Inter-patient variation in efficacy of five oncolytic adenovirus candidates for ovarian cancer...

THE JOURNAL OF GENE MEDICINE R E S E A R C H A R T I C L EJ Gene Med 2004; 6: 1333–1342.Published online 18 October 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.635

Inter-patient variation in efficacy of five oncolyticadenovirus candidates for ovarian cancer therapy

John T. Lam,1 AnnaKanerva,4,5,6 Gerd J.Bauerschmitz,1,7 KoichiTakayama,1,8Kaori Suzuki,1

Masato Yamamoto,1

Snehal M. Bhoola,2 Bin Liu,1

Minghui Wang,1 Mack N.Barnes,2 Ronald D. Alvarez,2

Gene P. Siegal,3 David T.Curiel,1 Akseli Hemminki4,6*1Division of Human Gene Therapy,Departments of Medicine, Pathologyand Surgery, and the Gene TherapyCenter2Department of Obstetrics andGynecology3Departments of Pathology, CellBiology, and Surgery, University ofAlabama at Birmingham,Birmingham, Alabama 35294, USA4Cancer Gene Therapy Group,Rational Drug Design, BiomedicumHelsinki, P.O. Box 63, 00014University of Helsinki, Finland5Department of Obstetrics andGynecology6Department of Oncology, HelsinkiUniversity Central Hospital, 00290Helsinki, Finland7Duesseldorf University MedicalCenter, Department of Obstetrics andGynecology, 40225 Duesseldorf,Germany8Research Institute for Diseases of theChest, Kyushu University, 812-8582Japan

*Correspondence to: AkseliHemminki, Cancer Gene TherapyGroup, Rational Drug DesignProgram, Department of Oncology,Biomedicum Helsinki, P.O. Box 63(Haartmaninkatu 8), 00014University of Helsinki, Finland.E-mail: [email protected]

Received: 13 February 2004Revised: 28 April 2004Accepted: 4 May 2004

Abstract

Background Gene therapy offers a new strategy for cancer treatment.Adenoviruses represent the most widely used gene therapy vector and featurean excellent safety record. Conditionally replicative adenoviruses (CRAds)effect solid tumor penetration and tumor selective oncolysis and consequentlyoffer potential efficacy for metastatic disease treatment. We evaluated fiveCRAds as candidate clinical agents for ovarian cancer therapy: RGDCRADcox-2R, Ad5VEGFE1, Ad5/3VEGFE1, Ad5-�24RGD, and Ad5/3-�24.

Methods DNA replication by these five CRAds, wild-type adenovirus, andan E1-deleted control was measured in purified primary ovarian cancer cellspheroids by quantitative PCR. CRAd-mediated oncolysis was quantified inovarian cancer cell monolayers and three-dimensional spheroids by cellularviability assays. The therapeutic efficacy of each CRAd was tested byintraperitoneal administration in mice with peritoneally disseminated humanovarian cancer.

Results An increase in viral DNA was noted in primary tumor cell spheroidsfor all replicative viruses tested. Variation was noted in viral DNA replicationbetween patient samples. All five CRAds induced remarkable oncolysis. Theyalso prolonged survival in vivo compared with the wild-type control group.

Conclusions All five CRAds tested showed robust DNA replication,oncolysis, and in vivo therapeutic efficacy. Each virus has potential for clinicaltesting, and such further testing will ultimately determine its safety andrelative usefulness. Variation of CRAd DNA replication between differentpatient samples suggests that target tissue features, such as surface receptorsand endogenous transcription factors, may affect CRAd infectivity andreplicativity. Evaluation of such factors may become important to optimizecancer therapy for individual patients. Copyright 2004 John Wiley & Sons,Ltd.

Keywords ovarian cancer; gene therapy; adenovirus; oncolytic virus;replication-competent viruses; translational research

Introduction

Advanced stage cancers still incur unacceptable high mortality rates.Gene therapy provides a novel approach for treating these difficultcases. In clinical gene therapy trials, adenoviruses (Ads) represent themost widely used vector. Prior to 2003, more than 600 clinical trials

Copyright 2004 John Wiley & Sons, Ltd.

1334 J. T. Lam et al.

featuring Ad vectors were approved [1,2]. Over 700cancer patients have been treated with Ads withouttherapy-related mortality, and nearly 300 of these havereceived replicative Ads [3]. Ads also remain the onlygene therapy vectors to show evidence of efficacy [4–6].

Despite this optimism, limited gene transfer stillrestrains clinical progress for cancer gene therapy [7–9].To increase transduction, non-replicative viruses requiremultiple administrations and large doses, which maylead to toxicity and immunogenicity. Replicative agentshave demonstrated augmented gene expression based ontheir capability to multiply their own genome [10,11].Moreover, replication-competent viruses can penetratesolid tumors [4,12]. The viral replication cycle itselfcauses oncolysis with subsequent virion release andinfection of neighboring cells [13].

To mitigate viral replication and injury in non-tumortissues, strategies to control Ad replication have beendeveloped. One method involves control of the replicationregulating E1A gene with tumor-specific promoters. Sinceintracellular transcription factors activate promoters,selecting promoters active only in target tissues to driveE1A gene expression can restrict viral replication to tumortissues. Cyclooxygenase-2 (cox-2) is highly expressedin a number of epithelial tumors including ovariancancer [14,15]. Accordingly, one conditionally replicativeadenovirus (CRAd) RGDCRADcox-2R incorporates thecox-2 promoter to drive E1A gene expression [16].Preclinical studies employing RGDCRADcox-2R haveshown robust replication and oncolysis in ovarian cancer[17]. Furthermore, because systemic Ad administrationcauses hepatic toxicity, the low liver activity of thecox-2 promoter holds important clinical implications[18]. The novel CRAds Ad5VEGFE1 and Ad5/3VEGFE1exploit the need of cancers for neovascularization byincorporating the vascular endothelial growth factor(VEGF) promoter [19]. Many malignancies includingovarian cancer increase VEGF expression to stimulateangiogenesis for growth and metastasis [20].

Another means to achieve tumor-specific viral replica-tion involves deletions in the viral genome that requirecompensation by cellular factors found only in target cells.The CRAds Ad5-�24RGD and Ad5/3-�24 each incorpo-rate deletions in their E1A genes [21–24]. Since this geneproduct binds cellular retinoblastoma (Rb) protein toinduce S-phase entry, these CRAds have limited ability toovercome the G1-S checkpoint in normal cells. In contrast,these viruses replicate efficiently in cells with mutant Rb,such as tumor cells, where this interaction is unnecessary.Since the Rb-p16 pathway has been reported defective inmost human tumors [25–27], these CRAds replicate withhigh specificity in malignant cells. Both Ad5-�24RGDand Ad5/3-�24 have undergone preclinical evaluationfor ovarian cancer [23,24], and the former is entering amulti-center phase I clinical trial for treatment of gliomaand ovarian cancer.

Complementing viral replication control strategiesare approaches to modify viral tropism that canlimit non-tumor tissue infection while augmenting

tumor transduction. RGDCRADcox-2R and Ad5-�24RGDincorporate the arginine-glycine-aspartic acid (RGD-4C)integrin binding peptide [16,22], a modification thatenhances infectivity of these CRAds in ovarian cancercells in vitro and in vivo [28]. Retargeting Ads can beachieved by viral knob modification. The adenovirusserotype 3 (Ad3) binds to a yet unidentified receptor[29] distinct from coxsackie-adenovirus receptor (CAR),the native receptor for Ad5. A chimeric vector geneticallymodified by replacing the Ad5 fiber knob domain with theAd3 knob showed increased gene transfer to ovariancancer cells in comparison with a conventional Ad5vector [30,31]. Ad5/3VEGFE1 and Ad5/3-�24 employthis fiber chimerism for retargeting [19,24]. Importantly,the murine biodistribution and toxicity of these chimericand RGD-4C modified viruses resemble those of tropismunmodified Ad5-based vectors [31], which have provensafety in clinical trials.

Despite the potential of replicative agents such asCRAds for effective gene transfer and tumor penetration,their transition to clinical application is limited by assaysubstrates. Evaluation of candidate agents could beconfounded by differences between primary tumor cellsand tumor cell lines, as discordance in their levels ofsurface receptors relevant for viral entry has been reported[32–35]. Moreover, the repeated passaging of cell linesselects only certain clones from the polyclonal in vivotumor. Assays for CRAds should employ substrates withAd infectivity profiles and other phenotypical attributesas similar as possible to those of primary tumor tissue.The importance of preserving Ad infectivity parametersis emphasized by the direct correlation of Ad infectivityto Ad-mediated oncolysis [36,37]. Use of primary tumorcells in CRAd assays, however, is limited by their frequentcontamination with irrelevant non-tumor cells. To addressthis problem, we developed an immunomagnetic tumorcell purifying system to achieve up to 96% tumorpurity from malignant ascites samples, a doubling oftumor cell concentration from malignant ascites samples[38].

Another problem with primary tumor cells is their lim-ited in vitro viability. In order to sustain primary tumortissue ex vivo for periods sufficient to observe viral repli-cation, we developed three-dimensional aggregates orspheroids of purified and unpassaged ovarian cancercells [39]. Spheroids also provide a three-dimensionalmodel that allows more accurate analysis of CRAddispersion and penetration. To apply the most strin-gent preclinical assays, we employed primary tumorspheroids in our analysis of CRAd replication and oncol-ysis. We measured the DNA replication and oncolyticpotency of five CRAds with high potential for treat-ment of ovarian cancer: Ad5VEGFE1, Ad5/3VEGFE1,RGDCRADcox-2R, Ad5-�24RGD, and Ad5/3-�24. Wealso tested these agents in survival studies utilizing anorthotopic model of peritoneally disseminated ovariancancer.

Copyright 2004 John Wiley & Sons, Ltd. J Gene Med 2004; 6: 1333–1342.

Replicating Adenoviruses for Ovarian Cancer 1335

Materials and methods

Cells and tissues

The human ovarian adenocarcinoma cell line SKOV3.ip1was obtained from Dr. Janet Price (University of Texas,M.D. Anderson Cancer Center). The 293 and A549cell lines were purchased from Microbix (Toronto,Canada) and the American Type Culture Collection(Manassas, VA, USA), respectively. The transformedhuman embryonic retinoblasts 911 were kindly providedby Dr. Alex van der Eb (Leiden University MedicalCenter, The Netherlands). RPMI-1640 growth medium(Mediatech, Herndon, VA, USA) was used for cultureof primary ovarian adenocarcinoma cells. All other cellswere cultured in the growth medium recommended bytheir manufacturers. All cells were cultured at 37 ◦C in5% CO2 atmosphere. Primary ovarian adenocarcinomacells (pathologically confirmed) were purified frommalignant ascites samples obtained with informed consentfrom patients at the University of Alabama Hospital.Malignant cells were purified with a previously describedimmunomagnetic-based method [38]. To create three-dimensional spheroids, cells were suspended in growthmedium in Costar ultra-low attachment 96- or 24-wellplates (Corning Inc., Corning, NY, USA) and incubated ona rocking platform.

Viruses

Since E1-deleted Ads generally have mitigated replicationcompetency, we employed RGDTKSSTR construct as acontrol virus (Table 1). RGDTKSSTR [40], RGDCRADcox-2R [16], Ad5luc1 [41], AdVEGFLuc [19], Ad5VEGFE1[19], Ad5/3VEGFE1 [19], Ad5-�24RGD [22], Ad5/3-�24 [24], and Ad5-�24RGDGFP [42] have beenpreviously described. Ad300wt, a wild-type humanAd5, was obtained from the American Type CultureCollection. AdVEGFLuc, Ad5VEGFE1 and Ad5/3VEGFE1were propagated on 911 cells. Ad5-�24RGD andAd5/3-�24 were propagated on A549 cells. The otherviruses were propagated on 293 cells, and all werepurified on cesium chloride gradients. The viral particle

(vp) concentration was determined at 260 nm andstandard plaque assay on 293 cells was performedto determine infectious particles. The vp/ml titer was2.82 × 1011, 8.7 × 1011, 1.28 × 1011, 1.01 × 1012, 3.41 ×1012, 6.33 × 1012, 6.3 × 1011, 2.6 × 1012, and 4.5 ×1012 for RGDTKSSTR, Ad300wt, Ad5luc1, AdVEGFLuc,Ad5VEGFE1, Ad5/3VEGFE1, RGDCRADcox-2R, Ad5-�24RGD, and Ad5/3-�24, respectively. The functionaltiters were 9.98 × 108, 5.7 × 1010, 4.7 × 1010, 1.5 × 1010,1.75 × 1010, 1.3 × 1010, 1.3 × 1010, 5.2 × 1010, and9.5 × 1011 plaque-forming units (pfu)/ml, respectively.

CRAd penetration of tumor cellspheroids

SKOV3.ip1 cells were seeded in a 24-well low adherenceplate at 1 × 105 cells/well. After a 1-week period to allowspheroid formation, 1 × 107 vp of Ad5-�24RGDGFP wereadded to the 1 ml of supernatant. Imaging by fluorescentmicroscopy to detect the expressed viral reporter gene,green fluorescent protein (GFP), was performed dailyafter infection until detection of GFP ceased.

Validation of VEGF promoter activity inovarian cancer

Monolayers were infected in quadruplicate with 1000vp/cell of AdVEGFLuc or Ad5luc1. After 24 h incubation,luciferase expression was quantified by the luciferaseassay system (Promega Corporation, Madison, WI, USA).Protein concentration was measured with the Bio-RadDC protein assay kit (Bio-Rad, Hercules, CA, USA) toallow normalization of the gene expression data for thenumber of cells. Background values were subtracted fromthe readings. Results are expressed as a percentage of theVEGF activity of the CMV promoter.

CRAd replication quantified in purifiedprimary tumor spheroids

Purified primary ovarian adenocarcinoma cell spheroidswere divided into aliquots of 105 cells in Costar

Table 1. Features of viruses used in study

Virus name E1A promoter E1A E3 Fiber

E1-deleted control del CMV-TK-pA-CMV-SSTR2-pA del RGD4C in HI loopAd5Luc1 del CMV-Luciferase-pA del wtAd5VEGFLuc del VEGF-Luciferase-pA del wtAd5 wild-type wt wt wt wtRGDCRADCox-2R Cox-2 wt (reverse orientation) wt RGD4C in HI loopAd5VEGFE1 VEGF wt del wtAd5/3VEGFE1 VEGF wt del serotype 5/3 chimeraAd5-�24RGD wt deletion of Rb binding site wt RGD4C in HI loopAd5/3-�24 wt deletion of Rb binding site wt serotype 5/3 chimera

del = deleted wt = wild-type adenovirus serotype 5 CMV = cytomegalovirus immediate early promoter TK = Herpes simplex virus thymidinekinase gene SSTR2 = human somatostatin type 2 receptor gene pA = polyadenylation signal



96-well ultra-low attachment plates, followed by 1 hinfection with 100 vp/cell of RGDTKSSTR, Ad300wt,RGDCRADcox-2R, Ad5VEGFE1, Ad5/3VEGFE1, Ad5-�24RGD, or Ad5/3-�24. Thereafter, enough fetal bovineserum (FBS) was added to each well to achieve a 10%concentration. Cells and growth medium were harvestedtogether and frozen at indicated time points. DNA waspurified with the DNeasy tissue kit (Qiagen Inc., Valencia,CA, USA). Quantitative PCR was performed as described[24,40]. The probe used was specific for the Ad E4 gene.Viral DNA copies detected were normalized to cellularβ-actin.

CRAd-mediated oncolysis quantified byMTS assay

SKOV3.ip1 cell monolayers in quadruplicate were infectedwith 100 vp/cell for 1 h at 37 ◦C in 50 µl of growthmedium with 2% FBS. Thereafter, cells were incubatedwith 5% growth medium. Cell viability was measuredusing the CellTiter 96 AQueous one solution cellproliferation assay (MTS assay, Promega, Madison, WI,USA) on days 4, 8 and 20. In Figure 4B, SKOV3.ip1 cellswere seeded into low adherence 96-well plates to formspheroids. After overnight incubation, spheroids wereinfected with 100 vp/cell in 2% growth medium for1 h at 37 ◦C. Then, FBS was added in cell suspension toachieve 5% concentration. MTS assay was performed asabove. All cell viability results are expressed as a fractionof viable cells compared with non-infected control at eachtime point.

CRAd treatment in orthotopic ovariancancer model

CB17 SCID mice (Charles River Laboratories, Wilmington,MA, USA) were injected with 5 × 106 SKOV3.ip1cells intraperitoneally (i.p.) on day 0. On day 10,mice received 1 × 108 vp i.p. of RGDTKSSTR (n = 7),Ad300wt (n = 7), Ad5VEGFE1 (n = 11), Ad5/3VEGFE1(n = 11), RGDCRADcox-2R (n = 11), Ad5-�24RGD (n =11), Ad5/3-�24 (n = 11), or no virus (n = 6) in 1 mlof growth medium with no FBS. Mice were inspecteddaily, and euthanasia was performed at the first sign ofdiscomfort or distress. Survival data were plotted into aKaplan-Meier curve, and, using the LIFETEST procedurein SAS v.8.2 (SAS Institute, Cary, NY, USA), the CRAdgroups were compared with the other groups with thelog-rank and χ2 tests. The distribution of the data bestfit the Weibull model, which was used for individualcomparisons between the CRAd groups and controls,using the χ2 test of SAS v.8.2 LIFEREG procedure.

Results

CRAd penetration of tumor cellspheroids

SKOV3.ip1 spheroids infected by Ad5-�24RGDGFP dis-played early expression of GFP restricted to the superficiallayer (Figure 1A). The reporter gene expression pro-gressed into the center of each spheroid until the entirecluster of tumor cells was transduced (Figures 1B–1E).

Figure 1. CRAd penetration of a tumor cell spheroid. (A) An SKOV3.ip1 spheroid infected with Ad5-�24RGDGFP shows transgeneexpression (GFP fluorescence) in only the most superficial layer of cells 10 days after infection. Subsequent imaging 12 (B), 14 (C),and 16 (D) days after infection shows progressive spread of GFP expression into the core of the spheroid. This progression suggeststhat this replicative virus completely penetrated the cell cluster. (E) The decrease in spheroid mass noted 18 days after infectionsuggests that continued viral replication caused extensive oncolysis and consequent spheroid disruption. (F) The absence offluorescence in uninfected spheroids confirms the absence of any confounding background fluorescence. Each white bar representsa distance of 100 µm



Spheroid size was remarkably smaller at the last timepoint with complete transduction of all cells (Figure 1E).No similar decrease in spheroid size and no back-ground fluorescence were noted in uninfected spheroids(Figure 1F).

Validation of VEGF promoter in ovariancancer

Since the VEGF promoter implemented in Ad5VEGFE1and Ad5/3VEGFE1 has not been previously validatedfor ovarian cancer, we employed AdVEGFLuc (fireflyluciferase gene in the E1 region driven by the VEGFpromoter) and Ad5luc1 (firefly luciferase gene in theE1 region driven by the CMV promoter) to assess VEGFpromoter activity relative to CMV promoter activity inovarian cancer. The percentage of VEGF promoter activityrelative to CMV promoter activity in the positive controlcell line H358 and negative control cell line OV4 [19] was0.40 and 0.09%, respectively. VEGF promoter activity inthe ovarian cancer cell lines Hey, SKOV3.ip1, OV3 andOvcar3 was 4.90, 4.97, 3.42 and 0.39%, respectively(Figure 2).

CRAd replication quantified in purifiedprimary tumor spheroids

Quantitative PCR detected the Ad E4 gene in infectedpurified primary ovarian adenocarcinoma cell spheroidsthroughout the duration of all experiments (up to 22 days)(Figure 3). The number of Ad DNA copies generallyincreased in the replicative virus groups but not in themock and E1-deleted groups during the first week of eachexperiment.

Figure 2. The VEGF promoter shows activity in ovarian cancercells. The H358 positive control, OV4 negative control, and fourovarian cancer cell lines were infected with two E1-deletedviruses, AdVEGFLuc and Ad5luc1, which incorporate fireflyluciferase gene expression cassettes driven by VEGF andCMV promoters, respectively. AdVEGFLuc transgene expressionshowed activity of the VEGF promoter in the ovarian cancer cellsrelative to CMV promoter activity

CRAd-mediated oncolysis quantified byMTS assay

SKOV3.ip1 monolayers and spheroids were infectedwith 100 vp/cell of the E1-deleted control, the wild-type Ad5, RGDCRADcox-2R, Ad5VEGFE1, Ad5/3VEGFE1,Ad5-�24RGD, or Ad5/3-�24. After, 4, 8 and 20 days, cellsurvival was determined by MTS assay and expressedas a fraction of the mock value (Figure 4). Datafrom the middle time point 8 days after infectionallowed the clearest comparison between oncolysisaffected by the five CRAds. Based on these values theCRAds killing the most tumor cells from highest tolowest in both monolayers and spheroids were: Ad5/3-�24, Ad5-�24RGD, Ad5/3VEGFE1, RGDCRADcox-2R,and Ad5VEGFE1. Rankings based on means fromall three time points were very similar (data notshown). Slight differences in cell killing patterns werenoted between the monolayers and spheroids. Overall,there seemed to be a tendency for more rapidkilling of monolayers, in particular for viruses lackinginfectivity enhancement. Further studies are neededto comprehensively demonstrate this, but the initialobservation is best exemplified by the wild-type andAd5/3-�24 groups.

CRAd treatment in orthotopic model ofmetastatic cancer

Using a murine model of i.p. ovarian carcinomatosiswe treated the mice with single i.p. doses of either novirus, E1-deleted control, wild-type Ad, or one of the fiveCRAds of interest (Figure 5). All mice in untreated andE1-deleted control groups expired before day 45. All ofthe wild-type Ad5 group, 8 of 11 in the Ad5VEGFE1,and 2 in the Ad5/3VEGFE1 group died within 2 weeksafter treatment. Microscopic examination of the liver ofone of these mice (Ad5/3VEGFE1 group) showed massivehepatic necrosis. All mice treated with RGDCRADcox-2R, Ad5-�24RGD, and Ad5/3-�24 survived until at least43, 67, and 58 days after treatment, respectively. Themedian survival times for groups treated with mock,E1-deleted control, wild-type Ad5, RGDCRADcox-2R,Ad5VEGFE1, Ad5/3VEGFE1, Ad5-�24RGD, and Ad5/3-�24 was 35, 39, 9, 73, 13, 97, 120, and >150 days,respectively. Statistical analysis with the log-rank and χ2

tests indicated that survival was significantly better inanimals treated with RGDCRADcox-2R, Ad5/3VEGFE1,Ad5-�24RGD, and Ad5/3-�24 (P < 0.001 in all cases)than those untreated. Similar analysis also showed thatmice treated with RGDCRADcox-2R, Ad5/3VEGFE1, Ad5-�24RGD, and Ad5/3-�24 lived longer than those treatedwith the E1-deleted control (P < 0.002 in all cases).Finally, these statistical tests revealed that treatmentwith all five tested CRAds allowed prolonged survivalcompared with treatment with wild-type Ad (P < 0.0001in all cases).



Figure 3. All five CRAds show robust DNA replication in primary ovarian cancer cells. (A–E) Purified primary ovarian cancer cellsfrom five patient malignant ascites samples were cultured as spheroids and infected with 100 vp of no virus, E1-deleted control,wild-type Ad5, RGDCRADcox-2R, Ad5VEGFE1, Ad5/3VEGFE1, Ad5-�24RGD, or Ad5/3-�24. Spheroids and growth medium wereharvested at indicated time points, and virus copy number was measured with quantitative PCR. This data was then transformedto cumulative virus DNA copy numbers normalized to cellular β-actin. All replicative viruses showed clear increases in viral DNAduring the first week. Notably, no single CRAd consistently demonstrated superior replicativity, but instead the behavior of eachagent varied between patient samples

Discussion

Advanced stage cancers require new treatment strategies,and gene therapy offers promising therapeutic options.Ads represent the most extensively utilized vector systemclinically [1,2]. Despite their excellent safety history[3] and preliminary evidence of efficacy [4–6], vectorsystems are limited by the formidable requirement foreffective tumor penetration and transduction of a largeportion of cells. Replicative agents, however, apparentlyeffect greater transduction than their non-replicativecounterparts due to local replication, dispersion, andlocal amplification of effect [4,10–12]. Moreover, viralreplication alone kills infected cells.

In this study, we show evidence of a replicative viruspenetrating three-dimensional spheroids, which wereutilized as crude models for solid tumors. Figure 1shows Ad reporter gene expression progressing from

the superficial cell layers of an ovarian cancer spheroidto its center. The marked disruption and decreasein spheroid size illustrates viral replication inducedoncolysis. These findings also emphasize the importanceof three-dimensional models for analyzing dynamic viralbehavior such as replication and oncolysis.

Assays for preclinical evaluation of therapeutic Adsshould also employ primary tumor substrates, sincevariations of viral infectivity in tumor cell lines comparedwith patient tumor could confound these analyses.This discrepancy is substantiated by reports of unequalexpression of surface receptors essential for Ad infectionbetween tumor cell lines and primary tumor cells[32–35]. To apply the most stringent preclinical assays,we employed primary tumor tissue and spheroids in ouranalysis of Ad replication and oncolysis. Furthermore, thepurification of our primary tumor samples allowed us tominimize infection of irrelevant non-tumor cells [38].



Figure 4. All five CRAds show potent ovarian cancer cell killing.(A) SKOV3.ip1 monolayers and (B) spheroids were infected with100 vp/cell of no virus, E1-deleted control, wild-type Ad5,RGDCRADcox-2R, Ad5VEGFE1, Ad5/3VEGFE1, Ad5-�24RGD, orAd5/3-�24 in quadruplicate. Tumor cell viability was assessedby MTS cell viability assay at 4 (white bars), 8 (grey bars), and20 days (black bars) after infection. Each error bar represents a95% confidence interval

Our objective in this study was to compare fivenovel CRAds with potential for treatment of peri-toneally disseminated ovarian cancer: RGDCRADcox-2R,Ad5VEGFE1, Ad5/3VEGFE1, Ad5-�24RGD, and Ad5/3-�24. RGDCRADcox-2R, Ad5VEGFE1, and Ad5/3VEGFE1feature tumor-specific promoters that induce robust repli-cation in tumor tissue and minimal replication in non-tumor tissues [16,19]. We demonstrated that the VEGFpromoter has activity in ovarian cancer cells (Figure 2).Presently, it is not known what is the optimal levelfor effective replication and oncolysis. However, ourresults suggest that an in vitro activity of 5% may beenough. In vivo, the presence of ascites (which usuallycontains lysophosphatidic acid) may further induce VEGF

Figure 5. The CRAds tested show remarkable therapeuticefficacy in an orthotopic model of metastatic ovarian cancer.Ten days after i.p. injection of 5 × 106 SKOV3.ip1 tumorsin SCID mice, i.p. injections of either no virus or 1 × 108

vp of E1-deleted control, wild-type Ad5, RGDCRADcox-2R,Ad5VEGFE1, Ad5/3VEGFE1, Ad5-�24RGD, or Ad5/3-�24 wereperformed. Survival was improved by treatment with each ofthe four capsid-modified CRAds when compared with treatmentwith E1-deleted control

promoter activity in ovarian cancer cells [45]. The othertwo CRAds, Ad5-�24RGD [22] and Ad5/3-�24 [24],selectively replicate in cells defective in the Rb-p16 path-way, such as most tumor cells. In addition to replicationcontrol mechanisms, these CRAds tested also exemplifytropism-modifying features. RGDCRADcox-2R [16] andAd5-�24RGD [22] incorporate the RGD-4C integrin bind-ing motif into the fiber HI loop, an alteration shown toaugment transduction to ovarian cancer both in vitro andin vivo [28]. Ad5/3VEGFE1 [19] and Ad5/3-�24 [24]feature a genetic modification replacing the Ad5 fiberknob domain with the Ad3 knob. This retargeting strat-egy has been shown to increase Ad infectivity in primaryovarian cancer cells approximately 10-fold [30,31].

Purified primary ovarian cancer cell spheroids wereinfected with the five CRAds and controls, and AdDNA replication was quantified with real-time PCR.With knowledge that viral infectivity directs the extentof replication and oncolysis [36,37], we noticed thatdifferent types of tropism modification correlated withdistinct replicativity profiles in different patient tumorsamples. Experiments using samples from patients A andB showed relatively greater replication of Ad5/3-�24 thanAd5-�24RGD (Figure 3). These results suggest that tumorcells from patients A and B may express relatively more ofthe Ad3 receptor. In contrast, samples from patients C andE showed no clear distinction between Ads with serotype 3knob chimerism and those without. Furthermore, viruseswith native Ad5 knob showed greater replication thanviruses with Ad3 knob in sample cells from patient D.Presumably, these tumor cells from patient D may haverelatively more Ad5 than Ad3 receptor, and tumor cellsfrom patients C and E might express comparable amountsof each receptor. These findings agree with evidence that



ovarian adenocarcinomas from different patients expressvariable amounts of receptors relevant to Ad infectivity[32].

Variation between tumors from different patients wasalso noticed when comparing CRAds with different E1promoters. Ads with the VEGF tumor-specific promotershowed greater replication than those with the cox-2promoter in all five patient samples and especially intumor cells from patient C. These results suggest thatthe sample from patient C might have relatively highVEGF promoter activity. In samples from patients A andE, the CRAds with the VEGF and cox-2 promoters hadonly marginal replicativity compared with wild-type Ad.In contrast, cancer cells from patients B and D showmore replication of CRAds with the VEGF and cox-2promoters. Therefore, samples from patients A and E mayhave relatively low VEGF and cox-2 promoter activities,and these promoters might have high activity in samplesfrom patients B and D. The substantial replication ofAd5-�24RGD and Ad5/3-�24 in all samples indicates apotential efficacy to a wide range of clinical cases. Mostimportantly, all five CRAds tested demonstrated robustDNA replication comparable to that of wild-type Ad5.

Clearly, it would be of interest to directly correlatereceptor and promoter expression with the degreeof oncolysis on clinical specimens. Unfortunately, thiswas not possible with the samples studied here,as the whole sample was necessary for obtainingenough spheroids for the analyses performed. However,we have demonstrated previously, that on cell linesin vitro and in vivo, there is good correlation betweenprimary receptor density, infectivity and oncolysis[16,22–24,30,31,36,37]. For promoter driven CRAds,there is also close correlation between expression of thepromoter and replicativity/oncolysis [16–18].

Cell viability assays showed that Ad5-�24RGD andAd5/3-�24, especially the latter, effected the most oncol-ysis overall in SKOV3.ip1 monolayers and spheroids(Figure 4). The chimeric CRAds with Ad3 knobs demon-strated superior cell killing, a finding that concurs withevidence that SKOV3.ip1 cells have high Ad3 receptorexpression [30]. Again, when compared with wild-typeAd, all five CRAds tested showed strong tumor cell killing.

The cell viability assays also showed slight temporaldifferences between CRAd-mediated oncolysis patterns inspheroids and monolayers. In general, the more oncolyticviruses killed tumor cells faster in monolayers, an obser-vation best seen in the wild-type Ad5 and Ad5/3-�24data. The culture of SKOV3.ip1 cells as spheroids perse does not alter the surface receptors needed for Adinfection [39]. Instead, the discrepancies in oncolysis pat-terns might be attributed to the morphologic differencesbetween two- and three-dimensional models. These dif-ferences could affect CRAd infectivity, dispersion, andoncolysis, which further substantiates the need for three-dimensional model systems for assay of CRAds and otherreplicative agents.

The survival studies show that all the CRAds tested,except Ad5VEGFE1, had high therapeutic efficacy as

anticancer agents in vivo (Figure 5). In an orthotopicmodel of disseminated ovarian cancer, mice treatedwith RGDCRADcox-2R, Ad5/3VEGFE1, Ad5-�24RGD,and Ad5/3-�24 had prolonged survival compared withuntreated mice (P < 0.001 in all cases). Also, these dataseemed to highlight Ad5/3-�24 as the most effectivetreatment in studies employing SKOV3.ip1. The rankingof therapeutic efficacy for the five CRAds treating this cellline in the oncolysis and survival studies was identical.

The deaths observed within 2 weeks of treatmentprobably related to viral toxicity, as massive hepaticnecrosis was documented with histological analysis. Thisfinding concurs with our previous experiences withsimilarly timed deaths in mice treated i.p. with wild-type Ad5 at comparable doses [17]. An excessive virusdose should have killed mice within a few days aftertreatment instead of allowing a 1–2 week survival.Therefore, the observed delay could be attributed toviral replication within the engrafted human cancerwith subsequent systemic distribution and liver injury[43]. Notably, both viruses associated with the mostsevere toxicity (Ad5VEGFE1 and Ad5 wild-type) featuredunmodified Ad5 fibers. If these findings are confirmed inhuman cancer studies, they may support the usefulnessof targeting approaches for reducing liver tropism andconsequent liver damage.

In summary, Ad5VEGFE1, RGDCRADcox-2R, Ad5/3VEGFE1, Ad5-�24RGD, and Ad5/3-�24 all showedhigh levels of DNA replication and oncolysis in ovariancancer. The latter four also prolonged survival ina model of peritoneally disseminated human ovariancancer. Interestingly, the clinical samples data suggestedvariability in CRAd promoter activity and in CRAd relevantsurface receptor expression. Therefore, assessment ofpromoter activity and surface receptor expression foreach patient’s tumor tissue may aid selection of anoptimal CRAd agent for each patient. Furthermore,identifying several candidate agents for each patientmight aid repeated administrations or combinationtreatments. Either approach may help avoid selectionof resistant clones. Also, alternative treatment optionscould help in the event of relapse. Importantly, theanti-viral immune response is mostly targeted againstspecific capsid configurations [31,44]. Therefore, utilizingviruses with distinct capsids prevents neutralization withrepeated rounds of treatment or high initial antibody titers[28,40]. In conclusion, this study included primary tumortissue, spheroids, and an orthotopic model of humanovarian cancer to evaluate five CRAds that show promisefor treatment of peritoneally metastatic ovarian cancer.Clinical trials will ultimately determine their safety andefficacy in humans.

Acknowledgements

Supported by the Sohlberg Foundation, Sigrid Juselius Foun-dation, Emil Aaltonen Foundation, Maud Kuistila Foundation,Finnish Medical Foundation Duodecim, Academy of Finland,Finnish Cancer Society, Ida Montin Foundation, Biomedicum



Helsinki Foundation, Biocentrum Helsinki internal funds ofthe University of Helsinki, Helsinki University Central HospitalResearch Funds, Research and Science Foundation of Farmos,Instrumentarium Research Fund, and the NCI (RO1 CA83821,P50 CA83591, P50 CA89019, and RO1 CA93796). Gene transferassays were performed in part at the UAB Gene Therapy CenterCorrelative Laboratories for Human Clinical Trials. The authorsthank Drs. H. Inoue and T. Tanabe of the National Cardiovas-cular Center Research Institute, Suita, Japan, for providing theplasmid phPES2 containing the cox-2 promoter.

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