Preclinical Evaluation of Telomerase-Specific Oncolytic ... · Cancer Therapy: Preclinical Preclinical Evaluation of Telomerase-Specific Oncolytic Virotherapy for Human Bone and Soft

Cancer Therapy: Preclinical

Preclinical Evaluation of Telomerase-Specific Oncolytic Virotherapyfor Human Bone and Soft Tissue Sarcomas

Tsuyoshi Sasaki1, Hiroshi Tazawa2,3, Jo Hasei1, Toshiyuki Kunisada1,4, Aki Yoshida1,Yuuri Hashimoto3, Shuya Yano3, Ryosuke Yoshida3, Futoshi Uno2,3, Shunsuke Kagawa2,3,Yuki Morimoto1, Yasuo Urata5, Toshifumi Ozaki1, and Toshiyoshi Fujiwara2,3

AbstractPurpose: Tumor-specific replication-selective oncolytic virotherapy is a promising antitumor therapy

for induction of cell death in tumor cells but not of normal cells. We previously developed an oncolytic

adenovirus, OBP-301, that kills human epithelial malignant cells in a telomerase-dependent manner.

Recent evidence suggests that nonepithelial malignant cells, which have low telomerase activity, maintain

telomere length through alternative lengthening of telomeres (ALT). However, it remains unclear whether

OBP-301 is cytopathic for nonepithelial malignant cells. Here, we evaluated the antitumor effect of OBP-

301 on human bone and soft tissue sarcoma cells.

Experimental Design: The cytopathic activity of OBP-301, coxsackie and adenovirus receptor (CAR)

expression, and telomerase activity were examined in 10 bone (OST, U2OS, HOS, HuO9, MNNG/HOS,

SaOS-2, NOS-2, NOS-10, NDCS-1, andOUMS-27) and in 4 soft tissue (CCS, NMS-2, SYO-1, andNMFH-1)

sarcoma cell lines. OBP-301 antitumor effects were assessed using orthotopic tumor xenograft models. The

fiber-modified OBP-301 (termedOBP-405) was used to confirm an antitumor effect onOBP-301–resistant

sarcomas.

Results: OBP-301 was cytopathic for 12 sarcoma cell lines but not for the non–CAR-expressing OUMS-

27 and NMFH-1 cells. Sensitivity to OBP-301 was dependent on CAR expression and not on telomerase

activity. ALT-type sarcomas were also sensitive to OBP-301 because of upregulation of human telomerase

reverse transcriptase (hTERT) mRNA following virus infection. Intratumoral injection of OBP-301

significantly suppressed the growth of OST and SYO-1 tumors. Furthermore, fiber-modified OBP-405

showed antitumor effects on OBP-301–resistant OUMS-27 and NMFH-1 cells.

Conclusions: A telomerase-specific oncolytic adenovirus is a promising antitumor reagent for the

treatment of bone and soft tissue sarcomas. Clin Cancer Res; 17(7); 1828–38. �2011 AACR.

Introduction

Bone and soft tissue sarcomas are annually diagnosed in13,230 patients in the United States (1). They are the thirdmost common cancer in children and account for 15.4% ofall childhood malignancies. Treatment of patients with

bone and soft tissue sarcomas requires a multidisciplinaryapproach that involves orthopedic oncologists, musculos-keletal radiologists and pathologists, radiation oncologists,medical and pediatric oncologists, and microvascular sur-geons (2, 3). Despite major advances in the treatment ofbone and soft tissue sarcomas, such as neoadjuvant andadjuvant multiagent chemotherapy and aggressive surgery,about one fourth of the patients show a poor response toconventional therapy, resulting in subsequent recurrenceand leading to a poor prognosis (1). Therefore, the devel-opment of a novel therapeutic strategy is required to curepatients with bone and soft tissue sarcomas.

Recent advances in molecular biology have fosteredremarkable insights into the molecular basis of neoplasia.More than 85% of all human cancers, but only a fewnormal somatic cells, show high telomerase activity (4–6). Telomerase activity has also been detected in 17% to81% of bone and soft tissue sarcomas (7–10). Telomeraseactivation is considered to be a critical step in cancerdevelopment, and its activity is closely correlated withthe expression of human telomerase reverse transcriptase

Authors' Affiliations: 1Department of Orthopaedic Surgery, OkayamaUniversity Graduate School of Medicine, Dentistry and PharmaceuticalSciences; 2Center for Gene and Cell Therapy, Okayama University Hos-pital; Departments of 3Gastroenterological Surgery and 4Medical Materialsfor Musculoskeletal Reconstruction, OkayamaUniversity Graduate Schoolof Medicine, Dentistry and Pharmaceutical Sciences, Okayama; and5Oncolys BioPharma, Inc., Tokyo, Japan

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Toshiyoshi Fujiwara, Department of Gastroenter-ological Surgery, Okayama University Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku,Okayama 700-8558, Japan. Phone: 81-86-235-7257; Fax: 81-86-221-8775. E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-10-2066

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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(hTERT; ref. 11). Recently, telomerase-specific replication-selective oncolytic virotherapy has emerged as a promisingantitumor therapy for induction of tumor-specific celldeath. We previously developed an oncolytic adenovirus,OBP-301, in which the hTERT promoter drives the expres-sion of the E1A and E1B genes linked to an internalribosome entry site (IRES; ref. 12). We determined thatOBP-301 efficiently induced the selective killing of a varietyof human malignant epithelial cells, such as colorectal,prostate, and non–small cell lung cancers, but not ofnormal cells (12, 13). Furthermore, a phase I clinical trialof OBP-301, which was conducted in the United States onpatients with advanced solid tumors, indicated that OBP-301 is well tolerated by patients (14).There are 2 known telomere-maintenance mechanisms

in human malignant tumors (15, 16): telomerase activa-tion (4–6) and telomerase-independent alternative length-ening of telomeres (ALT; ref. 17–19). The ALT-typemechanism is more prevalent in tumors arising from none-pithelial tissues than in those of epithelial origin (20, 21).Therefore, ALT-type nonepithelial malignant cells fre-quently show low telomerase activity, suggesting that theyhave a low sensitivity to OBP-301, which kills cancer cellsin a telomerase-dependent manner. However, it remains tobe determined whether OBP-301 can exert an antitumoreffect on human nonepithelial and on epithelial malig-nancies.Adenovirus infection is mainly mediated by interaction

of the virus with the coxsackie and adenovirus receptor(CAR) expressed on host cells (22). Therefore, while CAR-expressing tumor cells are the main targets for oncolytic

adenoviruses, tumor cells that lack CAR can escape frombeing killed by oncolytic adenoviruses. It has been reportedthat CAR is frequently expressed in human cancers ofvarious organs such as the brain (23), thyroid (24), eso-phagus (25), gastrointestinal tract (26), and ovary (27).Bone and soft tissue sarcomas also express CAR (28–30).However, some populations of tumor cells lack CARexpression, suggesting a requirement for the developmentof a novel antitumor therapy against CAR-negative tumorcells. We recently developed fiber-modified OBP-301(termed OBP-405), which can bind to not only CAR butalso integrinmolecules (avb3 andavb5) and efficiently killCAR-negative tumor cells (31).

In the present study, we first investigated the in vitrocytopathic efficacy of OBP-301 against 14 human bone andsoft tissue sarcoma cells. Next, the relationship between thecytopathic activity of OBP-301, CAR expression, and telo-merase activity in human sarcoma cells was assessed. The invivo antitumor effect of OBP-301 was also confirmed usingorthotopic animal models. Finally, the antitumor effect ofOBP-405 against OBP-301–resistant sarcoma cells wasevaluated in vitro and in vivo.

Materials and Methods

Cell linesThe human osteosarcoma (HuO9; ref. 32), chondrosar-

coma (OUMS-27; ref. 33), and synovial sarcoma (SYO-1;ref. 34) cell lines were previously established in our labora-tory. The human osteosarcoma cell lines OST, HOS, andSaOS-2 were kindly provided by Dr. Satoru Kyo (KanazawaUniversity, Ishikawa, Japan). The human clear cell sarcomacell line CCS was maintained in our laboratory. Thesecells were propagated as monolayer cultures in Dulbecco’smodified Eagle’s medium (DMEM). The human osteosar-coma cell line U2OS was obtained from the American TypeCulture Collection (ATCC) and was grown in McCoy’s 5amedium. The human osteosarcoma cell line MNNG/HOSwas purchased from DS Pharma Biomedical and wasmaintained in Eagle’s minimum essential medium contain-ing 1% nonessential amino acids. The human osteosar-coma cell lines NOS-2 and NOS-10 (35), the humandedifferentiated chondrosarcoma cell line NDCS-1 (36),the human malignant peripheral nerve sheath cell lineNMS-2 (37), and the human malignant fibrous histiocy-toma cell line NMFH-1 (38) were kindly provided by Dr.Hiroyuki Kawashima (Niigata University, Niigata, Japan)and were grown in RPMI-1640 medium. The transformedembryonic kidney cell line 293 was obtained from theATCC and maintained in DMEM. All media were supple-mented with 10% heat-inactivated FBS, 100 units/mLpenicillin, and 100 mg/mL streptomycin. The cells weremaintained at 37�C in a humidified atmosphere with 5%CO2.

Recombinant adenovirusesThe recombinant tumor-specific, replication-selective

adenovirus OBP-301 (Telomelysin), in which the promoter

Translational Relevance

Bone and soft tissue sarcomas frequently occur inyoung children and show aggressive progression, resis-tance to conventional chemotherapy, and poor prog-nosis, indicating a requirement for novel antitumortherapy to improve the clinical outcome. Telomerase-specific replication-selective oncolytic virotherapy isemerging as a promising antitumor therapy. We devel-oped an oncolytic adenovirus, OBP-301, that efficientlykills human epithelial malignant cells in a telomerase-dependentmanner. However, alternative lengthening oftelomeres (ALT)-type nonepithelial malignant cellsshow low telomerase activity, suggesting lower effec-tiveness of OBP-301 in these cells. Here, we showed thatOBP-301 has antitumor effects on both non–ALT-typeand ALT-type sarcoma cells through upregulation ofhuman telomerase reverse transcriptase mRNA. Further-more, coxsackie and adenovirus receptor–negative sar-coma cells were efficiently killed by fiber-modifiedOBP-301 (termed OBP-405) through virus–integrin binding.Thus, a telomerase-specific oncolytic adenovirus wouldgreatly improve the clinical outcome of young patientswith advanced sarcomas.

Oncolytic Virotherapy for Sarcoma

www.aacrjournals.org Clin Cancer Res; 17(7) April 1, 2011 1829




element of the hTERT gene drives the expression of E1A andE1B genes linked with an IRES, was previously constructedand characterized (12, 13).OBP-405 is a telomerase-specificreplication-competent adenovirus variant that was pre-viously generated to express the RGD peptide in the fiberknob of OBP-301 (31). The E1A-deleted adenovirus vectordl312 andwild-type adenovirus serotype 5 (Ad5) were usedas the control vectors. Recombinant viruseswere purified byultracentrifugation using cesium chloride step gradients,and their titers were determined by a plaque-forming assayby using 293 cells and they were stored at �80�C.

Cell viability assayCells were seeded on 96-well plates at a density of 1 �

103 cells/well 20 hours before viral infection. All cell lineswere infected with OBP-301 or OBP-405 at multiplicity ofinfections (MOI) of 0, 0.1, 1, 10, 50, or 100 plaque formingunits (PFU)/cell. Cell viability was determined on days 1, 2,3, and 5 after virus infection, using a Cell Proliferation kit II(Roche Molecular Biochemicals) that was based on an XTT,sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate, assay,according to the manufacturer’s protocol. The ID50 value ofOBP-301 for each cell line was calculated using cell viabilitydata obtained on day 5 after virus infection.

Flow cytometric analysisThe cells (5� 105) were labeled with mousemonoclonal

anti-CAR (RmcB; Upstate Biotechnology), anti-humanintegrin avb3 (LM609; Chemicon International), or anti-human integrin avb5 (P1F6; Chemicon International)antibody for 30 minutes at 4�C. The cells were thenincubated with fluorescein isothiocyanate (FITC)-conju-gated rabbit anti-mouse IgG second antibody (ZymedLaboratories) and were analyzed using flow cytometry(FACS Array; Becton Dickinson). The mean fluorescenceintensity (MFI) of CAR and integrin avb3 or avb5 for eachcell line was determined by calculating the differencebetween the MFI in antibody-treated and nontreated cellsfrom 3 independent experiments.

Quantitative real-time PCR analysisU2OS cells, seeded on 6-well plates at a density of 5� 105

cells/well 20 hours before viral infection, were infectedwithAd5, OBP-301, or dl312 at an MOI of 10 or 100 PFUs/cell.Mock-infected cells were used as controls. Furthermore, toconfirm the modulation of hTERT mRNA expression byOBP-301 infection, CAR-positive and hTERTmRNA-expres-sing human sarcoma cell lines were seeded on 6-well platesat a density of 5 � 104 cells/well 20 hours before viralinfection andwere infectedwith OBP-301 at anMOI of 100PFUs/cell. Total RNA was extracted from the cells 2 daysafter virus infection by using the RNA-Bee reagent (Tel-TestInc.). After synthesis of cDNA from100 ng of total RNA, thelevels of hTERT and glyceraldehyde-3-phosphate dehydro-genase (GAPDH) mRNA expression were determined usingquantitative real-time PCR and a Step One Plus Real TimePCR System (Applied Biosystems) and TaqMan Gene

Expression Assays (Applied Biosystems). The relative levelsof hTERT mRNA expression were calculated by using the2�DDCt method (39) after normalization with reference tothe expression of GAPDH mRNA.

To compare the E1A copy number between OBP-301-and Ad5-infected U2OS cells, U2OS cells, seeded on 6-wellplates at a density of 5� 105 cells/well 20 hours before viralinfection, were infected with OBP-301 or Ad5 at an MOI of10 PFUs/cell. Genomic DNA was extracted from seriallydiluted viral stocks, and tumor cells were infected withOBP-301 or Ad5 by using the QIAmp DNA Mini Kit(Qiagen). E1A copy number was also determined usingTaqMan real-time PCR systems (Applied Biosystems).

In vivo OST and OUMS-27 xenograft tumor modelsAnimal experimental protocols were approved by the

Ethics Review Committee for Animal Experimentation ofOkayama University School of Medicine. The OST andOUMS-27 cells (5 � 106 cells per site) were inoculatedinto the tibia or the flank of female athymic nude miceaged 6 to 7 weeks (Charles River Laboratories). Palpabletumors developed within 14 to 21 days and were permittedto grow to approximately 5 to 6 mm in diameter. At thatstage, a 50 mL volume of solution containing OBP-301,OBP-405, dl312, or PBS was injected into the tumors.Tumor size was monitored by measuring tumor lengthand width by using calipers. The volumes of OUMS-27tumors were calculated using the following formula: (L �W2)� 0.5, where L is the length andW is the width of eachtumor. The volumes of OST tumors were calculated usingthe formula: (L þ W) � L � W � 0.2618, as previouslyreported (40).

X-ray examinationThe formation of osteolytic lesions was monitored using

radiography (FUJIFILM IXFR film; FUJIFILMCo.) and an X-ray system (SOFTEX TYPE CMB; SOFTEX Co.).

Histopathologic analysisTumors were fixed in 10% neutralized formalin and

embedded in paraffin blocks. Sections were stained withhematoxylin/eosin (H&E) and analyzed by light micro-scopy.

Statistical analysisData are expressed as means � SD. Student’s t test was

used to compare differences between groups. Pearson’sproduct–moment correlation coefficients were calculatedusing PASW statistics version 18 software (SPSS Inc.).Statistical significance was defined when the P value wasless than 0.05.

Results

In vitro cytopathic efficacy of OBP-301 against humanbone and soft tissue sarcoma cell lines

To evaluate the in vitro cytopathic effect of OBP-301against nonepithelial malignant cells, 14 tumor cell lines

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derived from human bone and soft tissue sarcomas wereinfected with various doses of OBP-301. The cell viability ofeach cell line was assessed over 5 days after infection by theXTT assay. OBP-301 infection induced cell death in a time-dependent manner in all sarcoma cell lines except for theOUMS-27 and NMFH-1 cell lines (Fig. 1A). Calculation ofthe ID50 values revealed that, of the 12 OBP-301–sensitivesarcoma cell lines, MNNG/HOS and SaOS-2 cells wererelatively less sensitive than the other 10 sarcoma cell lines(Fig. 1B). Furthermore, to rule out the possibility thatcytopathic effect of OBP-301 is due to nonspecific toxicitybased on the high uptake of virus particles into tumor cells,we examined the cytopathic activity of replication-deficientdl312 in U2OS and HOS cells. dl312 did not show anycytopathic effect in U2OS and HOS cells, even when thesecells were infected with dl312 at high dose (50 and 100MOIs; Supplementary Fig. S1). These results indicate thatOBP-301 is cytopathic formost human bone and soft tissuesarcoma cells line but that some sarcoma cell lines areresistant to OBP-301.

Expressions of the adenovirus receptor and hTERTmRNA on human bone and soft tissue sarcoma celllinesBecause adenovirus infection efficiency depends mainly

on cellular CAR expression (22), we determined the expres-

sion level of CAR on the 14 sarcoma cell lines by flowcytometry. The 12 OBP-301–sensitive sarcoma cell linesshowed CAR expression, determined as MFIs, at variouslevels, whereas the OBP-301–resistant OUMS-27 andNMFH-1 cells did not express CAR (Fig. 2A and Supple-mentary Fig. S2).

OBP-301 contains the hTERT gene promoter, whichallows it to tumor specifically regulate the gene expres-sion of E1A and E1B for viral replication. Thus, OBP-301 can efficiently replicate in human cancer cells withhigh telomerase activity but not in normal cells withouttelomerase activity (12). Recently, some populationsof human sarcoma cells have been shown to possesslow telomerase activity and to maintain telomerelengths through an ALT mechanism (17–19). Thus, itis probable that OBP-301 cannot efficiently replicate in,and kill, ALT-type human sarcoma cells because of theirlow telomerase activity. To assess whether the telomer-ase activity of human sarcoma cells affects the cyto-pathic activity of OBP-301, we analyzed hTERT mRNAexpression levels in the 14 sarcoma cell lines by quan-titative real-time reverse transcriptase PCR (RT-PCR)analysis. Thirteen of the sarcoma cell lines had detect-able hTERT mRNA expression at variable levels, andonly SaOS-2 cells did not express hTERT mRNA(Fig. 2B).

Figure 1. Cytopathic effect ofOBP-301 on human bone andsoft tissue sarcoma cell lines. A,cells were infected with OBP-301at the indicated MOI, and cellsurvival was quantified over5 days using the XTT assay. Thecell viability of mock-treatedgroup on each day wasconsidered 1.0, and the relativecell viability was calculated.Data are means � SD. The typesof tumor except for osteosarcomawere shown in parentheses.B, the 50% inhibiting doses ofOBP-301 on cell viability 5 daysafter infection were calculatedand are expressed as ID50 values.






We next investigated the relationship between CAR andhTERT mRNA expressions and the cytopathic activity ofOBP-301 among the 11 CAR-positive sarcoma cell lineswith hTERT gene expression. CAR expression levels signifi-cantly (r¼ –0.834; P¼ 0.01) correlated with the cytopathicactivity of OBP-301 against 8 of the bone sarcoma cell lines(Fig. 2C). CAR expression in 3 of the soft tissue sarcoma celllines also correlated (r ¼ –0.99) with the cytopathic effectof OBP-301, but the differences did not reach significance(P ¼ 0.1) because of the low number of cell lines assayed.In contrast, there was no significant correlation betweenhTERT mRNA expression and the cytopathic activity ofOBP-301 (Fig. 2D). These results indicate that the cyto-pathic activity of OBP-301, at least in part, depends on CARexpression.

Furthermore, SaOS-2 and U2OS cells have already beenshown to be ALT-type sarcoma cell lines with low telomer-

ase activity (9, 17). Among these ALT-type sarcoma cells,U2OS cells showed a sensitivity to OBP-301 that wassimilar to that of non–ALT-type sarcoma cells such asHOS and NOS-10 (Fig. 1B). These results indicate thatALT-type human sarcoma cells are sensitive to OBP-301and that a low telomerase activity does not detract from thecytopathic activity of OBP-301.

Enhanced virus replication and cytopathic activity ofOBP-301 through hTERT mRNA upregulation in ALT-type sarcoma cell lines

The high sensitivity of ALT-type sarcoma cells to OBP-301 prompted us to hypothesize that OBP-301 mayactivate the hTERT gene promoter, thereby enhancingthe viral replication rate and subsequently inducing cyto-pathic activity in ALT-type sarcoma cells. Furthermore,it has been previously shown that the adenoviral E1A

Figure 2. Relationship between the expression levels of CAR and hTERT mRNA and the cytopathic activity of OBP-301 against human bone and soft tissuesarcoma cell lines. A, the MFI of CAR expression on human bone and soft tissue sarcoma cells. The cells were incubated with a monoclonal anti-CAR (RmcB)antibody, followed by flow cytometric detection using an FITC-labeled secondary antibody. B, expression of hTERT mRNA in human bone and soft tissuesarcoma cells by quantitative real-time PCR. The relative levels of hTERT mRNA were calculated after normalization with reference to the expression ofGAPDHmRNA. C, correlation between the MFI of CAR and the ID50 of OBP-301 on human bone and soft tissue sarcoma cells. D, correlation between hTERTmRNA expression and the ID50 of OBP-301 on human bone and soft tissue sarcoma cells. Statistical significance was determined as P < 0.05, after analysis ofPearson's correlation coefficient.

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protein can activate the promoter activity of the hTERTgene (41, 42). Therefore, to determine whether OBP-301infection activates hTERTmRNA expression, we examinedthe expression level of hTERT mRNA in ALT-type U2OScells after infection with OBP-301 at MOIs of 10 and 100PFUs/cell (Fig. 3A). Compared with mock-infected U2OScells, OBP-301–infected U2OS cells showed a 6- to 8-foldincrease in hTERT mRNA expression in a dose-dependentmanner. Ad5 infection also increased hTERT mRNAexpression in U2OS cells, whereas there was no increasein U2OS cells infected with E1A-deleted dl312. Theseresults suggest that OBP-301 is cytopathic for ALT-typesarcoma cells through E1A-mediated activation of thehTERT gene promoter.We next compared viral replication rates after infection

of ALT-type U2OS cells with OBP-301 or Ad5. As expected,the viral replication rate of OBP-301 was significantly

higher than that of Ad5 (Fig. 3B). Furthermore, the cyto-pathic activity of OBP-301 was significantly higher thanthat of Ad5 against the ALT-type U2OS cells (Fig. 3C).Finally, to determine whether OBP-301 activates hTERTmRNA expression in both ALT-type and non–ALT-typehuman sarcoma cell lines, we infected 11 CAR-positivehuman sarcoma cells with OBP-301 at 100MOI. Ten of the11 CAR-positive human sarcoma cell lines showed anincrease in the expression level of hTERT mRNA afterOBP-301 infection that ranged from a 1.1- to 50.0-foldincrease (Fig. 3D and Supplementary Table S1). In addi-tion, the expression level of hTERT mRNA was also upre-gulated when OST cells were infected with 5 or 50 MOI ofOBP-301 (Supplementary Fig. S3). These results suggestthat OBP-301 is cytopathic for both ALT-type and non–ALT-type human sarcoma cells through activation of thehTERT gene promoter.

Figure 3. Upregulation of hTERT gene expression in ALT-type human sarcoma cell lines enhances the replication and the cytopathic effect of OBP-301.A, expression of hTERT mRNA in U2OS cells that were mock-infected or were infected with OBP-301, Ad5, or dl312. The cells were infected withOBP-301, Ad5, or dl312 at the indicated MOIs for 48 hour, and hTERT mRNA expression was analyzed using quantitative real-time RT-PCR. The value ofhTERTmRNA expression in the mock-infected cells was set at 1, and relative mRNA levels were plotted. B, quantitative measurement of viral DNA replicationin U2OS cells infected with OBP-301 or Ad5. The cells were infected with OBP-301 or Ad5 at an MOI of 10 PFUs/cell, and E1A copy number was analyzedover the following 2 days by quantitative real-time PCR. The value of the E1A copy number at 2 hours after infection was set at 1, and relative copy numberswere plotted. C, comparison of the cytopathic effect of OBP-301 and Ad5 in U2OS cells. The cells were infected with OBP-301 or Ad5 at the indicatedMOIs, and cell survival was quantified 5 days after infection by using an XTT assay. D, expression of hTERTmRNA after infection of human bone (left) and softtissue (right) sarcoma cell lines with OBP-301 at an MOI of 100 PFUs/cell. Statistical significance (*) was determined as P < 0.05 (Student's t test).






Antitumor effect of OBP-301 against 2 orthotopictumor xenograft models

To evaluate the in vivo antitumor effect of OBP-301against human bone and soft tissue sarcomas, we used 2types of orthotopic tumor xenograft models: the OSTbone sarcoma xenograft and the SYO-1 subcutaneous softtissue sarcoma xenograft. We first identified a dose of OBP-301 that was suitable for induction of an antitumor effect inthe subcutaneous OST bone sarcoma xenograft model(determined as >107 PFUs; Supplementary Fig. S4). Wenext assessed the antitumor effect of OBP-301 on theorthotopic OST bone sarcoma xenograft model. OBP-301 was injected into the tumor once a day for 3 days,with 108 PFUs per day (10). Replication-deficient adeno-virus dl312 or PBS was also injected into control groups.Tumor growth was significantly suppressed by OBP-301injection compared with injection of dl312 or PBS(Fig. 4A). Macroscopic analysis of the tumors indicatedthat OBP-301–treated tumors were consistently smallerthan dl312- or PBS-treated tumors on day 28 after treat-ment (Fig 4B). We further determined whether OBP-301–

treated tumors were less destructive to surrounding normaltissues than control tumors, using X-ray and histologicanalyses (Fig. 4C and D). X-ray examination revealed thatOBP-301–treated tumors resulted in less bone destructionthan dl312- or PBS-treated tumors. Histologic findingswere consistent with the X-ray results, showing that sometumor tissue had penetrated over the growth plate cartilagein dl312- and PBS-treated tumors but not in OBP-301–treated tumors.

With future clinical application in mind, we soughtto establish a suitable protocol for repeated intratumo-ral injection of OBP-301 by using an orthotopic SYO-1soft tissue sarcoma xenograft model. Doses of OBP-301that were suitable for induction of an antitumor effecton SYO-1 tumors (>108 PFUs) were determined in amanner similar to that of OST bone sarcoma cells(data not shown). OBP-301 was injected 3 times intothe tumor, with 109 PFUs and intervals of 1 day, 2 days,or 1 week between injections (Supplementary Fig. S5).A total of 3 OBP-301 injections, with intervals of 2 daysor 1 week between injections, induced a significant

Figure 4. Antitumor effect of OBP-301 in an orthotopic OST bone sarcoma xenograft model. A, athymic nude mice were inoculated intratibially withOST cells (5 � 106 cells/site). Fourteen days after inoculation (designated as day 0), OBP-301 (&) or OBP-405 (~) was injected into the tumor, with 1 � 108

PFUs on days 0, 2, and 4. PBS (*) was used as a control. Four mice were used for each group. Tumor growth was expressed as mean tumor volume � SD.Statistical significance (*) was determined as P < 0.05 (Student's t test). B, macroscopic appearance of OST tumors in nude mice on days 0 and 28 aftertreatment with PBS, dl312, or OBP-301. Tumor masses are outlined by a dotted line. C, X-ray photographs of mice bearing OST tumors. The white arrowheadsindicate the space occupied by the tumor mass. D, histologic analysis of the OST tumors. Tumor sections were obtained 28 days after inoculation of tumorcells. Paraffin-embedded sections of OST tumors were stained with H&E. The black arrowheads indicate growth plate cartilages. a, c and e, are low-magnification images and b, d and f are high-magnification images of the area outlined by a white square. Left scale bar, 5 mm. Right scale bar, 500 mm.

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suppression of tumor growth, whereas intervals of 1 daybetween injections were not effective. These results sug-gest that an interval of more than 2 days between injec-tions is necessary to efficiently suppress tumor growth byrepeated injections of OBP-301.

Antitumor effect of OBP-405 on OBP-301–resistantsarcoma cell linesOUMS-27 and NMFH-1 cells are resistant to OBP-301

because they lack CAR expression (Fig. 1A and Supplemen-tary Fig. S2). We previously developed a fiber-modifiedOBP-301, termed OBP-405, which can enter not only CAR-positive cancer cells but also CAR-negative cancer cellsthrough binding to the cell surface integrins avb3 andavb5 (31). We therefore sought to evaluate the antitumoreffect of OBP-405 on the OBP-301–resistant OUMS-27 andNMFH-1 cells. We first examined the expression levels of

the integrins avb3 and avb5 on the surface of these cells byflow cytometry (Fig. 5A). OUMS-27 and NMFH-1 cellsexpressed both integrin molecules. We next examinedthe effect of OBP-405 on OUMS-27 and NMFH-1 cellviability by using the XTT assay (Fig. 5B). OBP-405 effi-ciently suppressed cell viability of both of these cell lines ina dose- and time-dependent manner. We further assessedwhether OBP-405 has an in vivo antitumor effect by assay-ing the effect of 3 intratumoral injections of OBP-301or OBP-405, with 108 PFUs or of control PBS, into sub-cutaneous OUMS-27 tumor xenografts. As shown inFigure 5C, administration of OBP-405 resulted in signi-ficant suppression of tumor growth compared with OBP-301- or PBS-treated tumors 28 days after treatment. Theseresults suggest that fiber-modified OBP-405 is a potentialantitumor reagent that is effective against CAR-negativehuman sarcoma cells.

Figure 5. In vitro and in vivo antitumor effects of OBP-405 on OBP-301–resistant human sarcoma cell lines. A, expression of the integrins avb3 (left)and avb5 (right) on OUMS-27 and NMFH-1 cells. The cells were incubated with a monoclonal anti-avb3 integrin (LM609) or an anti-avb5 integrin(P1F6), followed by flow cytometric detection using an FITC-labeled secondary antibody. The gray histogram represents integrin antibody staining. Thenumber at the top right-hand corner of each graph is the MFI. B, cytopathic effect of OBP-405 on OUMS-27 and NMFH-1 cells. The cells were infectedwith OBP-405 at the indicated MOI values, and cell survival over 5 days was quantified using an XTT assay. C, antitumor effect of OBP-405 in a subcutaneousOUMS-27 xenograft tumormodel. Athymic nudemice were inoculated subcutaneously with OUMS-27 cells (5� 106 cells/site). Fourteen days after inoculation(designated as day 0), OBP-301 (~) or OBP-405 (&) was injected into the tumor, with 1 � 108 PFUs on days 0, 2, and 4. PBS (*) was used as acontrol. Ten mice were used for each group. Tumor growth was expressed as the mean tumor volume � SD. Statistical significance (*) was determinedas P < 0.05 (Student's t test).






Discussion

Telomerase-specific replication-selective oncolytic ade-noviruses are emerging as promising antitumor reagentsfor induction of tumor-specific cell death. We previouslyreported that OBP-301 has a strong antitumor effect on avariety of human epithelial malignant cells that have hightelomerase activity (12, 13). However, nonepithelialmalignant cells often show low telomerase activity andinsteadmaintain telomere length through anALTmechan-ism (20, 21). The effect of OBP-301 on human bone andsoft tissue sarcoma cells has not been extensively exam-ined. In this study, we showed that OBP-301 induced celldeath in 12 of 14 human bone and soft tissue sarcoma celllines (Fig. 1) and that the cytopathic activity of OBP-301significantly correlated with tumor CAR expression(Fig. 2A). Furthermore, 2 ALT-type sarcoma cells showedlow hTERT mRNA expression (Fig. 2B) but a similarsensitivity to OBP-301 compared with non–ALT-type cellsbecause of hTERT mRNA upregulation by OBP-301 infec-tion (Fig. 3). In contrast, 2 OBP-301–resistant sarcomacells that lack CAR expression were highly sensitive toOBP-405, which can infect cells by binding to surfaceintegrin molecules (Fig. 5). On the basis of these results,and with future clinical application in mind, we estab-lished a therapeutic strategy for the use of telomerase-specific oncolytic adenoviruses to treat patients with boneand soft tissue sarcomas (Fig. 6). This strategy involvesassessment of the expression levels of CAR, hTERT, andintegrins on human sarcoma cells, which would thenallow easy selection of the most effective protocol forthe treatment of patients by using oncolytic adeno-viruses. Furthermore, as OBP-301 and OBP-405 show

the profound antitumor effect in the combination ofvarious chemotherapeutic agents (43, 44), further evalua-tion for the strategy using OBP-301 and OBP-405 incombination with chemotherapy should be warranted.

The cytopathic activity of OBP-301 significantly cor-related with CAR expression, but not with telomeraseactivity, of human sarcoma cells (Fig. 2). These resultssuggest that the cytopathic activity of OBP-301 dependsprimarily on infection efficiency rather than virus replica-tion. Primary epithelial and nonepithelial malignanttumors frequently express CAR (23–30). However, CARexpression can often be downregulated by tumor progres-sion (45, 46) or under hypoxic conditions (47), possiblyleading to a low infection efficiency and resistance toOBP-301. Thus, for future clinical application of OBP-301, it may be necessary to overcome the resistance toOBP-301 that arises during tumor progression. A histonedeacetylase (HDAC) inhibitor has been previouslyshown to enhance CAR expression on human cancer cells(48–50). Therefore, for the treatment of OBP-301–resistantsarcomas, it may be necessary to either upregulate CARexpression on tumor cells in combination with an HDACinhibitor or use OBP-405 to kill tumor cells in an integrin-dependent manner (31).

ALT-type sarcoma cells that express a low level of hTERTmRNA showed sensitivity to OBP-301 that was similar tothat of non–ALT-type sarcoma cells (Figs. 1 and 2). Wefurther showed that OBP-301 infection upregulateshTERT gene expression and subsequently activates virusreplication and cytopathic activity in ALT-type sarcomacells (Fig. 3). These results suggest that the hTERT genepromoter is a useful tool for enhancement of the onco-lytic adenoviruses not only because it induces tumor-specific virus replication but also because it enhancesvirus replication after infection. Indeed, the ALT-typesarcoma SaOS-2 cells that lack hTERT gene expressionwere relatively less sensitive to OBP-301 than the otherALT-type sarcoma U2OS cells that express low levels ofhTERT mRNA (Figs. 1 and 2). We further observed thathTERT mRNA expression was not upregulated afterOBP-301 infection of SaOS-2 cells (data not shown).These results suggest that if hTERT gene expression can-not be detected in tumor cells, then ALT-type sarcomacells should be treated with high doses of OBP-301, orwith OBP-405, to enhance OBP-301 infection efficiency(Fig. 6).

It is also worth noting in terms of future clinicalapplication that an interval of more than 2 days betweeninjections is necessary in order for repeated injections ofOBP-301 to induce a strong antitumor effect in an SYO-1animal xenograft model (Supplementary Fig. S5). Wefirst expected that continuous injection of OBP-301 atintervals of 1 day, when tumors are of a minimum size,might be more effective in inducing an antitumor effectthan injection at intervals of 2 days or 1 week. Surpris-ingly, continuous injection of OBP-301 at intervals of 1day, for 3 days, could not induce an antitumor effect.There are 2 possible explanations for these results. The

Figure 6. Outline of a therapeutic strategy for the use of telomerase-specific replication-selective oncolytic adenoviruses for human bone andsoft tissue sarcoma cells. Assessment of CAR expression in tumor cellscould serve as an indicator for OBP-301 or OBP-405 treatment. Of the 14human sarcoma cell lines shown, the 12 CAR-expressing sarcoma cells(86%) should be treated with OBP-301 and the 2 sarcoma cells (14%) thatlack CAR expression should be treated with OBP-405. The tumorexpression level of hTERT mRNA would be useful in deciding the dose ofOBP-301 to be used for treatment. The expression level of integrins on thetumor surface should be confirmed prior to OBP-405 treatment.

Sasaki et al.





first possibility is that 3 days of continuous injectionsmay not provide enough time for OBP-301 to replicateand reach the minimal dose required for induction of anantitumor effect within tumor tissues. The second pos-sibility is that OBP-301 may be less effective againstmore slowly proliferating tumor cells than it is againstrapidly proliferating tumor cells because its replicationrate would be lower in the more slowly proliferatingcells. Although it remains unclear why continuous injec-tion of OBP-301 was less effective, it is clear thatrepeated infection with OBP-301 at intervals of morethan 2 days would be sufficient to exert an antitumoreffect against human sarcoma tissues.In conclusion, we have clearly shown that OBP-301 has

strong in vitro and in vivo antitumor effects against humanbone and soft tissue sarcoma cells. Telomerase-specificreplication-selective oncolytic virotherapy would providea new platform for the treatment of patients with bone andsoft tissue sarcomas.

Disclosure of Potential Conflict of Interest

Y. Urata is an employee of Oncolys BioPharma, Inc., the manufacturer ofOBP-301(Telomelysin). The other authors disclosed no potential conflictsof interest.

Acknowledgments

The authors thank Dr. Satoru Kyo (Kanazawa University) for providingthe OST, HOS, and SaOS-2 cells, Dr. Hiroyuki Kawashima (Niigata Uni-versity) for providing the NOS-2, NOS-10, NDCS-1, NMS-2, and NMFH-1cells, and Tomoko Sueishi for her excellent technical support.

Grant Support

This study was supported by grants-in-aid from the Ministry of Educa-tion, Science, and Culture, Japan (T. Fujiwara) and grants from the Ministryof Health and Welfare, Japan (T. Fujiwara).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 1, 2010; revised November 4, 2010; accepted November15, 2010; published OnlineFirst February 16, 2011.

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2011;17:1828-1838. Published OnlineFirst February 16, 2011.Clin Cancer Res Tsuyoshi Sasaki, Hiroshi Tazawa, Jo Hasei, et al. for Human Bone and Soft Tissue SarcomasPreclinical Evaluation of Telomerase-Specific Oncolytic Virotherapy

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