868 targeting of CRAd agent to human squamous cell carcinomas of the head and neck (SCCHN)

868 Targeting of CRAd Agent to Human Squamous Cell Carcinomas of the Head and Neck (SCCHN) 866 Development of Peptide Targeted Adenovirus with the pFex System Ping Wu, TaranaA Kudrolli, WasimH Chowdhu[.]

866 Development of Peptide Targeted Adenovirus with the pFex System Ping Wu, TaranaA Kudrolli, Wasim H Chowdhury, Shawn E LupoId, Ronald Rodriguez I Department ofUrology Johns Hopkins University School of Medicine , Baltimore, MD Introduction: Adenoviral gene therapy vectors offer a safe, alternative platform for developing anti-cancer therapeutics The most recent "next generation" vectors have improved transduction and bio-distribution by mutating receptor binding domains (de-targeting) and by inserting targeting peptides (re-targeting) into viral coat proteins, such as fiber Methods: Weapply a novel adenoviral vector system, pFex, to generate virus with altered tropism through Fiber-displayed peptides These virus were generated with an HI Loop altered Fiber-gene recombined into replicating adenovirus through uni-directional Cre recombinase-mediated cassette exchange The adenoviral vector was mutated by deleting fiberamino acids T489AYT492 for ablation of Fiber-Coxsackie-Adenovirus Receptor (CAR) mediated infection, and/or addition offiber amino acid Y477A mutation for ablation of Fiber-blood-factor-binding (de-targeting) Then the de-targeted viruses were retargeted by displaying an integrin targeting motif (RGD) or Prostate Specific Membrane Antigen (PSMA) targeting peptides in the Fiber 1-11loop These viruses have been evaluated for re-targeting ability by infecting various cell lines using different concentration (MOl = I to 10) The viruses then were removed and cells were continually cultured for 48 hours The number of infected cells was quantified by fluorescent microscopyand flowcytomctry,Results: Our results have shown that de-targeted viruses were unable to infect cells, and the peptide re-targeted viruseswere capableofre-directing infection through non-CAR mediated pathways These data demonstrated that cell binding and internalization are mediated by re-targeted peptides Conclusions: This novel pFex vector system is highly efficientand capable of generatingtestable Fiber-modified adenovirus Fiber-CARde-targetedand specificpeptide re-targeted viruses are capable of infecting cells Ablation of Fiber-CAR binding, the natural attachment of virus with cells, can be rescued by using re-targeting peptides This vector system can be used to generate novel prostate-specific adenovirus as targeted gene therapy vector for treatment of prostate cancer that increase the expression of RGD-specific integrins on DC will therefore enhance the response to RGD-AdV-PSCA-based vaccines All-trans retinoicacid (ATRA), a biologicallyactive retinoid that influences the maturation and function of DC was therefore evaluated (day 9) for its capacity to modulate integrinexpressionon mouse and human DC at high or low concentration, in presence of GM- GM-CSF and IL-4 or GM-CSF alone DC generated with the combination ofGM-CSF and IL4, in absence of ATRA, expressed 0: integrins on only 37.4% of the resulting DC The addition of I~w dose ATRA (10.12 M) at the initiation of the BM-DC culture with GM-CSF increased the population of a, integrin expressing DC to 68% Similarly,addition of a higher concentration of ATRA (10-8 M) at later stages of the culture with GM-CDF and IL-4 (day 5) resulted in 62.7% of DC expressing O:v integrin The need for higher concentrations of ATRA in this setting may be due to an IL-4-induced decrease in the expression of rctinoic acid receptors (RAR) as previously reported Similar studies were carried out with human monocyte-derived DC (CDI Ic+/CD86+) generated in presence of GM-CSF and IL-4 for seven days 56% of control DC generatedwithGM-CSFand IL-4expressed a,.~~ AdditionofATRA (10-8 M) on day five increased the population of av~~-expressing DC to 70% Similarly, only 15.4% of control DC expressed O:v~, whereas I% of DC generated in the presence of ATRAexpressed O:"~3' ATRAresulted in a three-fold increase in DC expressing both the O:v~~ and O:V~ integrin receptors on their surface Expression of HLA-DR was stable or slightly increased when DC were exposed to ATRA We propose that exposure of DC to ATRA either in vitro or in vivo, will significantly enhance integrin expression and the efficiency of RGD-modified adenoviral cancer vaccines These hypotheses will be investigated using transgene-specific assays in vitro and in transgene-specific tumormodelsusingRGD-AdV-PSCA in vivo Supported by RL Kirschstein NRSA (AH), UCLA SPORE in Prostate Cancel; NIHINCI (MDR) and UCLA Human Gene Medicine Gram (SKB) 868 Targeting of CRAd Agent to Human Squamous Cell Carcinomas of the Head and Neck (SCCHN) Zeng B Zhu,' Michael Mathis,' Sharmila K Makhija,' Baogen Lu,' Minghui Wang,' Gene P Siegal,' David T Curiel.' Airi Harui, Michael D Roth, Saroj K Basak 'Pathology; University ofAlabama at Birmingham, Birmingham, AL; lLSU Health Scince Center; LSUHSC, Shreveport; 'Division ofGynecology Oncology, University ofAlabama at Birmingham, Birmingham; 'Dept ofPathology, Cell Biology, and Surgery and Gene Therapy Center; University ofAlabama at Birmingham, Birmingham Dendritic cells (DC) along with adenoviral (AdV) vectors are used for stimulating transgene-specific vaccine responses However, DC lack high affinity CAR receptors but express low affinity integrinreceptors (av~, and a,.~~) that interactionwithArg-Gly-Asp (RGD) sequences on AdV that are primarily responsible for AdV transduction Wchave reportedthat AdV-spccific intcgrin receptors arc upregulated on DC when cultured in the presence ofGM-CSF and IL-4 The resulting DC can be subdivided into two groups, one expressing high levels of integrin receptors (DChi) and the second expressing low levels of receptors (DClo) The DChi subpopulation is preferentiallytransduced byAdV, is primarily responsible for the activation of antigen specificT cells and is susceptible to modified RGD-AdV vectors that express RGD sequences on their capsid Using an RGD-AdV-PSCA vector that we constructed to express Prostate Stem CellAntigen (PSCA) as a vaccine for prostate cancer patients, we demonstrated that RGD-AdV-PSCA preferentially transduces the DChi population in vitro Wehypothesizethat factors Humansquamouscellcarcinomasofthe headand neck(SCCHN) are resistant to standard treatments, thereby requiring new therapeutic strategies Conditionally replicating adenoviruses (CRAds) represent a promising new modality for the treatment of solid neoplastic diseases, including SCCHN Specifically, following cellular infection, the virus replicates selectively in the infected tumor cells and kills the cells by cytolysis This is followed by the progeny virions infecting a new population of surrounding target cells, replicatinggain and eradicating the infectedtumor cells while leaving normal cells unaffected However, to date there have been two limitations to successful clinical application of these CRAd agents; i.e poor infectivity and poor tumor specificity Here we compared tumor specific promoters (the Cox-2, MK, VEGF, SLPI, TERSTS, CXCR4 and Survivin promoters) and two adenovirus capsid modifications(RGD and F513) in four different human head and neck cell lines (FaDu, SCC6, SCC22A and SCC27 cell lines) and in primary cells from three patients.The data showed the CRAd-CXCR4.F5/3 improved both the viral infectivity and tumor specificity as evaluated by viral binding efficacy, viral replication 867 All-Trans Retinoic Acid (ATRA) Enhances Adenovirus Specific Integrin Receptor Expression on Dendritic Cells I Deapartment ofMedicine, University ofCalifornia, Los Angeles, Los Angeles, CA Molecular Therapy Volume15 Supplement I ~ br 2007 Copyright © T he American Soci ety o r G ene Therapy S331 rate and oncolysis in both human tumor cell linesand primary cells from patients In this adenoviralvector,the CXCR4promoterdriving EI is responsible for the increased tumor specificity while the F5/3capsidmodification yieldsenhancementof viral infectivity As an added benefit, the activity of the CXCR4 promoter was low in human liver as compared to the three other promoters From these data, the CRAd-CXCR4.F513 appearsto be a promisingnovelagent for human SCCI-IN targeting with low host toxicity 869 Adenovirus Vectors Targeted to HER3/4 Receptors for Breast Cancer Gene Therapy Sheena 1-1 Macl.eod,' Mabrouk M ElgadiY Gianluca Bossi.>' Kate C Agopsowicz,' Frank L Graham," Mary M 1·litt.' 'Oncology; University ofAlberta Edmonton AB, Canada; 2Biology, McMaster University, Hamilton, ON, Canada; "lmmunology/ Virology/Respiratory Medicine, Boehringer lngelheim (Canada) Ltd., Burlington, ON Canada; "Molecular Oncogenesis Laboratory Regina Elena Cancer Institute , Rome, Italy; 'Pathology & Molecular Medicine, McMaster University, Hamilton, ON, Canada The primary receptor for most adenovirus serotypes is the coxsackie-adenovirus receptor(CAR) CAR is expressedon many cell types, but is underexpressed in many breast cancer cells, resulting in lowerlevelsof infectionoftumour cells by wild type adenovirus To construct an improvedadenoviral vector for breast cancer gene therapy we have genetically modified the virus to target the cell surface receptors HER3 and HER4, which arc overexpressed in a number of established breast cancer cell lines as well as in patient tissue samples The vector was modified in the HI loop of the fibre knob by insertingsequencescorrespondingto the EGF-likebinding domain of heregulin, the ligand for HER3 and HER4 This virus also encodes a luciferase reporter gene and can be compared to a virus with wild type capsid that contains the same reporter gene Total levels of HER3, HER4 and CAR in a panel of breast cancer cell lines were assessed by western blotting, and cell surface levels were measured by flow cytometry of breast cancer cell lines tested expressed high levels ofHER3 on the cell surface while of had low levels of CAR In general, western blotting results were in good agreement with flow cytometry results Using a luciferase reportergene assay,the levelofinfectivity of the modified viruscan be comparedto a viruswith wildtype capsidina panelof breastcancer cell lines.As expected, infectivity of the modified virus in breast cancer cclilines expressinghigh levelsofHER3/4 was increasedin comparisonto the wild type virus.The levelsof reporterexpression with the modified virus were similar to those with the wild type virus in cells expressing low levels ofHER3/4 , consistent with the predictionthat the insertioninto the HI loop did not affect the CAR bindingsiteofthe virus.Preliminary competition assayswithsoluble adenovirus fibre knob and heregulin suggest the virus interaction with the CAR and HER3 receptors is specific Specificity of this modified virus is being confirmed by two different approaches: I) testing infectivity following specific knockdown of HER3 and/or CAR receptorsin breast cancercell lines usingRNAinterference2) testing infectivity following transfection of the hamster CHO cell lines with ErbB3 and/or CAR expression plasmids These studies will provide a strong platform for the development of adenovirus vectors that no longer recognize CAR and instead bind to HER3/4 receptors on breast cancer cells 870 Effect of Coagulation Factor X on the Infectivity of Adenoviruses Pseudotyped with Fibers from Subgroup D Alan L Parker,I John H Mcvey,' Simon N Waddington,'Suzanne M K Buckley,' Jessica H Doctor,' Oscar Lopez-Franco,' Menzo J Havenga,~ Stuart A Nicklin,I Andrew H Baker.' I BHF Glasgow Cardiovascular Research Centre, University of Glasgow Glasgow United Kingdom; 2Haemostasis and Thrombosis, Imperial College London, London, United Kingdom; JDepartment ofHaematology; Royal Free and University College Medical School London, United Kingdom; 'Department ofVlrol0ID', Crucell Ltd Leiden, Netherlands Recentevidencesupportsa rolefor vitamin Kvdepcndent coagulationzymogcnsinadenovirusserotype5 (Ad5, subgroup C) infection of'hepatocytes' Bindingofcoagulation factors suchas FactorX (FX) to theAd capsid"bridges" the virusto cellular heparinsulphateproteoglycans(HSPGs),providinga CAR-independent routeof cellular uptake.Forefficientretargetingof Ad5-basedgene deliveryvectors to alternate sites (c.g disseminatedcancersor alternate organs)it is likelythat modulation of this pathwaywill be requiredsince binding of coagulationfactorsto thc virusappearshighlyefficient'.A simple method to alter susceptibility to coagulation factor binding may be to use fibers derived from alternate serotypes and pseudotyped onto the Ad5 capsid Here,we assessed the effectofvirus:zymogen interaction on cellulartransductionusinga paneloffiber(f)-pseudotyped virusesderivedfromsubgroup D(fl7, f24, 00, 03, f45, f47) Many of the receptorsfor adenoviruses from this subgroup remain to be isolated and those that have been characterised often bind at relatively low affinity Each pseudotyped virus bound dircetly to FX in a Ca2+-dependent manner as determined by surface plasmon resonance(SPR).This resulted in enhanced cell surface binding of virus, evaluated by quantitative PCR analysis of surface binding (p