imagingageneticallyengineeredoncolyticvacciniavirus (glv-1h99 ... · (glv-1h99 or glv-1h68) as well...

Imaging, Diagnosis, Prognosis

Imaging a Genetically Engineered Oncolytic Vaccinia Virus(GLV-1h99) Using a Human NorepinephrineTransporter Reporter GenePeter Brader,1,7 Kaitlyn J. Kelly,2 Nanhai Chen,8,9 Yong A. Yu,8,9 Qian Zhang,8,9 Pat Zanzonico,3

Eva M. Burnazi,6 Rashid E. Ghani,4 Inna Serganova,5 Hedvig Hricak,1 Aladar A. Szalay,8,9,10

Yuman Fong,2 and Ronald G. Blasberg1,5

Abstract Purpose: Oncolytic viral therapy continues to be investigated for the treatment ofcancer, and future studies in patients would benefit greatly from a noninvasivemodality for assessing virus dissemination, targeting, and persistence. The purposeof this study was to determine if a genetically modified vaccinia virus, GLV-1h99,containing a human norepinephrine transporter (hNET) reporter gene, could besequentially monitored by [123I]metaiodobenzylguanidine (MIBG) γ-camera and [124I]MIBG positron emission tomography (PET) imaging.Experimental Design: GLV-1h99 was tested in human malignant mesothelioma andpancreatic cancer cell lines for cytotoxicity, expression of the hNET protein usingimmunoblot analysis, and [123I]MIBG uptake in cell culture assays. In vivo [123I]MIBGγ-camera and serial [124I]MIBG PET imaging was done in MSTO-211H orthotopic pleuralmesothelioma tumors.Results: GLV-1h99 successfully infected and provided dose-dependent levels of trans-gene hNET expression in human malignant mesothelioma and pancreatic cancer cells.The time course of [123I]MIBG accumulation showed a peak of radiotracer uptake at48 hours after virus infection in vitro. In vivo hNET expression in MSTO-211H pleuraltumors could be imaged by [123I]MIBG scintigraphy and [124I]MIBG PET 48 and 72 hoursafter GLV-1h99 virus administration. Histologic analysis confirmed the presence ofGLV-1h99 in tumors.Conclusion: GLV-1h99 shows high mesothelioma tumor cell infectivity and cytotoxicefficacy. The feasibility of imaging virus-targeted tumor using the hNET reportersystem with [123I]MIBG γ-camera and [124I]MIBG PET was shown in an orthotopicpleural mesothelioma tumor model. The inclusion of human reporter genes intorecombinant oncolytic viruses enhances the potential for translation to clinicalmonitoring of oncolytic viral therapy.

Malignant pleural mesothelioma and pancreatic cancer arehighly aggressive diseases. The annual incidence in the UnitedStates was estimated to be ∼40,000 cases for pancreatic cancerand ∼4,000 cases for malignant mesothelioma in the year 2004

(1). The increasing incidence of mesothelioma worldwide, es-pecially in industrialized nations, is due to the etiology of thisdisease from asbestos exposure (2). Both of these tumors arehighly resistant to current therapy, with 5-year survival rates

Authors' Affiliations: Departments of 1Radiology, 2Surgery, 3Medical Physics,4Cyclotron and Radiochemistry Core Facility, 5Nuclear Pharmacy, 6Neurologyand, Memorial Sloan-Kettering Cancer Center, New York, New York;7Department of Radiology, Division of Pediatric Radiology, MedicalUniversity Graz, Graz, Austria; 8Genelux Corp., San Diego Science Center,San Diego, California; and 9Institute for Biochemistry, Biocenter and Institutefor Molecular Infectious Biology and 10Virchow Center for BiomedicalResearch, School ofMedicine,University ofWuerzburg,Wuerzburg,GermanyReceived 12/13/08; revised 2/24/09; accepted 3/4/09; published OnlineFirst5/26/09.Grant support: NIH grants R25-CA096945 and P50 CA86438 and Depart-ment of Energy grant FG03-86ER60407. Technical services were providedby the Memorial Sloan-Kettering Cancer Center Small-Animal ImagingCore Facility, supported in part by NIH Small-Animal Imaging Research Pro-gram grant R24 CA83084 and NIH Center grant P30 CA08748.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Requests for reprints: Ronald G. Blasberg, Departments of Neurologyand Radiology, MH (Box 52), Molecular Pharmacology and ChemistryProgram, Sloan-Kettering Institute, Memorial Sloan-Kettering CancerCenter, 1275 York Avenue, New York, NY 10065. Phone: 646-888-2211;Fax: 646-422-0408; E-mail: [email protected] or Aladar A.Szalay, Institute of Biochemistry and Institute for Molecular InfectiousBiology, University of Wuerzburg, Wuerzburg, Germany and GeneluxCorp., San Diego Science Center, 3030 Bunker Hill Street, Suite 310,San Diego, CA 92109. Phone: 858-483-0024; Fax: 49-931-888-4422;E-mail: [email protected].

F 2009 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-08-3236

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of only 5% for pancreatic cancer and 9% for mesothelioma (3).Even with combined surgery, chemotherapy, and radiation,only a small minority of patients are rendered disease-free fora prolonged period of time (4).Oncolytic viral therapy has been studied and tested over

the past century, and many viral types, including adenovirus,herpes simplex virus, Newcastle disease virus, myxoma virus,vaccinia virus, and vesicular stomatitis virus, are being investi-gated as novel agents for the treatment of human cancer (5).Adenovirus H101 was approved in 2005 for the treatment ofhead and neck cancer in China (6). Importantly, virusesgenerally kill cancer cells that are high in ribonucleotidereductase, high in DNA repair enzymes, and resistant toapoptosis, characteristics that tend to make tumor cells resistantto chemotherapy and radiation therapy (7).Vaccinia viruses are particularly attractive agents for oncolytic

therapy because versions of this virus have been given tomillions of humans in the eradication of smallpox. Vacciniavirus is also an excellent vector because its large genome allowsfor insertion of multiple foreign genes (8). In addition, it ishighly immunogenic and able to induce strong host immuneresponses against virus-infected cells (9). Although the accep-tance of oncolytic viral therapy has been mixed in the medicalcommunity, a substantial amount of data has been reportedfrom clinical trials with vaccinia virus in cancer patientsshowing good safety and promising responses (9–12).Future human oncolytic viral therapy studies would benefit

greatly from the ability to track and monitor viral distribution,tumor targeting, proliferation, and persistence by noninvasiveimaging (13). It would provide important safety, efficacy, andtoxicity correlations. Such real-time tracking would also provideuseful viral dose and administration schedule information foroptimization of therapy and would obviate the need formultiple and repeated tissue biopsies. The virus used in thecurrent study is GLV-1h99. This is a recombinant vaccinia vi-rus expressing transgenes for the human norepinephrine trans-porter (hNET) and β-galactosidase. hNET is a cell-membrane

transporter that is highly expressed in many neuroendocrinetumors and can be imaged by radiolabeled metaiodobenzyl-guanidine (MIBG). The use of hNET reporter gene imaging isparticularly attractive from a clinical investigative standpointbecause (a) hNET is a human protein that should minimizeimmunogenicity and (b) MIBG can be radiolabeled with 123Ior 131I for single-photon emission computed tomography(SPECT) and γ-camera imaging and also with 124I for positronemission tomography (PET) imaging. Currently, [123I]MIBGis a clinically approved radiolabeled probe for imaging hNETexpression.In this study, we show that insertion of the hNET reporter

gene into a recombinant vaccinia virus does not alter tumorkilling; GLV-1h99 retains excellent tumor specificity and cyto-toxic efficacy. In addition, we show the feasibility of usingthe hNET reporter system for in vivo noninvasive imaging ofoncolytic viral therapy in an orthotopic pleural mesotheliomatumor model by [123I]MIBG γ-camera imaging and [124I]MIBGPET imaging.

Materials and Methods

Cell lines. Human pancreatic carcinoma cell lines PANC1, BxPC-3,HS766T, and MiaPaCa-2, the mesothelioma cell line MSTO-211H,and the human neuroblastoma cell line SK-N-SH, which expresseshNET, were obtained from the American Type Culture Collection.JMN cells were a kind gift from Dr. Frank Sirotnik (Memorial Sloan-Kettering Cancer Center, New York, NY). H2052 and H2373 cell lineswere a kind donation from Dr. Pass (Karmanos Cancer Institute,Wayne State University, Detroit, MI).

All cells were grown in appropriate medium, maintained in a humid-ified incubator at 37°C supplied with 5% CO2, and subcultured twiceweekly.

Virus strains. GLV-1h68 (non–hNET-containing virus) is a replica-tion-competent, recombinant vaccinia virus derived from the LIVPstrain (Lister strain from the Institute for Research on Virus Prepara-tions, Moscow, Russia), and its construction was previously described(14).

GLV-1h99 (hNET-expressing virus) was derived from GLV-1h68 byreplacing the Renilla luciferase-green fluorescent protein expressioncassette at the F14.5L locus with a hNET expression cassette throughin vivo homologous recombination.

Cytotoxicity assay. Cells were plated at 2 × 104 per well in 12-wellplates in 1 mL of medium per well. After incubation for 6 h, cells wereinfected with GLV-1h99 or GLV-1h68 at multiplicities of infection(MOI) of 1.0, 0.10, 0.01, and 0 (control wells). Viral cytotoxicitywas measured daily for 7 d. Cells were washed with PBS and lysedin 200 μL per well of 1.5% Triton X-100 (Sigma) to release intracel-lular lactate dehydrogenase, which was quantified with a CytoTox 96kit (Promega) on a spectrophotometer (EL321e, Bio-Tek Instruments)at 490 nm. Results are expressed as the percentage of surviving cells.This percentage was determined by comparing the measured lactatedehydrogenase of each infected sample with that in uninfected controlcells. All samples were analyzed in triplicate.

Immunoblot analysis. To evaluate the level of hNET protein expres-sion in cells (H2052, MSTO-211H, and PANC1) infected with virus(GLV-1h99 or GLV-1h68) and in the neuroblastoma cell lineSK-N-SH at 12, 24, 48, and 72 h after infection, immunoblot analysiswas done. A purified mouse antibody against hNET (NET17-1; MAbTechnologies, Inc.) was used at a final dilution of 1:500 and incubatedfor 12 h at +4°C. The secondary antibody (peroxidase-conjugated anti-mouse IgG; Vector Laboratories, Inc.) exposure was for 1 h at a 1:2,000dilution. Peroxidase-bound protein bands were visualized using the en-hanced chemiluminescence method (Amersham Pharmacia Biotech).

Translational Relevance

Oncolytic viral therapy dates back more than acentury but has had a mixed and fluctuating levelof acceptance in the medical community. Neverthe-less, it continues to be investigated and still holdspromise as a biological treatment for resistantcancers, such as malignant pleural mesotheliomaand pancreatic cancer. Future oncolytic viral trialsin patients would benefit greatly from a noninvasiveimaging modality for assessing virus dissemination,targeting, and persistence. The purpose of this studywas to determine if a genetically modified vacciniavirus, GLV-1h99, containing a human norepineph-rine transporter reporter gene, could be sequentiallymonitored by [123I]metaiodobenzylguanidineγ-camera and [124I]metaiodobenzylguanidinepositron emission tomography imaging in anorthotopic pleural mesothelioma animal model. Thisimaging paradigm could be directly translated tohuman studies.

3792Clin Cancer Res 2009;15(11) June 1, 2009 www.aacrjournals.org




In vitro radiotracer assay. [123I]MIBG radiotracer uptake studieswere done in MSTO-211H and PANC1 cells after infection with virus(GLV-1h99 or GLV-1h68) as well as in the neuroblastoma cell lineSK-N-SH using previously described methods (15). Briefly, cells wereplated at 1 × 106 per well in six-well plates in 2 mL of medium perwell. After incubation for 6 h, cells were infected with GLV-1h68 orGLV-1h99 at MOIs of 1.0 and 0 (control wells). Following 12-, 24-,48-, and 72-h incubation periods with virus at 37°C and 5% CO2,the medium was aspirated and the cells were washed with PBS (pH7.4). [123I]MIBG uptake was initiated by adding 2 mL of DME con-taining 0.0185 MBq/mL (0.5 μCi/mL) carrier-free [123I]MIBG. Cellswere harvested after a 60-min incubation period, and the cell pel-let-to-medium activity ratio (cpm/g of pellet/cpm/mL of medium)was calculated from the radioactivity measurements assayed in a

gamma counter (Packard, United Technologies). All studies weredone in triplicate.

Malignant pleural mesothelioma xenograft model. Athymic nu/nu fe-male mice were purchased from the National Cancer Institute (Bethes-da, MD) and were housed five per cage and allowed food and waterad libitum in the Memorial Sloan-Kettering Cancer Center Vivarium for1 wk before tumor cell implantation. All animal studies were donein compliance with all applicable policies, procedures, and regulatoryrequirements of the Institutional Animal Care and Use Committee,the Research Animal Resource Center of Memorial Sloan-KetteringCancer Center, and the NIH “Guide for the Care and Use of Labora-tory Animals.” All animal procedures were done under anesthesia in-duced by inhalation of 2% isoflurane. After the studies, all animalswere sacrificed by CO2 asphyxiation.

Fig. 1. GLV-1h99 (hNET-expressing virus) and GLV-1h68 (non–hNET-containing virus) cytotoxicity in PANC1 and MSTO-211H cells. A, comparativeGLV-1h99 and GLV-1h68 cytotoxicity at a MOI of 1.0. B, percentage cell survival of PANC1 and MSTO-211H cells at MOIs of 0.01, 0.10, and 1.0. C, cell lysis in% ± SD of the four mesothelioma and four pancreatic cancer cell lines at day 7 with a MOI of 0.1 and 1.0.


Imaging of GLV-1h99 Using PET and γ Camera



An incision 3 to 5 mm in length was made over the fourth to fifthintercostal space of the right chest. The underlying inflating anddeflating lung was thereby easily visualized through the thin fascia.Slowly, 100 μL of MSTO-211H malignant mesothelioma cellular sus-pension (5 × 106 cells) were injected. After the injection, the skin wasclosed with surgical staples and mice were returned to their cages.Intrapleural treatment with virus was done in a similar fashion as

described above 10 d after tumor cell instillation into the pleural cav-ity. GLV-1h99 or GLV-1h68 (1 × 107 plaque-forming units) was ad-ministered in 100 μL PBS and animals were gently rotated from sideto side to help distribute the virus throughout the pleural cavity. Con-trol animals (no virus) received only 100 μL PBS.

MIBG synthesis. Clinical-grade [123I]MIBG was obtained from MDSNordion. The average radiochemical purity was in excess of 97%(determined by MDS Nordion using the Sep Pak cartridge method),and the specific activity was ∼320 MBq/μmol (8.7 mCi/μmol) accord-ing to the vendor.

[124I]MIBG was prepared using minor modifications to the reportednucleophilic isotopic exchange method (16), following a procedure pre-viously reported by Moroz and coworkers (17). The radiochemical pu-rity of the final product was >95% with an overall yield of >80% andthe specific activity was 18.5 ± 5.2 MBq/μmol (0.5 ± 0.14 mCi/μmol).The maximum specific activities (no carrier-added synthesis) for the123I- and 124I-labeled compounds were 8.9 and 1.2 TBq/μmol (241and 33 Ci/μmol), respectively, due to the 7.4-fold difference in thedecay rate of the two isotopes.

Clinical-grade [18F]fluorodeoxyglucose (FDG) was obtained fromIBA Molecular with a specific activity of >41 MBq/μmol (>11 mCi/μmol) and a radiochemical purity of >98%.

[123I]MIBG γ-camera in vivo imaging. Each animal was injectedi.v. with ∼18.5 MBq (500 μCi) of [123I]MIBG 48 h after intrapleuralGLV-1h99 injection and imaged on a X-SPECT dedicated small-animalγ-camera SPECT-CT scanner (GammaMedica). A photopeak energywin-dow of 143 to 175 keV and a low-energy high-resolution parallel-hole

Fig. 2. Immunoblot analysis in PANC1 and MSTO-211H cells. A, hNET expression in SK-N-SH cells as well as in H2052, MSTO-211H, and PANC1 cells24 h after GLV-1h99 (hNET-expressing virus) infection at a MOI of 1.0. B, hNET expression in MSTO-211H cells at various time points after GLV-1h99 infectionat a MOI of 1.0. hNET expression 24 h after GLV-1h99 infection at MOIs of 0.1, 1.0, 5, and 10 in MSTO-211H (C) and PANC1 (D) cells (immunoblots onA were not run on the same gel but were normalized to β-actin expression; the immunoblots on B-D were run on the same gel).





collimator was used to acquire 10-min 123I images at 2 h after [123I]MIBG administration.The X-SPECT γ-camera system was calibrated by imaging a mouse-

size (30 mL) cylinder filled with an independently measured concen-tration (MBq/mL) of 99mTc using a photopeak energy window of 126to 154 keV and low-energy high-resolution collimation. The resulting99mTc images were exported to Intefile and then imported into theASIPro (Siemens Pre-clinical Solutions) image processing softwareenvironment. By region of interest analysis, a system calibration factor(in cpm/pixel per MBq/mL) was derived. Animal images were likewiseexported to Intefile and then imported into ASIPro and parameterizedin terms of the decay-corrected percentage injected dose per gram(%ID/g) based on the foregoing calibration factor, the administeredactivity, the time after administration of imaging, and the imageduration. Implicit in the foregoing analysis is the reasonable assump-tion that the sensitivities of the X-SPECT γ-camera system for 123I and99mTc are comparable.

[124I]MIBG micro-PET in vivo imaging. In a group of five animals(10 d after MSTO-211H tumor cell instillation into the pleural cavity),each animal was injected via the tail vein with 9.25 MBq (250 μCi) of[18F]FDG. [18F]FDG PET scanning was done 1 h after tracer adminis-tration using a 10-min list-mode acquisition. Animals were fasted 12h before tracer administration and kept under anesthesia betweenFDG injection and imaging.In a group of 16 animals, 4 subgroups of 3 to 5 animals each were

studied (5 animals in subgroups 1 and 2; 3 animals in subgroups 3 and4). Each animal was injected via the tail vein with 9.25 MBq (250 μCi)

of [124I]MIBG. Animals in subgroups 1 and 2 were injected with GLV-1h99 48 and 72 h before [124I]MIBG administration. Subgroup 3 ani-mals received GLV-1h68 48 h before radiotracer administration;subgroup 4 animals were not injected with virus, receiving only100 μL PBS. Potassium iodide was used to block the uptake of radio-active iodine by the thyroid. [124I]MIBG PET was done for 10 min, 1 h,2 h, and 4 h after tracer administration, for 15 min at 12 h, for 30 minat 24 h, and for 60min at 48 h. After tracer administration and betweenimaging time points, the animals were allowed to wake up andmaintain normal husbandry.Imaging was done using a Focus 120 micro-PET dedicated small-

animal PET scanner (Concorde Microsystems, Inc.). Three-dimension-al list-mode data were acquired using an energy window of 350 to 700keV for 18F and 410 to 580 keV for 124I, respectively, and a coincidencetiming window of 6 ns. These data were then sorted into two-dimen-sional histograms by Fourier rebinning. The image data were correctedfor (a) nonuniformity of scanner response using a uniform cylindersource-based normalization, (b) dead time count losses using asingle-count rate-based global correction, (c) physical decay to the timeof injection, and (d) the 124I branching ratio. The count rates in the re-constructed images were converted to activity concentration (%ID/g)using a system calibration factor (MBq/mL per cps/voxel) derived fromimaging of a mouse-size phantom filled with a uniform aqueoussolution of 18F.Image analysis was done using ASIPro. At all acquired scans

(including [18F]FDG PET, serial [124I]MIBG PET, and [123I]MIBGγ-camera), regions of interest were manually drawn over tumor,

Fig. 3. In vitro [123I]MIBG uptake in PANC1 and MSTO-211H cells. [123I]MIBG uptake in MSTO-211H (A) and PANC1 (B) cells at various times after infectionwith GLV-1h99, 48 h after infection with GLV-1h68 (non–hNET-containing virus), and in SK-N-SH neuroblastoma cells. Corresponding total cell proteinfrom [123I]MIBG uptake in MSTO-211H (C) and PANC1 (D).





lung, liver, and skeletal muscle. For each tissue and time point afterinjection, the measured radioactivity was expressed as %ID/g. Themaximum %ID/g value was recorded for each tissue, and from these,tumor-to-organ ratios for lung, liver, and skeletal muscle were thencalculated.

Immunohistochemistry. After the final image, the animals were sacri-ficed and the tumors were harvested and frozen in Tissue-Tek OCT com-pound (Sakura Finetek USA, Inc.). Tissues were cut into 5-μm-thicksections andmounted on glass slides. Cryosections were fixed and stainedwith H&E and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

Fig. 4. [124I]MIBG and [18F]FDG PET imaging of MSTO-211H pleural tumors. A, axial, coronal, and sagittal views of [124I]MIBG PET 48 h after GLV-1h99(hNET-expressing virus) injection (images at 2 and 4 h after radiotracer administration) and a pretreatment [18F]FDG PET (images at 1 h after traceradministration). B, tumor radioactivity (%ID/g) of tumors injected with GLV-1h99 48 and 72 h before radiotracer administration (red and orange squares) aswell as injected with GLV-1h68 (non–hNET-containing virus) or no virus (light and dark blue symbols, respectively). C, photograph of a MSTO-211H pleuraltumor; the white plaques on the lung surface and chest wall are malignant pleural mesothelioma (the heart is removed in this photograph).





(1 mg/mL) in an iron solution of 5 mmol/L K4Fe(CN)6, 5 mmol/L K3Fe(CN)6, and 2 mmol/L MgCl2, as previously described (18), to identifyvirally mediated lacZ expression.

Statistics. A two-tailed unpaired t test was applied to determinethe significance of differences between values using the MS Office2003 Excel 11.0 statistical package (Microsoft).

Results

Cytotoxicity assays in vitro showed dose-dependent lytic activity.Four mesothelioma and four pancreatic cancer cell linesshowed lytic cytotoxicity following exposure to GLV-1h99(hNET-expressing virus) and to GLV-1h68 (non–hNET-con-taining virus). Similar cytotoxicity was observed with GLV-1h99 and GLV-1h68 at a MOI of 1.0 (Fig. 1A) and a dose-dependent lytic effect was also shown (Fig. 1B). At a MOI of0.1, all MSTO-211H and H2052 mesothelioma cells as wellas 80% of the PANC1 pancreatic cancer cells were dead atday 7. Oncolysis seemed to be more gradual over time inPANC1 cells compared with the more sigmoidal lytic timeprofile in MSTO-211H cells (Fig. 1). The mesothelioma cellline JMN and the pancreatic cancer cell line HS766T weremore resistant and showed only 80% cell death by day 7at a MOI of 1.0 (Fig. 1C). MiaPaCa2 and BxPC3 (pancreaticcancer cell lines) and H2373 (mesothelioma cell line) were

sensitive to the virus only at a higher MOI of 10 (data notshown).

Immunoblot analysis confirmed dose-dependent levels of trans-gene hNET expression. The two most sensitive mesotheliomacell lines (MSTO-211H and H2052) and the most sensitivepancreatic cancer cell line (PANC1), based on the cytotoxicityassays, were chosen for immunoblot analysis and comparedwith the endogenous hNET-expressing neuroblastoma cell line,SK-N-SH. The levels of hNET expression 24 hours after GLV-1h99 (hNET-expressing virus) viral infection at a MOI of 1.0were investigated (Fig. 2A). In addition to the ∼80-kDa hNETband, two low-molecular weight immunoreactive bands (∼50-55 kDa and ∼37-40 kDa, respectively) are seen in the blots ofthe GLV-1h99–infected cells; these two low-molecular weightbands are barely visible in the blots of the SK-N-SH neuroblas-toma cells. Similar to the cytotoxicity assay, there was a viraldose-dependent expression of hNET at different MOIs (0.1,1.0, 5, and 10; shown for MSTO-211H cells in Fig. 2B). StronghNET expression was found in the MSTO-211H and PANC1cell lines by 12 hours after GLV-1h99 viral infection, peakingat 24 hours followed by a gradual decline over 72 hours(shown for MSTO-211H cells in Fig. 2C and D). A similar pat-tern of hNET expression was observed in the other cell lines,although the hNET immunoblot bands were less intense (datanot shown). The neuroblastoma cell line (SK-N-SH), expressing

Fig. 5. Tumor-to-organ radioactivity ratios. Tumor-to-organ (lung, liver, and muscle) ratios calculated from PET image data (as described in Materialsand Methods) were obtained 1, 2, 4, and 12 h after [124I]MIBG injection. GLV-1h99 (hNET-expressing virus) injected 48 and 72 h before radiotraceradministration as well as GLV-1h68 (non–hNET-containing virus) injected 48 h before radiotracer administration and no virus studies at 1, 2, 4, and 12 h after[124I]MIBG injection are presented and are compared with 1-h [18F]FDG studies.





endogenous hNET, served as a positive control for the immu-noblot analysis and radiotracer uptake studies; the GLV-1h68virus-infected (non–hNET-containing virus) and the uninfectedmesothelioma and pancreatic cancer cell lines served as nega-tive controls (Fig. 2B).In vitro [123I]MIBG uptake showed peak of radiotracer uptake

48 hours after virus infection. The time course of [123I]MIBGaccumulation was studied in PANC1 and MSTO-211H cells fol-lowing infection with the GLV-1h99 (hNET-expressing virus)virus at a MOI of 1.0. [123I]MIBG accumulation in noninfectedMSTO-211H and PANC1 cells was low (Fig. 3A and B, respec-tively). There was no significant increase in radiotracer uptake24 hours after infection of the cells with GLV-1h68 (non–hNET-containing virus, negative control). In contrast, therewas a significant (P < 0.01) increase in [123I]MIBG accumula-tion in both cancer cell lines at all time points (12, 24, 48,and 72 hours) after infection with GLV-1h99 (Fig. 3A and B).Peak radiotracer uptake was observed at 48 hours after virusinfection in both cell lines. The natural hNET-expressing neuro-blastoma cell line (SK-N-SH) served as a positive control. Totalcell protein in the [123I]MIBG uptake assays was unchangedover the first 24 hours following GLV-1h99 infection comparedwith uninfected cells. At 48 and 72 hours after viral infection,there was a decrease in measured cell protein (Fig. 3C and D).

hNET expression imaging by [123I]MIBG scintigraphy and[124I]MIBG PET. Following direct injection of hNET-expressingGLV-1h99 virus into MSTO-211H orthotopic pleural tumors,viral localization was visualized by [124I]MIBG PET imagingof hNET expression in pleural tumors (Fig. 4A). [124I]MIBGwas i.v. administered 48 or 72 hours after intrapleural virus in-jection, and sequential PET imaging was done 1 to 48 hoursafter radiotracer administration. Tumor radioactivity values(%ID/g) were measured and tumor-to-organ ratios were calcu-lated. The highest levels of radioactivity in the pleural tumorswere found 48 hours after injection of GLV-1h99 (hNET-expres-sing virus) followed by tumors that were injected with GLV-1h99 72 hours before [124I]MIBG administration. Low levelsof radioactivity were observed in tumors that were injected with

GLV-1h68 (non–hNET-containing virus) and in tumors thatwere not injected with virus (Fig. 4B). Maximum activity inboth the pleural tumors and remote organs (background) wasobserved at the time of the initial measurement, 1 hour afterradiotracer administration. Tumor and remote organ activitydecreased over time (1-72 hours) in all four groups of animals.The decrease in tumor activity was more rapid over the first 12hours after [124I]MIBG administration in the two controlgroups: tumors injected with GLV-1h68 (non–hNET-containingvirus) or no virus.Tumor-to-organ (lung, liver, and muscle) ratios were calculat-

ed from the PET image data (as described in Materials andMethods) and the highest values were obtained for the groupof animals that were infected with GLV-1h99 (hNET-expressingvirus) 48 hours before radiotracer administration (Fig. 5). Com-paring the animals that were treated with GLV-1h99 48 hoursbefore [124I]MIBG administration with the animals that re-ceived no virus, the ratio differences were highly significant(P < 0.01) at the 2-hour imaging time point and significant(P < 0.05) at the 1-hour imaging time point. Nearly the samelow tumor-to-organ ratios were found for the two controlgroups of animals and the tumor-to-organ ratios decreased overtime.For localization of the tumors and for comparison with a

clinically used imaging technique, [18F]FDG PET imaging wasalso done. [124I]MIBG PET and [18F]FDG PET imaging werecompared (Figs. 4 and 5). The pleural tumors were visualizedby [18F]FDG PET imaging, but image contrast at 48 and 72hours after GLV-1h99 virus (hNET-expressing virus) injectionwas greater with [124I]MIBG PET compared with [18F]FDGPET. The [124I]MIBG and [18F]FDG tumor-to-lung, tumor-to-liver, and tumor-to-muscle ratios in control animals weresimilar.In vivo hNET expression in the pleural tumors after GLV-1h99

(hNET expressing) virus administration could also be imagedby [123I]MIBG planar scintigraphy. All GLV-1h99–injected ani-mals showed localized accumulation of [123I]MIBG radioactiv-ity in the virus-injected pleural tumors compared with the

Fig. 6. [123I]MIBG scintigraphy ofMSTO-211H pleural tumors.A, photograph of aMSTO-211H pleural tumor-bearingmouse.B, [123I]MIBG scintigrams (2 h aftertracer administration). TheGLV-1h99 virus-injected tumor is visualized (oval outline); the noninjected control tumor is not visualized.C, the tumor-to-backgroundradioactivity ratios were measured in the scintigrams. Orange column, GLV-1h99–injected tumors; blue column, noninjected control tumors.





control animals that received no virus (Fig. 6). The tumor-to-background ratios for the GLV-1h99–infected animals werewith 2.4 ± 0.2, significantly (P < 0.01) higher compared withthe group that received no virus, 1.5 ± 0.1.

Immunohistochemistry confirmed viral presence in tumors. Allanimals were sacrificed and examined to confirm the presenceof pleural tumors. All pleural lesions were shown to be malig-nant pleural mesothelioma on H&E staining (Fig. 7A and B). Inaddition, all tumors infected with vaccinia virus stained positivefor lacZ, confirming the presence of the virus in tumors andindicating that all tumors visualized by [124I]MIBG PET or[123I]MIBG scintigraphy reflect GLV-1h99 expression of a func-tional hNET transporter protein (Fig. 7C and D).

Discussion

Oncolytic viral therapy dates back more than a century (19)and has had a mixed and fluctuating level of acceptance in themedical community. Nevertheless, it still holds promise as abiological treatment for some standard therapy–resistant can-cers (11). Several viruses (e.g., adenovirus, herpes simplex virus,Newcastle disease virus, myxoma virus, vaccinia virus, and ve-sicular stomatitis virus) have been shown to infect and replicatein cancer cells and to selectively kill them (oncolysis). Oncolyticviral therapy has been used in cancer treatment and has evolvedfrom the use of wild-type viruses to genetically engineeredviruses that express therapeutic transgenes. Clinical and preclin-ical trials involving different viral strains and constructs haveshown to be safe and to have potent antitumor effects (10).Oncolytic viruses have also shown enhanced efficacy involvingcombination regimens with approved chemotherapeutics andradiotherapy (20). Two oncolytic viruses (G207 and H101)

have entered randomized phase III clinical testing (10), andmarketing approval was obtained for H101 in 2005 (6). Futurestudies are likely to focus on optimization of viral doses andadministration routes, interaction with the immune system,and in vivo monitoring through imaging. The ability to nonin-vasively and repetitively identify anatomic sites of viral target-ing and to measure the magnitude of viral infection couldprovide important safety, efficacy, and toxicity informationduring clinical studies of viral oncolysis.Vaccinia virus is perhaps the most widely administered

medical product in history; it is certainly the most successfulbiological product. Vaccinia also displays many of the qualitiesthought necessary for an effective antitumor agent and it is par-ticularly well characterized in humans due to its role in theeradication of smallpox. Vaccinia has a short life cycle andspreads rapidly; it has inherent systemic tumor targeting, a highpropensity to induce cell lysis, well-defined biology, and a largecloning capacity (12). The large insertional cloning capacityallows for the inclusion of several functional and therapeutictransgenes. With the insertion of reporter genes not expressedin uninfected cells, viruses can be localized and the course ofviral therapy can be monitored. A noninvasive, clinicallyapplicable method for imaging viruses in target tissue or specif-ic organs of the body would be of considerable value duringoncolytic viral therapy in patients.In this study, we describe the use of a genetically modified

vaccinia virus, GLV-1h99, which has been engineered for spe-cific targeted treatment of cancer and for noninvasive imag-ing. GLV-1h99 is able to efficiently infect, replicate in, andlyse a variety of human pancreatic and mesothelioma cancercell lines. The oncolytic potency of GLV-1h99 was shownto be similar to the non–hNET-containing parent virus,

Fig. 7. Immunohistochemistry H&E staining(A and B) confirms the histologic diagnosis ofmalignant pleural mesothelioma. lacZ staining(C and D) shows vaccinia viral infection of thetumor cells.





GLV-1h68, in eight pancreatic and mesothelioma cancer celllines. GLV-1h68 has also been shown to successfully treat anorthotopic animal model of mesothelioma with pleuraldisease (21).The reporter gene chosen for insertion into GLV-1h99 was

based on the very favorable PET and SPECT imaging character-istics of the hNET-MIBG reporter imaging system (17) and be-cause [123I]MIBG is an approved radiopharmaceutical forclinical imaging of neuroendocrine tumors (22, 23). In con-trast to a study published by McCart et al. (24) using an on-colytic vaccinia virus expressing the human somatostatinreceptor SSTR2, hNET is a transporter-based reporter gene sys-tem. Receptors usually have a 1:1 binding relationship with aradiolabeled ligand; transporters provide signal amplificationthrough transport-mediated concentrative intracellular accu-mulation of the radiolabeled substrate. hNET is a transmem-brane protein that mediates the transport of norepinephrine,dopamine, and epinephrine across the cell membrane (25).It is one of several human reporter genes that are currentlybeing used in preclinical studies (17, 26) and has a high po-tential for rapid translation into clinical reporter gene imagingstudies (27, 28).The hNET immunoblots showed protein expression in all

infected cell lines and the expression was time and dose de-pendent. The antibody used recognizes a degraded or lessglycosylated form of the protein (∼50-55 kDa and ∼37-40kDa) as well as a more highly glycosylated hNET protein(∼80 kDa). Interestingly, the low-molecular weight bandswere more intense early (12-24 hours) after GLV-1h99 infec-tion compared with later time points (48-72 hours; Fig. 2).These bands also appear in SK-N-SH neuroblastoma cells andin hNET-transduced cell lines (17) but at much lower inten-sity. We suspect that the prominence of the low-molecularweight bands is the effect of viral infection, replication, andlysis and that the low-molecular weight immunoreactive pro-tein may be nonfunctional with respect to MIBG transportand accumulation.In vitro [123I]MIBG uptake studies also showed time-depen-

dent radiotracer uptake, peaking at 24 to 48 hours after viralinfection (∼5-fold above control) for MSTO-211H cells. Theuptake levels were lower in the GLV-1h99–infected cells com-pared with the hNET-expressing neuroblastoma cells. In the vi-ral-treated cultures, it is likely that not all of the cells areinfected with virus and therefore not all express the reportergene during the early phase of viral infection. In addition, thehNET protein may not have been translocated and inserted intothe cell membrane to form a functional transporter during theinitial 12- to 24-hour period after infection. During the late,prelytic phase of viral infection (72 hours and beyond), thehNET transporter could be impaired, and following cell lysis,the accumulated MIBG radiotracer would be lost. Thus, thereseems to be a relatively narrow window, ∼24 to 48 hoursafter viral infection of MSTO-211H cells, during which thehNET reporter is maximally functional. These results reflectthe dynamic state between viral infection, replication, and lysisof tumor cells.This dynamic state of viral infectivity and functional hNET

expression was also observed in the in vivo imaging studies. Itshould be noted that the whole tumor is only partially infectedwith virus and tumor cells are at different stages of virus infec-tion at any given time (shown by immunohistochemistry).

Timing of [124I]MIBG PET imaging after GLV-1h99 virus injec-tion was very important in the in vivo studies. Better imagingresults were obtained at 48 hours compared with 72 hours afterviral injection. MIBG uptake in GLV-1h99–infected cells, bothin vitro and in vivo, is not exactly comparable with MIBG uptakein SK-N-SH neuroblastoma cells or in cells transduced withconstitutive hNET expression cassettes (17) where expressionlevels are constant. Nevertheless, the quantitative [124I]MIBGPET and [123I]MIBG SPECT studies showed that imaging ofGLV-1h99 viral infection of MSTO-211H pleural tumors isfeasible after direct tumor injection.Similarly, the timing of PET imaging after [124I]MIBG i.v.

injection was also shown to be important. Radioactivity levels(% dose/cm3) as well as tumor-to-organ ratios in GLV-1h99–infected tumors were highest during the first 4-hour period aftertracer administration. This differs from the findings of Moroz etal. (17) in a xenograft model, where constitutive expression ofthe hNET reporter occurred in all tumor cells and optimal im-aging results were obtained at late time points (48 and 72 hoursafter tracer administration). This time-dependent differenceprobably reflects the effect of increasing cell death resultingfrom viral oncolysis after [124I]MIBG injection. Oncolysis willresult in a loss of [124I]MIBG from the infected tumor cellsand tumor, consistent with the rapid decrease in the PET signalafter 4 hours (Fig. 4B). A similar pattern was observed in thein vitro immunoblot analysis and [123I]MIBG uptake studies,showing decreasing hNET expression and radiotracer uptakeat later time points after GLV-1h99 infection (Fig. 3B).Pancreatic cancer and malignant pleural mesothelioma re-

main largely unresponsive to standard treatments and are rap-idly fatal diseases in most cases. Thus, alternate treatmentoptions must be considered. Preclinical studies have shownthe efficacy of oncolytic herpes simplex viruses in treatmentof pancreatic cancer (29) and malignant mesothelioma (4).Oncolytic viral therapy has also been shown to be synergisticwith radiation and chemotherapy (30, 31). Kelly et al. (21)have shown effective killing in malignant mesothelioma celllines and xenografts using a genetically modified oncolyticvaccinia virus, GLV-1h68, the parent virus of GLV-1h99.

Conclusions

We have shown cytotoxic efficacy in vitro and tumor-specificimaging following GLV-1h99 infection of an orthoptic meso-thelioma tumor model. GLV-1h99 expresses the hNET humanreporter gene, which was imaged with a clinically approved ra-diopharmaceutical, [123I]MIBG, and with a positron-emittinganalogue, [124I]MIBG. This imaging paradigm could be directlytranslated to human studies, and clinical trials of oncolyticviral therapy would benefit from this noninvasive imagingparadigm.

Disclosure of Potential Conflicts of Interest

N. Chen, Y.A. Yu, Q. Zhang, and A.A. Szalay are employees of GeneluxCorp., the producer and patent holder of the genetically modified vacciniaviruses, GLV-1h99 and GLV-1h68, used in this study.

Acknowledgments

We thank Dr. Steven Larson (Memorial Sloan-Kettering Cancer Center)for his help and support.





References1. Soliman H, Agresta SV. Current issues in ado-lescent and young adult cancer survivorship.Cancer Control 2008;15:55–62.

2. Bianchi C, Bianchi T. Malignant mesothelioma:global incidence and relationship with asbestos.Ind Health 2007;45:379–87.

3. Jemal A, Siegel R, Ward E, et al. Cancer statis-tics, 2008. CA Cancer J Clin 2008;58:71–96.

4. Adusumilli PS, Stiles BM, Chan MK, et al. Imag-ing and therapy of malignant pleural mesothelio-ma using replication-competent herpes simplexviruses. J Gene Med 2006;8:603–15.

5. Woo Y, Adusumilli PS, Fong Y. Advances inoncolytic viral therapy. Curr Opin InvestigDrugs 2006;7:549–59.

6. Yu W, Fang H. Clinical trials with oncolytic ade-novirus in China. Curr Cancer Drug Targets 2007;7:141–8.

7. Adusumilli PS, Chan MK, Chun YS, et al. Cis-platin-induced GADD34 upregulation potentiatesoncolytic viral therapy in the treatment of malig-nant pleural mesothelioma. Cancer Biol Ther2006;5:48–53.

8. McFadden G. Poxvirus tropism. Nat Rev Micro-biol 2005;3:201–13.

9. Thorne SH, Hwang TH, Kirn DH. Vaccinia virusand oncolytic virotherapy of cancer. Curr OpinMol Ther 2005;7:359–65.

10. Liu TC, Galanis E, Kirn D. Clinical trial resultswith oncolytic virotherapy: a century of promise,a decade of progress. Nat Clin Pract Oncol 2007;4:101–17.

11. Liu TC, Kirn D. Systemic efficacy with oncolyticvirus therapeutics: clinical proof-of-concept andfuture directions. Cancer Res 2007;67:429–32.

12. Thorne SH, Bartlett DL, Kirn DH. The use ofoncolytic vaccinia viruses in the treatment ofcancer: a new role for an old ally? Curr GeneTher 2005;5:429–43.

13. Brader P, Stritzker J, Riedl CC, et al. Escheri-chia coli Nissle 1917 facilitates tumor detection

by positron emission tomography and opticalimaging. Clin Cancer Res 2008.

14. Zhang Q, Yu YA, Wang E, et al. Eradica-tion of solid human breast tumors in nudemice with an intravenously injected light-emitting oncolytic vaccinia virus. Cancer Res2007;67:10038–46.

15. Che J, Doubrovin M, Serganova I, Ageyeva L,Zanzonico P, Blasberg R. hNIS-IRES-eGFP dualreporter gene imaging. Mol Imaging 2005;4:128–36.

16. Eersels J, Herscheid J. Manufacturing I-123-labelled radiopharmaceuticals: pitfalls and so-lutions. J Labelled Compds Radiopharm 2005;48:241–57.

17.Moroz MA, Serganova I, Zanzonico P, et al. Im-aging hNET reporter gene expression with 124I-MIBG. J Nucl Med 2007;48:827–36.

18. Kelly K, Brader P, Rein A, et al. Attenuated mul-timutated herpes simplex virus-1 effectivelytreats prostate carcinomas with neural invasionwhile preserving nerve function. FASEB J 2008;22:1839–48.

19. Dock G. Influence of complicating diseasesupon leukemia. AM J Med Sci 1904;127:563–92.

20. Khuri FR, Nemunaitis J, Ganly I, et al. A con-trolled trial of intratumoral ONYX-015, a selec-tively-replicating adenovirus, in combinationwith cisplatin and 5-fluorouracil in patients withrecurrent head and neck cancer. Nat Med 2000;6:879–85.

21. Kelly KJ, Woo Y, Brader P, et al. Novel oncoly-tic agent GLV-1h68 is effective against malignantpleural mesothelioma. Hum Gene Ther 2008;19:774–82.

22. Sisson JC, Frager MS, Valk TW, et al. Scinti-graphic localization of pheochromocytoma. NEngl J Med 1981;305:12–7.

23. Valk TW, Frager MS, Gross MD, et al. Spec-trum of pheochromocytoma in multiple endo-crine neoplasia. A scintigraphic portrayal using

131I-metaiodobenzylguanidine. Ann Intern Med1981;94:762–7.

24. McCart JA, Mehta N, Scollard D, et al. Oncoly-tic vaccinia virus expressing the human somato-statin receptor SSTR2: molecular imaging aftersystemic delivery using 111In-pentetreotide. MolTher 2004;10:553–61.

25. Pacholczyk T, Blakely RD, Amara SG. Expres-sion cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter. Na-ture 1991;350:350–4.

26. Serganova I, Ponomarev V, Blasberg R. Hu-man reporter genes: potential use in clinicalstudies. Nucl Med Biol 2007;34:791–807.

27. Mullen CA, Petropoulos D, Lowe RM. Treat-ment of microscopic pulmonary metastases withrecombinant autologous tumor vaccine expres-sing interleukin 6 and Escherichia coli cytosinedeaminase suicide genes. Cancer Res 1996;56:1361–6.

28. Verzeletti S, Bonini C, Marktel S, et al. Herpessimplex virus thymidine kinase gene transfer forcontrolled graft-versus-host disease and graft-versus-leukemia: clinical follow-up and im-proved new vectors. Hum Gene Ther 1998;9:2243–51.

29.McAuliffe PF, JarnaginWR, Johnson P, DelmanKA, Federoff H, Fong Y. Effective treatment ofpancreatic tumors with two multimutated herpessimplex oncolytic viruses. J Gastrointest Surg2000;4:580–8.

30. Adusumilli PS, Chan MK, Hezel M, et al. Radi-ation-induced cellular DNA damage repair re-sponse enhances viral gene therapy efficacy inthe treatment of malignant pleural mesothelio-ma. Ann Surg Oncol 2007;14:258–69.

31. Eisenberg DP, Adusumilli PS, Hendershott KJ,et al. 5-Fluorouraciland gemcitabine potentiatethe efficacy of oncolytic herpes viral gene thera-py in the treatment of pancreatic cancer. J Gas-trointest Surg 2005;9:1068–77, discussion 77–9.





2009;15:3791-3801. Clin Cancer Res Peter Brader, Kaitlyn J. Kelly, Nanhai Chen, et al. Reporter Gene(GLV-1h99) Using a Human Norepinephrine Transporter Imaging a Genetically Engineered Oncolytic Vaccinia Virus

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