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`CancerTherapy: Preclinical
`
`Efficacy of Systemically Administered Oncolytic Vaccinia
`Virotherapy for Malignant Gliomas Is Enhanced by Combination
`Therapy with Rapamycin or Cyclophosphamide
`Xue Qing Lun,1,2 Ji-Hyun Jang,3 Nan Tang,3 Helen Deng,3 Renee Head,3 John C. Bell,6 David F. Stojdl,7
`Catherine L. Nutt,8,9 Donna L. Senger,1,2 Peter A. Forsyth,1,2 and J. Andrea McCart3,4,5
`
`Abstract Purpose: The oncolytic effects of a systemically delivered, replicating, double-deleted vaccinia
`virus has been previously shown for the treatment of many cancers, including colon, ovarian,
`and others.The purpose of this study was to investigate the oncolytic potential of double-deleted
`vaccinia virus alone or in combination with rapamycin or cyclophosphamide to treat malignant
`gliomas in vitro and in vivo.
`Experimental Design: Rat (RG2, F98, C6) and human (A172, U87MG, U118) glioma cell
`lines were cultured in vitro and treated with live or UV-inactivated vaccinia virus. Viral gene
`[enhanced green fluorescent protein (EGFP)] expression by fluorescence-activated cell
`sorting, relative cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
`(MTT), and assays for cytopathic effects were examined. S.c. murine tumor xenografts
`(U87MG, U118, C6) and i.c. (RG2, F98) tumor models in immunocompetent rats were treated
`with systemic administration of EGFP-expressing vaccinia virus (vvDD-EGFP), alone or in
`combination with rapamycin or cyclophosphamide, or controls. Tumor size, viral biodistribution,
`and animal survival were assessed. Lastly, the oncolytic effects of vvDD-EGFP on human malig-
`nant glioma explants were evaluated.
`Results: vvDD-EGFP was able to infect and kill glioma cells in vitro. A single systemic dose
`of vvDD-EGFP significantly inhibited the growth of xenografts in athymic mice. Systemic delivery
`of vvDD-EGFP alone was able to target solitary and multifocal i.c. tumors and prolong survival of
`immunocompetent rats, whereas combination therapy with rapamycin or cyclophosphamide
`enhanced viral replication and further prolonged survival. Finally, vvDD-EGFP was able to infect
`and kill ex vivo primary human malignant gliomas.
`Conclusions: These results suggest that vvDD-EGFP is a promising novel agent for human
`malignant glioma therapy, and in combination with immunosuppressive agents, may lead to
`prolonged survival from this disease.
`
`There has been minimal
`in the median
`improvement
`survival of patients with malignant gliomas, and these
`tumors remain largely incurable despite advances in surgery,
`radiation, and chemotherapy (1). The need for new
`treatment strategies for malignant gliomas has led to the
`emergence of oncolytic virotherapy, oncolytic viruses able
`to selectively destroy tumor cells while sparing normal
`tissue. Much effort to date has been achieved in preclinical
`
`models of malignant gliomas using several oncolytic viruses,
`including herpes virus (2), adenovirus (3),
`reovirus (4),
`poliovirus (5), myxoma virus (6), and vesicular stomatitis
`virus (7). Some of these have already been tested in early
`clinical trials (8 – 10), and a small number of responses were
`found. Efficacy may be limited by factors such as the host
`immune response against the virus (11, 12) and limitations
`of systemic delivery in brain tumor patients, which may
`
`Authors’ Affiliations: 1Departments of Oncology, Clinical Neurosciences,
`Biochemistry and Molecular Biology, Tom Baker Cancer Centre; 2Clark H. Smith
`Brain Tumor Center, University of Calgary, Alberta, Canada; 3Division of
`Experimental Therapeutics, Toronto General Research Institute; 4Department of
`Surgery, Mount Sinai Hospital; 5University of Toronto, Toronto, Ontario, Canada;
`6Centre for Cancer Therapeutics, Ottawa Health Research Institute; 7Apoptosis
`Research Centre, Children’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada;
`8Department of Pathology, Massachusetts General Hospital; and 9Harvard Medical
`School, Boston, Massachusetts
`Received 9/9/08; revised12/8/08; accepted12/9/08; published OnlineFirst 4/7/09.
`Grant support: National Cancer Institute of Canada with funds raised by the
`Canadian Cancer Society (P.A. Forsyth and D.L. Senger); a Program Project Grant
`from theTerry Fox Foundation (P.A. Forsyth, D.L. Senger, J.C. Bell, and D.F. Stojdl);
`the Clark Smith BrainTumor Center (P.A. Forsyth); a Grant Miller Cancer Research
`
`Grant from the Faculty of Medicine, University of Toronto (J.A. McCart); and
`commercial support from Jennerex (D.F. Stojdl and J.C. Bell).
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance
`with 18 U.S.C. Section 1734 solely to indicate this fact.
`Note: Supplementary data for this article are available at Clinical Cancer Research
`Online (http://clincancerres.aacrjournals.org/).
`P.A. Forsyth and J.A. McCart share senior authorship.
`Requests for reprints: J. Andrea McCart, Division of Experimental Therapeutics,
`Toronto General Research Institute, Room 4-408, 67 College Street, Toronto,
`Ontario, Canada. Phone: 416-586-4552; Fax: 416-586-8392; E-mail: amccart@
`uhnres.utoronto.ca.
`F 2009 American Association for Cancer Research.
`doi:10.1158/1078-0432.CCR-08-2342
`
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`were to determine the following: (a) the efficacy of an EGFP-
`expressing vaccinia virus (vvDD-EGFP) alone or in combina-
`tion with rapamycin or cyclophosphamide as an experimental
`therapeutic agent against malignant gliomas in vitro and in vivo,
`and (b) the distribution and clearance of the virus after systemic
`(i.v.) delivery in immunocompetent hosts. We found that
`vvDD-EGFP targeted malignant glioma tumors specifically and
`prolonged survival in immunocompetent animal models of
`malignant glioma. This was further improved by the addition of
`rapamycin and cyclophosphamide. Although no cures were
`seen, there was a dramatic improvement in median survival. We
`also found that systemic (i.v.) administration of vvDD-EGFP in
`rats was feasible, safe, and well tolerated. The results of this
`study suggest
`that
`further investigation of
`the utility of
`oncolytic viruses in the treatment of malignant gliomas is
`warranted.
`
`Materials and Methods
`
`Cell lines. Human and rat glioma cell lines (U87MG, U118MG,
`A172, F98, RG2, and C6), monkey kidney fibroblasts (CV1), and
`murine NIH3T3 cells were obtained from the American Type Culture
`Collection. All cell lines were grown in DMEM supplemented with 10%
`heat-inactivated FCS, 2 mmol/L glutamine, and 1% antibiotic-
`antimycotic (Gibco). Cell lines were maintained in an incubator at
`37jC with 5% CO2 and serially passaged every 3 to 4 d. Each cell line
`was tested routinely for mycoplasma contamination.
`Vaccinia viruses. The recombinant (WR strain) vaccinia viruses
`vvDD-EGFP (14) and vvDD-R2RLuc were used in these studies. vvDD-
`R2RLuc was constructed from the plasmid pTREX-R2RLuc, which
`contains the red fluorescent protein (mCherry; gene synthesized
`commercially) fused through a foot-and-mouth disease virus 2A
`peptide motif to the Renilla luciferase gene (Promega Corp.). The
`foot-and-mouth disease virus 2A motif allows for bicistronic expression
`of the two genes. The R2RLuc segment was subcloned into our vaccinia
`shuttle plasmid pVx – EGFP (14) upstream of the vaccinia synthetic
`early/late promoter, replacing EGFP. After homologous recombination
`with the parental virus VSC20 (ref. 29; a gift from Dr. B. Moss), vvDD-
`R2RLuc underwent five rounds of selection in mycophenolic acid. Both
`viruses are based on the double-deleted platform, which lacks
`thymidine kinase and vaccinia growth factor described previously
`(14). All viruses were expanded in HeLa cells, purified on a sucrose
`cushion, and tittered on CV1 cells (30).
`Flow cytometry analysis. Cells were infected with vvDD-EGFP
`or mock control at a multiplicity of
`infection (MOI) of 1 and
`harvested after 0, 24, 48, and 72 h. Cells were washed twice with
`
`1 PBS, resuspended in fluorescence-activated cell sortings (FACS)
`
`buffer (Optimized Sheath Fluid, BD Biosciences), and evaluated by
`FACS caliber (Becton Dickinson Canada, Inc.). Green fluorescent
`protein (FL1; 488 nm) and phycoerythrin (PE) (FL2; 488 nm;
`compensation, FL1-1.1% FL2) expression was analyzed using Cell-
`Quest software (BD Biosciences).
`Cell proliferation assays. For the MTT assay, cells were infected with
`vvDD-EGFP at an MOI of 1, seeded in complete medium (10% FCS)
`
`into 96-well plates (5  105 cells/well; triplicate), and incubated at
`37jC in a 5% CO2 humidified atmosphere. After 0, 24, 48, and 72 h,
`10 AL of the MTT labeling reagent (5 mg/mL in PBS; Cell Proliferation
`Kit 1, Roche Applied Science) was added to each well. After 4 h (37jC;
`5% CO2), 100 AL of solubilization solution (10% SDS in 0.01 mol/L
`HCL) was added and cells were incubated overnight. Microtiter plates
`were evaluated on a Vmax Kinetic microplate reader (Molecular Devices
`Corp.), and data analysis was done (SOFTMax software version 2.32,
`Molecular Devices Corp.). For the combined therapy group, rapamycin
`
`CancerTherapy: Preclinical
`
`Translational Relevance
`
`Malignant gliomas, an aggressive form of brain tumor,
`are currently incurable. In this study, we examine the activity
`of a novel therapeutic double-deleted vaccinia virus
`(vvDD). We show in animal models that vvDD is effective
`in prolonging survival from malignant gliomas and it is
`enhanced by the chemotherapy agents cyclophosphamide
`and rapamycin, which are now in clinical use. vvDD is
`safe in these models and is an excellent candidate to take
`forward in clinical trials for malignant glioma. This is
`an important step toward the future development of
`new treatments for malignant gliomas using this type of
`combination therapy.
`
`include the blood-brain barrier (13) and immune responses
`to virus in the vascular compartment.
`Vaccinia virus is a double-stranded, enveloped, lytic DNA
`virus (14) with several advantages over other oncolytic viruses.
`It is easy to manipulate genetically; its replication and spread
`are rapid; and it is motile (actin tail dependent) and has no
`potential capacity to integrate into foreign DNA. It is a clinically
`safe and well-known virus because of its widespread use as a
`vaccine in the small pox eradication program. Several poxvirus-
`based vaccination trials for cancer are currently under
`investigation (15, 16), highlighting the relevance of this virus
`for anticancer immunotherapy.
`Many strains of attenuated replicating vaccinia viruses have
`shown promising efficacy in the treatment of murine models of
`human gliomas (17) and other cancers (14, 18, 19), as well as
`primary cultures of human tumors (20). Because there may be a
`potential concern about the safety of a replicating vaccinia
`virus, a mutant ‘‘double-deleted’’ version of the Western Reserve
`(WR) strain [double-deleted vaccinia virus (vvDD)] with
`deletions of the thymidine kinase and vaccinia growth factor
`genes was created to enhance its safety (14). vvDD was
`nontoxic following i.v. delivery in nonhuman primates (21),
`which suggests that it may be a good candidate for the systemic
`oncolytic virotherapy of human tumors.
`As the efficacy of oncolytic virotherapy as a single agent has
`thus far been unsatisfactory (8 – 10), this suggests that new or
`multiple oncolytic viruses or other combination therapies might
`prove to be more effective against malignant gliomas. Others
`have investigated oncolytic viruses in combination with chemo-
`therapeutics, radiation therapy, or with suicide gene/prodrug
`systems, and have found enhanced efficacy of the oncolytic
`viruses toward brain tumors (22 – 24). Improved results have also
`been seen in immunocompetent animals using an oncolytic
`herpes virus in combination with the cyclophosphamide to
`suppress the immune response and provide for prolonged
`replication of the virus (25, 26). Recently, we and others have
`shown that the combination of myxoma virus plus rapamycin led
`to enhanced viral replication in vitro and in vivo, and improved
`efficacy against medulloblastomas (27) and melanoma (28). This
`led us to investigate the use of oncolytic vaccinia virus in
`combination with rapamycin or cyclophosphamide therapy for
`the treatment of experimental models of malignant gliomas.
`To date, vvDD has not been evaluated for its efficacy or
`toxicity in brain tumor models. The objectives of this study
`
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`Vaccinia Therapy of Malignant Glioma
`
`(1-10 nmol/L/mL) was added to the cells 1 h before treatment
`with virus.
`In vitro cytopathic effects (cytopathic effect assay) were visualized in
`six-well plates. Malignant glioma cell lines were plated until confluent
`and then infected with vvDD-EGFP (or mock control or UV-inactivated
`control) at an MOI of 1 for 2 h. At 24, 48, and 72 h after infection,
`cells were photographed under white light (original magnification,
`
`10; Nikon Eclipse TE200 microscope with a Hamamatsu ORCA100
`
`digital camera).
`Animals. CD-1 nude mice (female; 6-8 wks old) and Fisher 344 rats
`were purchased from Charles River Canada. The animals were housed
`in groups of three to five in a vivarium maintained on a 12-h light/dark
`schedule with a temperature of 22jC F 1jC and a relative humidity of
`50% F 5%. Food and water were available ad libitum. All procedures
`were reviewed and approved by the University of Calgary and
`University Health Network (Toronto, Canada) Animal Resource
`Centres.
`In vivo oncolysis in a s.c. xenograft model. To determine the
`antitumor effects of vaccinia virus in s.c. models, we injected C6, U87,
`and U118 (1  107 cells/mouse) malignant glioma cells into the right
`
`flank of female nude mice to establish s.c. tumors. Seven days later,
`when tumor volumes reached 0.10 cm3 for C6, 0.13 cm3 for U87, and
`0.06 cm3 for U118, the treatment group was injected i.p. with 109
`particle-forming unit of vvDD-EGFP in 2 mL of HBSS and the control
`group with 2 mL of HBSS i.p. Tumors were then measured twice a week
`by a blinded investigator, and tumor volume was calculated as (width)2
` length  0.52. Animals were sacrificed according to Animal Resource
`
`Centres guidelines, and tumor tissues were removed for histologic
`examination.
`Evaluation of toxicity of vvDD-EGFP in immunocompetent rats. To
`investigate the toxicity of vvDD-EGFP in non-tumor-bearing immuno-
`competent rats, Fischer 344 rats (n = 12) under anesthesia (isofluorane
`
`2%) received i.c. injections of increasing doses of vvDD-EGFP (1  102,
`1  104, 1  106, 1  108 particle-forming unit/rat; two rats per dose)
`using the guide-screw system (31) or 1  109 particle-forming unit/rat
`
`or vehicle control via tail vein. Animals were followed for 42 d and
`weighed twice per week. Animals losing 20% body weight or having
`other unacceptable symptoms were sacrificed as per our Institutional
`Animal Care Guidelines. After sacrifice, brains and major organs were
`taken out and processed for histologic examination.
`
`Fig. 1. vvDD-EGFP infects human and rat brain tumor cells in vitro. A, fluorescence-activated cell sorting analysis for EGFP of malignant glioma cell lines (C6, A172, RG2,
`U87MG, and F98) 24 to 72 h after infection with the vvDD-EGFP at an MOI of 1. B, MTTassay of malignant glioma cells (human: U87MG, U118MG, A172; rat: C6, RG2, F98)
`compared with uninfected controls over 72 h. C, cytopathic effect on malignant gliomas cells. Cells plated at confluence were infected the next day with vvDD-EGFP or
`UV/psoralen-inactivated virus (UV vvDD) at an MOI of 1. Microscopy was done 48 h after viral infection. Original magnification, 400.
`
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`Survival studies of orthotopic glioma models in immunocompetent
`hosts. To investigate the efficacy of vvDD-EGFP in orthotopic glioma
`animal models, female Fischer 344 rats under anesthesia were injected
`
`with 1  105 F98 cells in 3 AL of sterile PBS using the guide-screw
`system (31) or were fixed to a stereotactic apparatus, and 5  104
`RG2 cells in 3 AL of PBS were inoculated as described previously (6).
`Seven (F98) or five (RG2) days after tumor implantation, rats were
`i.v. administrated vvDD-EGFP (1  109 particle-forming unit/rat in
`100 AL HBSS) as a single dose or multiple doses (1  109 particle-
`
`forming unit/rat every 2 d for a total of three injections). Control
`animals were treated with HBSS. Rats were monitored daily until they
`lost >20% of body weight or had trouble ambulating, feeding, or
`grooming; then, they were sacrificed, and their brains and major organs
`were collected for histologic analysis.
`In vivo viral distribution studies in an orthotopic glioma model. To
`determine if vvDD-EGFP targets multifocal gliomas in the brain, we
`established a bilateral tumor model using RG2 tumor cells to mimic
`multifocal malignant glioma of patients. RG2 cells were implanted by
`stereotactic techniques as described above in immunocompetent Fischer
`rats. Ten days later, rats were administered i.v. vvDD-EGFP
`
`(1  109 particle-forming unit/rat in 100 AL HBSS). Animals (n = 3)
`
`were sacrificed at 1, 3, 5, 7, and 14 d after virus administration. When
`sacrificed, animals were anesthetized and perfused with 50 mL of saline,
`followed by 30 mL of phosphate-buffered 10% formalin via cardiac
`catheter. Following fixation, a whole brain picture was taken with a Leica
`MZ-FLIII fluorescence stereomicroscope using a standard green fluores-
`cent protein filter set and the brain, tumor, and other major organs were
`removed and frozen in liquid nitrogen for virus recovery assays.
`Combination therapy in vitro and in vivo. To determine whether
`pretreatment with rapamycin promotes vvDD oncolysis of rat cell
`lines in vitro, viral green fluorescent protein expression and cell viability
`were assessed 48 or 72 h postinfection in the presence or absence
`of rapamycin. F98 and RG2 Cells were pretreated with rapamycin
`(1 nmol/l) 1 h before virus infection, then infected with vvDD-EGFP
`(MOI, 0.1 or 1). After a 48-h incubation, green fluorescent protein
`expression was analyzed using a Zeiss inverted microscope (Axiovert
`200 M) with green fluorescent protein filter and a Carl Zeiss camera
`(AxioCam MRc). To assess the effects of combined therapy on cell
`viability, an MTT assay was done 72 h after infection. To determine the
`effect of rapamycin on viral replication, cells were pretreated for 2 h with
`rapamycin (10 nmol/L),
`infected with vvDD-EGFP (MOI, 0.01),
`incubated for 48 h, and lysed using three rounds of freeze thawing to
`extract viral particles. The viral titers of samples were determined using a
`standard plaque titration assay on CV1 cells.
`To evaluate the in vivo effects of combination therapy using vvDD-
`EGFP combined with rapamycin or cyclophosphamide, i.c. bilateral
`RG2 tumor – bearing animals (described above) were divided into
`the following four groups (n = 9-10 per group) 8 d after implantation:
`(a) dead virus control, (b) drug alone (i.p. administration of rapamycin
`5 mg/kg/d for 5 d or cyclophosphamide 60 mg/kg, one time only),
`
`(c) vvDD-EGFP (1  109 particle-forming unit/rat) alone, and (d)
`
`vvDD-EGFP plus drug (as above beginning 1 d before virus injection).
`Two animals (of nine) were sacrificed on day 7 to assess green
`fluorescent protein expression. Five or six animals (of 9 or 10) in each
`group were monitored daily and sacrificed when symptoms developed
`as described above.
`To assess the effects of rapamycin and cyclophosphamide on the
`immune response to viral replication, two animals (of nine) from each
`group were pretreated with drug and then treated with a second dose of
`vaccinia 3 d after the initial dose. The second virus expresses red
`
`fluorescent protein (vvDD-RFPLuc; 1  109 particle-forming unit/rat)
`
`to differentiate it from the initial injection. These mice were sacrificed
`3 d after the second virus was administered. The brains were removed
`and photographed using a fluorescent stereotactic microscope (Leica
`MZ-FLIII, Switzerland, Ltd.) to visualize green fluorescent protein – and
`red fluorescent protein – expressing virus, then frozen in liquid nitrogen
`for virus recovery assays.
`
`CancerTherapy: Preclinical
`
`Fig. 2. Systemic (i.p.) administration of vvDD-EGFP on growth of s.c. models of
`malignant glioma in nude mice. Female nude mice were injected with 1 107 C6,
`U87, and U118 cells s.c. in their right flanks. Seven days later, when tumor volumes
`reached f0.10 cm3 for C6, 0.13 cm3 for U87, and 0.06 cm3 for U118, a blinded
`experimenter administered the treatment group with 109 particle-forming units of
`vvDD-EGFP in 2 mL of HBSS i.p. and the control group with 2 mL of HBSS i.p.
`Tumors were then measured twice a week. A, C6 malignant gliomas in the treatment
`group had significantly (P = 0.03) smaller tumor volumes than the control
`group. B, the volume of U87 gliomas in the treatment group remained stable,
`whereas the tumor volume of the control group increased significantly (P = 0.01)
`with time. C, the volume of U118 malignant glioma also remained relatively
`stable over time in the treatment group, whereas the tumor volume of the control
`group increased significantly (P = 0.03).
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`Vaccinia Therapy of Malignant Glioma
`
`Immunohistochemistry. Paraffin embedded sections of rat brain
`
`were deparaffinized and rehydrated with 1 PBS after blocking, then
`
`exposed to primary antibody (murine monoclonal vaccinia virus
`antibody, Abcam, Inc.) at a 1:10 dilution in PBS overnight at 4jC.
`Biotinylated donkey anti – mouse IgG (1:2,000, Vector Laboratories)
`was used as the secondary antibody. Sections were then incubated with
`avidin conjugated to horseradish peroxidase (Vectastain ABC immu-
`nohistochemistry kit, Vector Laboratories), and staining was visualized
`by the addition of 3,3¶-diaminobenzidine substrate with hematoxylin
`counterstaining. Sections were mounted and viewed with a Zeiss
`inverted microscope (Axiovert 200M) and a Carl Zeiss camera
`(AxioCam MRc) to obtain images.
`For the CD68/CD163 staining, frozen sections of the brains were
`fixed with 4% paraformaldehyde for 15 to 20 mins, followed by two
`washes with PBS (for paraffin embedded sections were deparaffinized
`
`and rehydrated with 1 PBS). The sections were incubated with
`
`primary antibody [mouse anti – rat CD68 (1:500; Serotec; Cat MCA
`341R), CD163 (1:300; Serotec; Cat MCA 342R)] for 1 h at room
`temperature after blocking. Biotinylated anti – mouse IgG (1:500; Vector
`Laboratories; CatBA-2000) was used as a secondary antibody.
`
`Virus recovery assays. Rats were sacrificed; saline was immediately
`infused; and the tissues were extracted and homogenized in HBSS using
`a Pellet Pestles Kit (VWR International), followed by repeated freeze
`thawing to release virus from the cells. Supernatants were plaque
`tittered on CV1 cells, as previously described (29). Viral plaques were
`counted, and particle-forming unit were calculated by the number of
`plaques multiplied by the dilution factor and normalized to the weight
`of the tissues.
`Primary human glioma culture. Short-term cultures were established
`from patient samples of human gliomas (n = 8) obtained following
`brain tumor surgery at
`the Foothills Hospital (Calgary, Alberta,
`Canada). This study was approved by the Conjoint Medical Ethics
`Committee. Briefly, each patient specimen was split into two pieces for
`fixation in 10% formalin and short-term culture. For short-term
`cultures, the tissue was washed several times in sterile saline, cut into
`small pieces (f0.5-1 mm in diameter), and dissociated with trypsin
`(0.25%) and 50 Ag/mL DNase (Roche Diagnostics) for 30 mins at
`37jC. After filtering and washing with DMEM/F12 (containing 20%
`FBS), cells were resuspended in 20% FBS in DMEM/F12 and plated
`(at 10,000-100,000 cells per well) in 96-well plates. Cells were infected
`
`Fig. 3. Systemic (i.v.) administration of vvDD-EGFP prolonged survival of immunocompetent rats bearing i.c. F98 and RG2 gliomas. A, F98 tumor model. Kaplan-Meier
`survival analysis of rats implanted with F98 (1 105 cells/rat) and treated with either HBSS (n = 6) or vvDD-EGFP (n = 6; 1 109 particle-forming units/rat single i.v. injection
`on day 7 after tumor implantation). All Ps are two sided. Arrow, virus injections. B, representative immunohistochemistry of vvDD analysis in F98 tumors and normal brain
`7 d after i.v. administration of virus. Left column, mock control. Original magnification, 10 (top) and 40 (bottom). C, RG2 tumor model. Kaplan-Meier plot showing
`survival curves of rats harboring i.c. RG2 tumor treated with dead virus (n = 5) or a single i.v. administration of vvDD-EGFP (live virus single; n = 5; 1 109 particle-forming
`units/rat) or multiple i.v. administration of vvDD-EGFP (live virus multiple; n = 6; 1 109/rat every 2 d for three injections). All Ps are two sided. Arrows, day of virus
`administration. D, histologic analysis showed that all of the dead virus ^ treated mice and live virus ^ treated mice had large tumors in the brain. Most of animals treated with
`i.v. administration of vvDD-EGFP showed area of tumor necrosis. Original magnification, 25 (top) and 400 (bottom).
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`method. The log-rank test and ANOVA for repeated measures were used
`to compare the effect of different forms of treatment. The Student’s t test
`was used when appropriate. Data was expressed as means F SD. All Ps
`<0.05 were considered significant.
`
`Results
`
`Vaccinia virus (vvDD-EGFP) infects and kills malignant
`glioma in vitro. Malignant glioma cell lines were tested for
`susceptibility to infection by vvDD-EGFP using FACS analysis
`to quantitate the percentage of EGFP-positive cells. Malignant
`glioma cell lines (C6, A172, RG2, U87MG, and F98) were
`infected with vvDD-EGFP at an MOI of 1; the number of
`EGFP-positive cells was quantified 24, 48, and 72 hours after
`infection. All 5 malignant glioma cell lines were efficiently
`infected by vvDD-EGFP, with 70% to 99% of all cell lines
`infected by 24 hours (Fig. 1A).
`To confirm whether infection was leading to oncolysis and
`cell death, MTT and cytopathic effect assays were done. All six
`cell
`lines (U87, U118, A172, C6, RG2, and F98) were
`susceptible to killing, and extensive cell death was observed
`72 h after infection with an MOI of 1 (<40% of cells still viable
`compared with uninfected controls; Fig. 1B). Similar results
`were obtained with the cytopathic effect assay. There was no
`evidence of cytopathic effect in untreated (control) or dead
`virus – treated (UV vvDD) cells at an MOI of 1 at 48 hours
`postinfection (Fig. 1C).
`Antitumor effect of systemic administration of vvDD-EGFP on
`s.c. models of malignant glioma. We next investigated if vvDD-
`EGFP would infect and kill malignant glioma in a s.c. tumor
`model in nude mice. Nude mice bearing s.c. xenografts of
`U87MG, U118, or C6 malignant glioma cell lines were injected
`systemically (i.p.) with 109 particle-forming unit of vvDD-EGFP
`or HBSS control. We observed a statistically significant
`inhibition of tumor growth in virus-treated mice compared
`with the HBSS-treated control mice bearing either C6 (ANOVA;
`P = 0.03; Fig. 2A) or U87 (ANOVA; P = 0.01) tumors (Fig. 2B).
`The volume of U118 malignant glioma remained relatively
`stable over time in the treatment group, whereas the tumor
`volume of the control group increased significantly (Fig. 2C;
`ANOVA; P = 0.03).
`Toxicity of intracerebral and i.v. administration of vvDD-
`EGFP. We evaluated the toxicity of vvDD-EGFP in non-tumor-
`bearing immunocompetent rats when administered i.c. or i.v. A
`
`single dose of 1  108 particle-forming unit i.c. or 1  109
`
`particle-forming unit i.v. (the highest feasible dose we can
`prepare) of vvDD-EGFP was safe in normal non-tumor-bearing
`rats. At the highest i.c. dose administered, we saw some weight
`loss, which quickly recovered (Supplementary Fig. 1). There
`were no deaths and no neurologic symptoms noted. Surpris-
`ingly, when tumor-bearing rats were subsequently treated, all
`i.c. doses were extremely toxic in a non-dose-dependent
`manner. Necropsy showed gross hemorrhage and swelling of
`the tumor, presumably because of the rapid oncolysis by the
`
`virus (data not shown). I.v. administration of 1  109 particle-
`
`forming units to tumor-bearing rats was nontoxic and was the
`dose/route used in subsequent studies.
`Survival following systemic administration of vvDD-EGFP in
`immunocompetent rats bearing i.c. malignant glioma. To
`determine the efficacy of vvDD-EGFP oncolysis in immuno-
`competent models of i.c. malignant glioma, we implanted F98
`
`CancerTherapy: Preclinical
`
`Fig. 4. Distribution of i.v. administered vvDD-EGFP in immunocompetent rats
`bearing bilateral i.c. malignant gliomas. Rats bearing bilateral RG2 tumors (to mimic
`multifocal malignant glioma clinically) were treated with vvDD-EGFP i.v. at a dose
`of 1 109 particle-forming units rat 10 d after implantation of RG2 tumors. Animals
`were sacrificed at different time points (1, 3, 5, 7 d) and 14 d (data not shown)
`after viral administration. A, representative photomicrographs of EGFP ^ labeled
`virus in the brain tumor (n = 2 rat/group). Original magnification, 10 (top) and
`20 (bottom). Arrows, green fluorescent protein virus expression. B, titer of virus
`present in the bilateral tumors (n = 3 rats per time point).Tumors on each side of the
`brain (right or left) or resections of normal brain (brain) were harvested for virus
`extraction, and the samples were analyzed by virus recovery assay on CV1cells.
`C, titers of virus present in other organs in vivo following i.v. administration of
`vvDD-EGFP.
`
`the following day with vvDD-EGFP virus, live and UV inactivated, at
`MOIs of 0.1, 1, and 10. Cell viability was measured 96 h postinfection
`by MTT assay. Viral
`titers were obtained from patient short-term
`cultures after infection. Dissociated tumor cells from surgical samples of
`malignant gliomas were plated in six-well plates and infected the next
`day with vvDD-EGFP at an MOI of 0.1. Cells were collected at 0, 24,
`and 72 h after virus infection. These were then lysed using three rounds
`of freeze thawing to extract viral particles. U87 and NIH3T3 were used
`as positive and negative controls, respectively. The viral titers of samples
`were determined using a standard plaque titration assay on CV1 cells.
`Statistics. All statistics were generated using StatView Software
`(Abacus Concepts, Inc.) and GraphPad Prism (version 4; GraphPad
`Software, Inc.). Survival curves were generated by the Kaplan-Meier
`
`Clin Cancer Res 2009;15(8) April 15, 2009
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`www.aacrjournals.org
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`Replimune Limited Ex. 2022 - Page 6
`Transgene and Bioinvent International AB v. Replimune Limited
`PGR2022-00014 - U.S. Patent No. 10,947,513
`
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`

`Vaccinia Therapy of Malignant Glioma
`
`orRG2 cells into the brain of F344 rats to establish i.c. tumors.
`Rats were treated i.v. with single or multiple doses of vvDD-
`EGFP after tumor implantation. Animals were monitored daily.
`A single i.v. administration of vvDD-EGFP prolonged survival
`(Fig. 3A; long-rank test; P = 0.0475) of rats bearing i.c. F98
`tumors. The median survival of vvDD-EGFP – treated animals
`was 24.5 days [95% confidence interval (95% CI), 20-28 days],
`whereas contr