442 Examination of the Therapeutic Potential of an Engineered VEGF A Transcription Activator in a Spinal Cord Injury Model killing is based on targeting of adenoviral replication to cancer cells resul[.]
killing is based on targeting of adenoviral replication to cancer cells resulting in tumor oncolysis by a natural lytic mechanism This cancer therapy paradigm has rationalized the rapid translation ofCRAd agents to the context of human clinical trials Despite the demonstrated clinical safety, little clinical efficacy of CRAds was noted Understanding of the biological factors confounding CRAd function in the human clinical context has been limited by the lack ofadequate CRAd monitoring techniques This warranted development of CRAd imaging methods potentially allowing monitoring CRAd replication , amplification , and localization in the human clinical context However, the currently employed imaging modalities function solely as readouts for a reporter transgene expression in CRAd-infected cells and, thus, are incompatible with the cell killing-based mechanism ofCRAd function Besides, they not allow tracking of viral particles to provide a dynamic index of amplified viral mass and , thus, adequately monitor CRAd replication in vivo To circumvent this limitation we previously developed a novel Ad labeling system based on incorporation of imaging reporter motifs in the adenoviral capsid via their genetic fusion to Ad minor structural protein IX (pIX) On this basis we hypothesized that this approach can be applied to construct a first capsid-labeled human CRAd without perturbing the virion infectivity and/or CRAd's antitumor potency Furthermore, technical fcasibilitics established by our previous studies allowed us to further hypothesize that capsid incorporation of dual modality reporter fusions , embodying the capacity for SPECT/PET and highly sensitive fluorescent imaging detection systems simultaneously could provide a flexible framework to monitor viral replication by complementary modalities that are easily translatable to human applications To validate our dual imaging approach we constructed and successfully rescued the first capsid-modified CRAd carrying HSV thymidine kinase (TK)-monomeric Red Flourescent Proten (mRFP) imaging reporter fusion, covalently linked to C-terminus ofpIX In addition, the new imaging "delta 24"-CRAd derivative carried a genetically-modified chimera E5/3 fiber for enhancement of'the CRAd infectivity for tumors Our results demonstrate that the triple-component fusion pIX.TK-mRFP is efficiently incorporated in the CRAd's caps ids, as revealed by Western blot and fluorescence microscopy (mRFP) imaging of the purified virions, and does not significantly compromise the CRAd replication and cytotoxicity in culture Our results, thus, validate in vitro functionality of the infectivity enhanced capsid-labeled CRAd with dual-imaging modality and justify its further testing in animal models of cancer Further translation of this strategy in the human clinical context could become of an immense field-wide significance 441 Properties of Human Dendritic Cells after High Titer Infection with Oncolytic Adenoviruses Stephan Schicrcr,' Ina Muller,' Andrea Hesse ,' Jan Dorrie; Niels Schaft , I Alexander Steinkasserer, I Gerold Schuler, I Eckhart Kampgen, I Dirk M Nenelbeck.'-' I Dept ofDermatology, UniversityHospital Erlangen, Erlangen, Germany; lHelmllOltz-University Group Oncolytic Adenoviruses German Cancer Research Center and Heidelberg University Hospital, Heidelberg, Germany In spite ofintensivc efforts to modify oncolytic adenovirus (ads) for improved specificity and efficacy ofcancer cell lysis, it is becoming increasingly clear that the killing of non-infected cancer cells in parallel to viral cell lysis will be crucial for oncolytic ads to be effective in the clinic An attractive scenario towards this goal is the induction of systemic anti-tumor immunity by adenoviral oncolysis In order to establish the basis for the development of adenoviral oncolytic vaccination we examined the effects of oncolytic ads on the biology ofhuman dendritic cells (DCs) DCs are the most potent antigen presenting cells , key regulators of immune induction and Molecular Therapy Volume15 Supplement I ~ br 2007 Copyright © T he American Soci ety o r G ene "1l1f:r:lpy have been intensively exploited for various vaccination protocols The prime objective ofthis study was to investigate how optimized, melanoma-targeted oncolytic ads affect the viability and maturation of human monocyte-derived DC and the potency of such DCs to activate T cells Both immature DCs (iDCs) and mature DCs (mDCs) were transducible with luciferase encoding ads at high titers Fiberchimeric ads with an ad serotype derived knob domain (5/3fiber) showed an improved transduction efficacy compared with an ad with serotype (5fiber) fiber After infection ofiDC and mDC with oncolytic ads (5 or 5/3 fiber) at high titers (5000vp/cell), no significant toxicity was observed Oncolytic ads showed no or strongly attenuated DNA replication in human DCs, thus confirming their replication specificity As determined by staining of cell surface maturation markers of DCs two to three days post infection with oncolytic ads (5 or 5/3 fiber), we observed, dependent on the donor, no or a partial DC maturation With respect to the maturability of DCs with cytokines and/or LPS, no negative influence of infection with oncolytic adenovirus was documented However, we did find an increase in the IL-I2 secretion ofLPS resp LPS/IFNg matured DCs after infection with oncolytic ads Finally DCs, infected with oncolytic ads for days, matured and then loaded with peptide were still able to prime and stimulate specifically CD8 'l-cells with similar or superior efficacy compared to uninfccted DCs, dependent on the donor In conclusion, oncolytic ads not harm viability and maturability of monocyte derived DCs, but also not induce complete maturation ofthese cells Therefore, oncolytic ads might represent a promising tool for oncolysis-induced anti-tumor immune activation, however, this strategy might require additional maturation signals for DCs, for example by expression of immunostimulatory genes inserted into the genome of oncolytic ads 442 Examination of the Therapeutic Potential of an Engineered VEGF-A Transcription Activator in a Spinal Cord Injury Model Yang Liu,' S Kaye Spratt,' Gary Lee,' Michael G Fchlings.P" 'Cell and Molecular, Toronto Western Research Institute, Toronto, ON, Canada; lDevelopment, Sangamo BioSciences, Richmond, CA; 'Krembil Neuroscience Centre, University Health Network, Toronto ON, Canada; 'Division ofNeurosurgery, University of Toronto, Toronto, ON, Canada Spinal cord injury (SCI) results in the initiation of multiple secondary injury cascades, one of which is the disruption of spinal cord blood flow and the onset of spinal cord ischemia Vascular endothelial growth factor (VEGF) is a major regulator ofendothelial cell proliferation, angiogenesis and vascular permeability, a potent neurotrophic factor and glial cell growth factor An engineered zinc finger protein transcription (ZFP-TF) factor was designed to activate expression of all the isoforms of the endogenous VEGF gene The aim of this study was to evaluate the effect ofa recombinant adenoviral vector containing an engineered ZFP-VEGF-A transcriptional activator (AdV-ZFP-VEGF) after SCI in adult rats The adenovirus vectors encoding either Os-Red fluorescent protein or ZFP- VEGFA were delivered locally immediately after a 35g clip compression injury In Ad'V-Ds-Red injected rats, adenovirus-infected cells were observed in gray and white matter, and the vectors transduced neurons, astrocytes and oligodendrocytcs Adv-ZFP-VEGF treatment led to increased levels of VEGF protein , as revealed by Western blot and RT-PCR analysis three days after SCI Expression of the ZFP-VEGF-A was also confirmed by Western blot In the injured spinal cord area ten days after SCI and treatment, using immunohistochemistry, a significant increase in vascularization (assessed by RECA-l immunostaining) and decreased levels of apoptosis (assessed by TUNEL) occurred in rats treated with AdV-ZFP·VEG F compared with controls Our finding suggest that this engineered ZFP-VEGF-activating transcription factor led to upregulation of SI71 YEGF protein, protection/repair of blood vessels and decreased apoptosis, These data suggest that VEGF gene therapy using a ZFP transcription factor plasmid holds promise as a therapy for SCI and other forms of neurotrauma 443 Systemic Dissemination of the Replication-Competent Oncolytic Adenovirus Telomelysin (OBP-301) Following Intratumoral Injection Yu-An Zhang, 1,2 Alex W Tong,I,2,3 Padmasini Kumar.' Michael Ncrnunaitis.v' Minai Barvc.' Neil Scnzcr ,3 ,~ Hitoshi Kawamura ~ John J, Nemunaitis,2,~ " , 'Cancer Research, Cancer Immunology Research Laboratory, Dallas, TX 2Cancer Research, Mal) ' Crowley Cancer Research Centers, Baylor Sammons Cancer Center; Dallas, TX; ' Cancer Research, Murex Pharmaceuticals, lnc., Dallas, TX 'Cancer Research, Texas Oncology, P.A" Dallas, TX ~Cancer Research, Oncolys Biol'harma Inc" Tokyo, Japan A Phase I, dose escalation study was recently initiated to examine the safety of intratumoral injection with the novel conditional replicative adenovirus Telomelysin (0131'-301), Telomelysin is a modified , human adenovirus type that incorporates the human telomcrase reverse transcriptase gene (hTERT) promoter sequence in its EIA region, hence restricting replication to cancers cells that highly express hTERT Differential viral oncolytic activity is anticipated, since normal cells commonly lack hTERT expression To identify the systemic dissemination pattern of 0131'-301 alter intratumoral injection, patient plasma, urine , sputum and saliva was collected at scheduled time points before and after injection, A biopsy of the injected tumor was also obtained at day 28 post-treatment To date, one patient with squamous cell carcinoma of the head and neck and one patient with melanoma have completed qPCR analysis of 0131'-30 J Following a single intratumoral injection of Ix 1010 viral particles (vp), 0131'-301 was not detected in all fluid samples (plasma, saliva, urine and sputum) at all scheduled time points (pretreatment, I hr, hrs, day I, day and day 14 post-treatment), using a 0131'-301 specific qPCR assay with detection sensitivity as low as 5-10 vp per reaction Plaque forming assays arc currently underway to quantify viral titer and the presence ofanti-adenovirus neutralizing antibody titer at these post-treatment time points Together with the determination of hexon/El A expression at day 28 post-treatment tumor biopsies, these findings will allow us to define systemic viral phannacokinetic and evidence of 0131'-301 replication following intratumoral treatment Additional patients arc recently entered into trial Updated results will be presented 444 YB-1 as a Target for the Development of Oncolytic Adenoviruses Klaus Mantwill,' Regina Holzmueller,' Moritz Widmaier, I Emanuel Rognoni ,' Per S Holm ':' 'Institute ofExperimental Oncology, Klinikum Reclus derIsar; Technical University ofMunich, Munich Germany; 2X ViI; Therapeutics GmbH, Munich, Germany into the fiber knob (Ad-Del03 RGD) Finally we tested an YB-I triggered self promoting system by incorporating the YB-I-gene into the E3 region driven by the major late promoter (Ad-DeloYB-I RGD) All viruses tested showed strong antitumor activity in vitro and in vivo especially in combination with radiation- and chemotherapy Since the oncolytic activity of Ad dl520 or vectors of similar design is not limited to tumors of a particular tissue type, we expect a broad range of application 445 Effects of Exogenous p53 on Potency and Safety of Oncolytic Adenovirus Victor W van Beusechem,' Petra 13 van den Doel,' Juliette C 13I0kker,1 Winald R Gerritsen ' 'Dept ofMedical Oncology VU University Medical Center; Amsterdam, Netherlands, Virotherapy with oncolytic adenoviruses that selectively destroy cancer cells by lytic replication is a promising new way to treat cancer, Previously, we constructed an oncolytic adenovirus derived from AdLU4 (with a deletion in the pRb-binding E IA-CR2 domain) expressing functional human p53, The new virus Ad~4 -p53 was more effective than its parent AdL124 in killing most human cancer cell lines and primal)' cancer cells tested in vitro and xenograft tumors in vivo Here , we investigate if functional adenovirus E11355K, which binds p53, is required for p53-mediated efficacy enhancement To this end, we introduced in the genome ofAdL124 and Ad~4-p53 a mutation encoding a single amino acid substitution (R240A) in E1B55K that abrogates its capacity to bind p53, Relative cytotoxicity ofthc viruses was compared in vitro on p53 wild type U20S cancer cells, p53 mutant PC-3 cancer cells and non-malignant human endothelial (I-IMVEC-L) cells and fibroblasts Cells were infected with a 2-fold virus dilution titration (normalized by infectious units on E l-complementing 911 cells) and days later cell viability was measured by WST-I conversion, Two to four independent experiments in triplicate werc done on each cell type MOl 50 values were calculated using GraphPad Prism software, The two virus backbones were found similarly active against each cell type, suggesting that the E IB55K(R240A) mutation did not attenuate the virus As expected and confirming earlier observations, p53 expresagainst U20S and sion augmented oncolytic potency of Ad~4 PC-3 cells (in this experimental setting approximately and fold, was not more respectively) In contrast, Ad~4/55K(R240A)-p53 potent in killing cancer cells than its parent Ad~4/55K(R240A), suggesting that oncolysis enhancement was abolished by the R240A mutation No clear differences between any of the viruses were observed on HMYEC-L cells Surprisingly, AdL124-p53 was 12-fold less toxic than AdL124 to normal fibroblasts This elTect was lost if EII355K was mutated Adenovirus-encoded p53 thus seems not only to improve oncolytic adenovirus potency, but also safety, at least on fibroblasts Both effects require EIB55K capable of binding p53, suggesting that temporal regulation ofp53 activity during oncolytic adenovirus replication by E IB55K is important This makes oncolytic adcnoviruses with intact E IB55K gene and expressing p53 promising agents for cancer treatment and warrants their further evaluation The transcription factor YB-I is ovcrexpressed in many solid tumors and one of'the highest overexpressed genes in drug-resistant tumors We discovered that nuclear localization ofYI3-1 facilitates E l-independent replication of serotype adenoviruses (Cancer Research 64, 322-328, January I, 2004) and studied the effect of the otherwise non replicative Ad d1520, which carries a deletion in E 1A and does not express the large 289 amino acid long E 1A protein on a broad spectrum of tumor cells in vitro and in vivo To improve the cell killing capacity ofAd d1520, we introduced a delet ion in the antiapoptotic E I13 19K gene and expanded the tropism ofAd-Oel03 toward nv integrins by insertion of an Arg-Gly-Asp (RGD) motif SIn Molecular Therapy Volume 15.Supplcrnenr I ,\by 2007 Copyright © ' 111C American Society of Gene Tllt'r.lP)' ... injected tumor was also obtained at day 28 post-treatment To date, one patient with squamous cell carcinoma of the head and neck and one patient with melanoma have completed qPCR analysis of 0131''-30... ~Cancer Research, Oncolys Biol''harma Inc" Tokyo, Japan A Phase I, dose escalation study was recently initiated to examine the safety of intratumoral injection with the novel conditional replicative... dissemination pattern of 0131''-301 alter intratumoral injection, patient plasma, urine , sputum and saliva was collected at scheduled time points before and after injection, A biopsy of the injected