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-tar-geting) and by insertingtargeting 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

ex-change.The adenoviral vectorwas 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

-11-loop 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-targetedviruses were unable to infect cells,and

the peptidere-targeted viruseswerecapableofre-directing infection

through non-CAR mediated pathways These data demonstrated

that cell binding and internalization are mediated by re-targeted

peptides Conclusions: This novelpFex vector system is highly

efficientand capableof generatingtestable Fiber-modified

adenovi-rus Fiber-CARde-targetedand specificpeptide re-targetedviruses

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

867 All-Trans Retinoic Acid (ATRA) Enhances

Adenovirus Specific Integrin Receptor Expression

on Dendritic Cells

Airi Harui, Michael D Roth, Saroj K Basak

I Deapartment ofMedicine, University ofCalifornia, Los Angeles,

Los Ang eles, CA

Dendriticcells (DC) along with adenoviral (AdV) vectors are

used for stimulating transgene-specific vaccine responses

How-ever, DC lack high affinity CAR receptors but express low affinity

integrinreceptors(av~, anda,.~~) that interactionwithArg-Gly-Asp

(RGD) sequences on AdV that are primarily responsible for AdV

transduction Wchave reportedthat AdV-spccific intcgrinreceptors

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 primarilyresponsible 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 prostatecancer

patients, we demonstrated that RGD-AdV-PSCA preferentially

transducesthe DChipopulationin vitro Wehypothesizethat factors

Molecular Therapy Volume15 SupplementI ~ br 2007

C opyright © T he Ameri can So ci ety o r G ene Therapy

that increase the expression of RGD-specific integrins on DC will therefore enhance the response to RGD-AdV-PSCA-based vac-cines.All-transretinoicacid (ATRA),a biologicallyactive retinoid that influences the maturation and function of DC was therefore evaluated(day9) for its capacity to modulateintegrinexpressionon 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(10.1 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 (10-8M) 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-4expresseda,.~~. AdditionofATRA (10-8M) on day five increased the population ofav~~-expressing

DC to 70% Similarly,only 15.4% of control DC expressedO:v~, whereas 2 I% of DC generated in the presence of ATRAexpressed

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-modifiedadenoviral cancer vaccines These

hypotheses will be investigated using transgene-specific assays in

vitroand in transgene-specific tumormodelsusingRGD-AdV-PSCA

in vivo Supported by RL Kirschstein NRSA (AH), UCLA SPORE

in Prostate Cancel; NIH INCI (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,'Michael1 Mathis,'Sharmila K Makhija,' Baogen Lu,' Minghui Wang,' Gene P Siegal,' DavidT Curiel.'

'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.

Humansquamouscellcarcinomasofthe headand neck(SCCHN) are resistant to standard treatments, thereby requiring new thera-peutic 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 eradicatingthe 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 7 tumor specific promoters (the Cox-2, MK, VEGF, SLPI, TERSTS, CXCR4 and Survivin promoters) and two adeno-virus capsid modifications(RGD andF513)in four different human head and neck cell lines (FaDu,SCC6, SCC22A and SCC27 cell lines) and in primarycells 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

S331

rate and oncolysis in both human tumorcell linesand primarycells

from patients In this adenoviralvector,the

CXCR4promoterdriv-ing EI is responsible for the increasedtumor 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 targetingwith low host toxicity

869 Adenovirus Vectors Targeted to HER3/4

Receptors for Breast Cancer Gene Therapy

Sheena1-1.Macl.eod,' MabroukM ElgadiY GianlucaBossi.>'

Kate C Agopsowicz,'Frank L.Graham,"Mary M.1·litt.'

'Oncology; University ofAlberta Edmonton AB, Canada;

2Biol-ogy, McMaster University, Hamilton, ON, Canada; "lmmunology/

Ltd., Burlington, ON Canada; "Molecular Oncogenesis

& Molecular Medicine, McMaster University, Hamilton, ON,

Canada.

The primary receptor for most adenovirus serotypes is the

cox-sackie-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 capsidthat 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 7 of 9 breast cancer cell lines

tested expressed high levels ofHER3 on the cell surface while 6 of

9 had low levels of CAR In general, western blotting results were

in good agreement with flow cytometry results Using a luciferase

reportergene assay,the levelofinfectivityof the modified viruscan

be comparedto a viruswithwildtype capsidina panelof

breastcan-cer cell lines.As expected,infectivity of the modified virus inbreast

cancercclilines 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 loopdid 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 confirmedby two different approaches:I)

testing infectivity following specific knockdown of HER3 and/or

CAR receptorsin breastcancercell linesusingRNAinterference2)

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

S332

870 Effect of Coagulation Factor X on the Infectivity of Adenoviruses Pseudotyped with Fibers from Subgroup D

AlanL.Parker,IJohn H Mcvey,' Simon N

Waddington,'Su-zanne M K Buckley,'Jessica H Doctor,' Oscar Lopez-Franco,'

MenzoJ Havenga,~ StuartA Nicklin,IAndrew H Baker.'

Glasgow Glasgow United Kingdom; 2Haemostasis and

JDepartment ofHaematology; Royal Free and University College

Recentevidencesupportsa roleforvitaminKvdepcndent coagula-tionzymogcnsinadenovirusserotype5 (Ad5,subgroupC) infection of'hepatocytes'.Bindingofcoagulation factors suchas FactorX (FX)

to theAd capsid"bridges" the virusto cellularheparinsulphatepro-teoglycans(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 assessedthe effectofvirus:zymogen interaction on cellulartransductionusinga paneloffiber(f)-pseudo-typedvirusesderivedfromsubgroupD(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+-dependentmanner 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<O.OI).lnfection of HepG2 cells was promoted by FX (p<0.05),

as was transduction of CHO-KI cells (p<0.05), whilst infection

of CHO-pgsA745 heparan sulphate proteoglycan deficient cells was blocked in the presence of FX (p<0.05) We have previously described a method of depleting functional Vitamin K-dependent coagulation factorsfromthe circulation of normalmice' Micewere injected,subcutaneously, with I mg/kg warfarinsuspendedin pea-nut oil three and one days before intravenousadenovirus injection Using 1'47 pseudotyped Ad5, bioluminescence imaging of hepatic transduction 48 hours post virus infusion in warfarinised mice in-dicatcd significantly Icss hepatic transduction (p<O.OI) compared

to non warfarinisedmice.Takentogcthcrthis suggests a broad role lor FX in dictating adenovirus infectivity in vitro andin vivo for adenovirusespseudotyped with fibers from subgroup D 'Parker et

al, Blood pp.2554-2561 (2006)

871 Characterization of Coagulation Factor IX Interactions with Human Adenovirus Fiber Knob Domains Using Tandem Mass Spectrometry and Surface Plasmon Resonance

Oleksandr Kalyuzhniy,'Catalin Doneanu,' Martin Sadilek,' Jan Seebacher,' Dmitry M Shayakhrnetov,'

"Medictne, University of Washington Seattle, WA; 2Medicinal

Over the last few years it has been demonstratedthat blood co-agulation factors playa significant role in targetingAd vectors to hepaticcells in vivo One ofthe possible mechanismsforAd infec-tion ofliver cells reliesondirect bindingof the fiberknobdomainto bloodfactors,whenvirus particlesare applied intravenously Inthis study we quantitatively characterized interactions between human

Molecular Therapy Volume15 Supplement I, \by 2007

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