death among American men and advanced metastatic disease remains incurable Therefore, there is a great need to develop alternative treatment strategies to improve these outcomes One approach has been exploration of virolytic approaches that target cancer cells We have developed a novel strategy that combines a conditionallyreplicative adenovirus (CRAd) vector with an immunotherapy approach We propose to advance this CAdVex vector into clinical testing for prostate cancer The vector design employs a “dual-lock” mechanism to control expression of both the E1 and E4orf6 genes necessary for replication We have found that using the probasin promoter to control expression of these genes results in enhanced replication that is specic for prostate cancer cells When injected into human prostate cancer tumors (50-100 mm3) in nude mice, virus employing this double lock mechanism was efcacious in blocking tumor growth In contrast, a “single-lock” virus with only the E1 genes under probasin control exhibited only a limited effect on tumor growth These data indicate the potent activity of the double-lock CAdVex is a significant improvement for CRAd design To further enhance the efcacy of these vectors we have engineered expression of immune enhancing cytokines and tumor antigens into the dual-lock CAdVex One of the vectors we have evaluated expresses TRAIL controlled by the probasin promoter in the dual-lock CAdVex backbone The rationale of our approach is that intratumoral injection of this vector has the ability to eliminate prostate cancer cells by a combination of mechanisms: 1) Replication of the virus among cancer cells will induce apoptosis leading to ablation of the primary tumor 2) Induction of apoptosis will be further expanded by production and release of TRAIL 3) Immune responses induced to antigens expressed by the replicating virus will lead to killing of vector-transduced cells 4) The pro-inammatory environment created by viral replication will enhance immune activation against tumor antigens that are poorly immunogenic in the context of the intact tumor The immune response to tumor antigens may be critical for reducing metastasis Details of the vector design and progress in preliminary evaluation will be presented Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy 505 A Probasin Promoter, Conditionally Replicating Adenovirus that Expresses the Sodium Iodide Symporter (NIS) for Radiovirotherapy of Prostate Cancer Miguel A Trujillo,1 Michael J Oneal,2 Samantha McDonough,1 Rui Qin,3 John C Morris.1 Endocrinology, Mayo Clinic, Rochester, MN; 2Department of Molecular Medicine, Mayo Clinic, Rochester, MN; 3Department of Health Sciences Research, Mayo Clinic, Rochester, MN The sodium iodide symporter (NIS) directs the uptake and concentration of iodide in thyroid cells We have extended the use of NIS-mediated radioiodine therapy to other types of cancer, and have transferred and expressed the sodium-iodide symporter (NIS) gene in prostate, colon, and breast cancer cells using adenoviral vectors To improve vector efciency we have developed a conditionally replicating adenovirus (CRAd) in which the E1a gene is driven by the prostate specic promoter, Probasin and the cassette RSV promoter-human NIScDNA-bGH polyA replaces the E3 region (CRAd Ad5PB_RSV-NIS) In vitro infection of the prostate cancer cell line LnCaP resulted in virus replication, cytolysis, and release of infective viral particles Conversely, the prostate cancer cell line PC-3 (androgen receptor negative) and the pancreatic cancer cell line Panc1 were refractory to the viral cytopathic effect and did not support viral replication Radioiodine uptake was readily measurable in LnCaP cells infected with Ad5PB_RSV-NIS 24 hours post-infection, conrming NIS expression In vivo, LnCaP tumor xenografts in nude mice injected intratumorally with Ad5PB_RSV_NIS CRAd expressed NIS actively as evidenced by 99Tc uptake and imaging Administration of therapeutic 131I after virus injection signicantly increased survival probability in mice carrying xenografted LnCaP tumors compared to virotherapy alone The data indicate that Ad5PB_RSV_NIS replication is stringently restricted to androgen positive prostate cancer cells and results in effective NIS expression and uptake of radioiodine This construct may allow multimodal therapy, combining cytolytic virotherapy with radioiodine treatment, to be developed as a novel treatment for prostate cancer 506 Adenovirus Vectors Transductionally Retargeted to Her2/neu for Prostate Tumour Treatment Maria K Magnusson,1 Robert Kraaij,2 Corrina M A De Ridder,2 Leif Lindholm.3 Dep for Microbiology and Immunology, University of Gothenburg, Inst for Biomedicine, Gothenburg, Sweden; 2Department of Urology, Erasmus MC, Rotterdam, Netherlands; 3Got-A-Gene AB, Kullavik, Sweden Preclinical and clinical studies have demonstrated that adenovirus (Ad) gene transfer vectors have great potential for the treatment of tumor diseases However, several signicant hurdles currently limit the efciency of gene transfer with adenovirus-based vectors One of these hurdles is that the virus infects many different cells in the body while cells relevant for targeting (such as cancer cells) are often refractory to infection This fact necessitates the development of Ad vectors that are targeted to selected cells We have previously reported a replication competent Ad vector specic for Her2/neu by inserting a tandem repeat of the Her2/neu reactive AfbodyTM molecule (ZH) in the HI-loop of a CAR binding ablated ber genetically modied to contain sequences for exible linkers between the ZH and the knob sequences (Ad5/FibZH/1) ZH is an AfbodyTM molecule specic for the extra-cellular domain of human epidermal growth factor receptor (Her2/neu) that is over-expressed in many carcinomas To further de-target the single ablated virus Ad5/FibZH/1 we changed the RGD motif in the penton base to EGD and substituted the KKTK motif in the third shaft S195 ... Carolina, Charleston, SC; 2GenPhar, Inc., Mount Pleasant, SC Prostate cancer remains the second leading cause of cancer death among American men and advanced metastatic disease remains incurable... serum analysis, comparison between the mda- 7 and the CMV-CEA cohorts, the only clinically signicant difference noted in the levels of alanine aminotransferase (ALT) and alkaline phosphatase (ALP)... is that intratumoral injection of this vector has the ability to eliminate prostate cancer cells by a combination of mechanisms: 1) Replication of the virus among cancer cells will induce apoptosis