Epidermal Growth Factor Receptor (EGFR

© The American Society of Gene Therapy

original article

Epidermal Growth Factor Receptor (EGFR)– Retargeted Measles Virus Strains Effectively Target EGFR- or EGFRvIII Expressing Gliomas Georgia Paraskevakou1, Cory Allen1, Takafumi Nakamura1, Paula Zollman1, C David James2, Kah Whye Peng1, Mark Schroeder1, Stephen J Russell1 and Evanthia Galanis1 1 Mayo Clinic and Mayo Foundation, Rochester, Minnesota, USA; 2Department of Neurological Surgery, University of California, San Francisco, California, USA

A retargeted measles virus strain MV-GFP-HAA-scEGFR was generated by engineering the MV-NSe Edmonston vaccine strain to incorporate both CD46 (Y481A) and signaling lymphocyte activation molecule (SLAM) (R533A) ablating mutations in the hemagglutinin protein in combination with the display of a single-chain antibody against epidermal growth factor receptor (EGFR) at the C terminus of hemagglutinin. The unmodified MV-GFP virus was used as a positive control. Specificity of the EGFR retargeted virus was demonstrated in non-permissive Chinese hamster ovary (CHO) cells stably transfected to express either the natural receptors CD46 or SLAM or the target receptors EGFR and EGFRvIII. In vitro, the retargeted virus had potent antitumor activity against EGFR- or EGFRvIII-overexpressing primary glioblastoma multiforme (GBM) cell lines that was comparable to the activity of the unmodified MVGFP virus. Intratumoral administration of MV-GFP-HAAscEGFRvIII in orthotopic GBM12 xenografts resulted in tumor regression, as demonstrated by bioluminescence imaging and significant prolongation of survival, that was comparable to the effect of the unmodified strain. In contrast to MV-GFP, central nervous system administration of the targeted MV-GFP-HAA-scEGFR virus in measles replication–permissive Ifnarko CD46 transgenic mice resulted in no neurotoxicity. In conclusion, EGFRretargeted measles virus strains have comparable therapeutic efficacy to the unmodified virus in glioma cells overexpressing EGFR or EGFRvIII in vivo and in vitro, and improved therapeutic index, a finding with potential translational implications in glioma virotherapy. Received 25 October 2006; accepted 14 December 2006; advance online publication 13 February 2007. doi:10.1038/mt. sj.6300105

Introduction Gliomas are the most common primary brain tumor in adults and have dismal prognosis despite aggressive use of multimodality treatment consisting of surgery, radiotherapy, and chemotherapy.1

Novel therapeutic approaches based on our understanding of molecular alterations in gliomas are urgently needed to improve the outcome of glioma patients. Because gliomas rarely metastasize, they are excellent candidates for gene therapy/virotherapy approaches. We have previously demonstrated that derivatives of the measles Edmonston vaccine strain have potent oncolytic activity against gliomas.2 Measles virus (MV) enters cells through attachment of its hemagglutinin (H) protein to viral receptors expressed on the cell surface, which results in conformational changes followed by membrane fusion mediated by the fusion (F) protein.3 Two natural receptors for MV are known in humans: CD46, which is ubiquitously expressed on nucleated cells and overexpressed in tumors, and SLAM (signaling lymphocyte activation molecule), which is predominantly expressed on T and B lymphocytes, macrophages and dendritic cells.4,5 We and others have recently identified H protein mutations that ablate entry via CD46 or SLAM.6,7 In glioma virotherapy, retargeting MV strains against a glioma-specific receptor with ablation of entry via the natural receptors could have two potential advantages: it could increase specificity against tumor cells and also allow us to overcome possible variability in the expression of the natural receptor CD46 in a therapeutic setting.8,9 The most frequent genetic alteration associated with highgrade gliomas, and especially glioblastoma multiforme (GBM), is the amplification of the epidermal growth factor receptor (EGFR) gene, which results in overexpression of EGFR. Epidermal growth factor receptor is a 170 kd transmembrane tyrosine kinase recep tor, amplification of which leads to stimulation of a signal transduction cascade that promotes cellular proliferation. It is amplified in 30–50% of GBM and therefore has significant potential as a glioma-specific molecular target.10–12 EGFR gene amplification in gliomas is often accompanied by gene rearrangements, including deletions within the coding region. The most common EGFR mutant, EGFRvIII, results from deletion of exons 2–7, and it is expressed in 40–60% of GBM with EGFR amplification.11,13,14 We therefore generated the EGFR-retargeted virus MV-GFPHAA-scEGFR by combining ablation of entry via the natural receptors CD46 or SLAM with the display of a single-chain antibody recognizing EGFR at the C terminus of H.15 The H protein of

Correspondence: Evanthia Galanis, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA. E-mail: [email protected] Molecular Therapy vol. 15 no. 4, 677–686 april 2007

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EGRF Retargeted Measles Virus Strains in Glioma Treatment

MV-GFP-HAA-scEGFR contains alanine substitutions in aminoacid residues 481 and 533, which ablate viral entry through CD46 and SLAM, respectively.16 We demonstrated that EGFR-retargeted MV strains can selectively enter glioma cells via either EGFR or the EGFRvIII receptor, without loss of its oncolytic efficacy in vitro or in vivo but with an improved therapeutic index, a finding with potential translational implications in glioma virotherapy.

Results Construction of MV-GFP-HAA-scEGFR

Construction of the MV-GFP-HAA-scEGFR virus was performed as previously described.7 This virus is derived from the Edmonston vaccine lineage NSe strain; it was rescued using the pseudoreceptor STAR (six-his tagging and retargeting) system and propagated on Vero-HIS cells.7 The viral H protein contains the CD46-ablating mutation Y481A and the SLAM-ablating mutation R533A.7 It also contains the green fluorescent protein (GFP) gene in position 1 (Figure 1), which facilitates viral rescue and allows visualization of infection in vitro and in vivo. A schematic representation of the retargeted viral genome is presented in Figure 1. MV-GFP containing the unmodified H of the Edmonston NSe strain and the GFP gene in position 1 was used as the control virus. To determine the specificity of the EGFR-retargeted virus, we developed Chinese hamster ovary (CHO) transfectants expressing CD46, SLAM, EGFR, and EGFRvIII receptors. Receptor expression was confirmed by fluorescence-activated cell sorting analysis (data not shown). We infected CHO parental cells and transfectants with either MV-GFP or MV-GFP-HAA-scEGFR at a multiplicity of infection of 1.0. The parental CHO line does not express CD46, SLAM, EGFR, or EGFRvIII, and as expected no infection was observed. The CHO-CD46 and CHO-SLAM cell lines expres sing the natural MV receptors were effectively transduced by the unmodified virus MV-GFP, but not by the EGFR-retargeted strain (Figure 2). In contrast, the EGFR-retargeted strain MV-GFP-HAAscEGFR resulted in transduction and cytopathic effect in CHO cells expressing the EGFR or the EGFRvIII receptor. Entry of the retargeted MV-GFP-HAA-scEGFR virus via the EGFR and via the mutant receptor EGFRvIII was comparable, as indicated by the comparable GFP positivity and cytopathic effect in CHOEGFR and CHO-EGFRvIII cells.

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Figure 1 Schematic presentation of the epidermal growth factor receptor (EGFR)–retargeted measles viral genome. The hemagglutinin (H)-protein contains both D46 and signaling lymphocyte activation molecule (SLAM) ablating mutations (Y481A and R533A, respectively). A single-chain antibody against EGFR is displayed on the C terminus of H. GFP, green fluorescent protein.

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In vitro infection of EGFR or EGFRvIII tumor cells with retargeted strains results in significant cytopathic effect To assess the in vivo efficacy of our retargeted strain, we employed primary lines derived from GBM patients and kept as subcutaneous xenografts in nude mice.17 These lines maintain the molecular characteristics of the human tumor they derived from, including EGFR amplification and EGFRvIII mutation. Levels of EGFR and EGFRvIII expression in the primary lines GBM6, GBM8, GBM12, GBM14, and GBM39 were characterized by western blot analysis. EGFR (170 kd) was expressed in the primary glioma line GBM12. GBM8 and GBM39 expressed both EGFR and EGFRvIII (140 kd), whereas GBM6 expressed only EGFRvIII. In GBM14, neither receptor was present (Figure 3). The pattern of GFP positivity after infection with MV-GFP-HAA-scEGFR confirmed EGFR and EGFRvIII receptor–specific transduction (Figure 4A). We next investigated the efficacy of our virus in these primary GBM cell lines. Cytotoxicity was compared using trypan blue exclusion assays at multiple time points. MV-GFP-HAA-scEGFR had significant antitumor efficacy (comparable to the unmodified strain MV-GFP) against the EGFR-amplified primary GBM cell line GBM12, the GBM39 and GBM8 cells expressing both EGFR and EGFRvIII, and the EGFRvIII-expressing glioma cell line GBM6. In GBM14, which did not express EGFR or EGFRvIII, treatment with MV-GFP-HAA-scEGFR did not result in infection or cytopathic effect (Figure 4B). To assess viral replication in vitro, we generated one-step viral growth curves. Viral replication of the EGFR-retargeted strain MV-GFP-HAA-scEGFR was comparable to that of the unmodified MV-GFP strain in the primary glioma lines GBM12 and GBM8 overexpressing EGFR, GBM39 and GBM8 expressing both EGFR and EGFRvIII, and GBM6 overexpressing EGFRvIII. Replication of the unmodified strain MV-GFP was at least two logs higher than that of the retargeted strain in GBM14 cells expressing neither EGFR nor EGFRvIII receptor (Figure 5). EGFR–retargeted strains show significant antitumor efficacy in vivo against EGFR-overexpressing xenografts Development of EGFR-targeted therapeutics has been hampered by the lack of animal models maintaining EGFR alterations in vivo. Our group has recently described the development of animal models using tumors derived from glioblastoma patients and passaged subcutaneously in mice. After orthotopic implantation, these xenografts maintain in vivo the molecular alterations and invasiveness of the primary human tumor from which they were derived.17 The GBM12 model that overexpresses EGFR was employed for our in vivo study. To facilitate non-invasive monitoring of intracranial tumor growth and treatment response, we engineered the primary GBM cells to stably express firefly luciferase (Fluc). In all animals, tumor implantation was confirmed by assessing a bioluminescent imaging signal 1 week after tumor implantation; subsequent images were obtained weekly (Figure 6A). Treatment was initiated at 7 days, upon verification of successful implantation, and animals were randomized into three groups of 8–10 animals each. www.moleculartherapy.org vol. 15 no. 4, april 2007



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Figure 2 Chinese hamster ovary (CHO) transfectants were infected with MV-GFP or MV-GFP-HAA-scEGFR at a multiplicity of infection of 1. There was efficient and specific entry of MV-GFP-HAA-scEGFR in CHO-EGFR and CHO-EGFRvIII cells as demonstrated by GFP positivity. There was no viral entry of the retargeted strain in CHO-CD46, CHO-SLAM expressing the natural receptors, or the parental CHO line.

Quantification of photons released by GBM12 orthotopically implanted tumors demonstrated that mice treated with MV-GFP or MV-GFP-HAA-scEGFR-H6 exhibited a significant decrease in tumor burden compared to animals treated with the UVinactivated virus (UV-MV) (Figure 6B). In addition, treatment with MV-GFP or the EGFR-retargeted strain MV-GFP-HAA-scEGFR resulted in prolongation of animal survival (P = 0.001) (Figure 6C). There was statistically significant improvement of survival in mice treated with MV-GFP-HAAscEGFR as compared with UV-MV (P < 0.0001). There was also significant prolongation of survival in mice treated with MV-GFP Molecular Therapy vol. 15 no. 4, april 2007

as compared with UV-MV (P < 0.0001). There was no significant difference in therapeutic efficacy between MV-GFP and MV-GFPHAA-scEGFR, however (P = 0.88). The characteristic cytopathic effect of MV, with abundant formation of syncytia, was observed in tumors treated with the retargeted strain MV-GFP-HAAscEGFR (data not shown).

MV-GFP-HAA-scEGFRvIII causes no neurotoxicity after intracerebral administration Ifnarko CD46 Ge mice are susceptible to MV infection, and intracerebral administration of the MV Edmonston strain results in 679



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Actin control Figure 3 Western blot analysis of tumor cells with an anti-epidermal growth factor receptor (anti-EGFR) antibody. There is EGFR expression (170 kd band) in the primary glioma cell line GBM12, both EGFR and EGFRvIII (145 kd band) expression in GBM39 and GBM8 cells, and EGFRvIII expression in GBM6 cells. There was no EGFR or EGFRvIII expression in GBM14 glioma cells. Immunoblotting for actin was used as loading control.

the development of lethal encephalitis.18,19 These mice have been used to characterize the virulence of different MV strains.18 The retargeted strain MV-GFP-HAA-scEGFRvIII and the unmodified strain MV-GFP were administered using the same stereotactic parameters as for the efficacy study. MV-GFP administration in the central nervous system of Ifnarko CD46 Ge mice at a total dose of 2 × 105 50% tissue culture infective dose (TCID50) resulted in significant neurotoxicity, which necessitated euthanasia of all five treated animals. In contrast, there was no neurotoxicity after central nervous system administration of the EGFR-retargeted strain, indicating an improved therapeutic index (Figure 7). There was abundant rescue of MV from the brains of Ifnar mice, treated with MV-GFP virus in Vero cell overlays. In contrast, no virus was recovered in Vero-HIS overlays from the brains of mice treated with the retargeted MV-GFP-HAA-scEGFR virus.

DISCUSSION We have previously demonstrated that oncolytic MV strains have significant antitumor activity against gliomas.2 In addition, we have recently begun a phase I clinical trial of intratumoral and resection-cavity administration of an MV derivative genetically engineered to produce CEA (MV-CEA) in recurrent glioblastoma multiforme patients. Development of retargeted MV strains has the potential to increase the versatility of this novel viral technology in the management of gliomas, especially if systemic delivery is contemplated, as it could help us overcome possible toxicity issues arising from ubiquitous (although lowlevel) expression of CD46 in normal cells and immunosuppression owing to recognition of the MV SLAM receptor in B and T cells and lymphocytes.4,5 EGFR is attractive for glioma-targeted therapy because it is overexpressed in a large percentage of high-grade gliomas (50–60% of GBM).1 It is a 170 kd glycoprotein with intrinsic tyrosine protein kinase activity and an extracellular domain that contains two functional domains linked by a transmembrane region. Tumors with EGFR amplification frequently (40–60%) also express a mutated, constitutively activated, form of EGFR, EGFRvIII.11 The high frequency of EGFR overexpression in GBM and its low expression level only in subgroups of neurons and glial cells in adult brain makes EGFR an appealing approach for glioma-specific retargeting of oncolytic MV strains.12,20 The epitope recognized by the single-chain antibody we utilized to retarget the MV is 680

contained within the 110 kd EGF-binding fragment, which includes the entire extracellular domain,15 and it is preserved in the mutant variant EGFRvIII,21–22 allowing both EGFR- and EGFRvIII-specific entry of the MV. Furthermore, systemic biodistribution studies performed with F(ab′)2 fragments of this antibody have shown preferential localization in glioma tumor tissue and sparing of normal tissues, despite low levels of EGFR expression in organs such as kidney tubules, squamous cells, and sweat glands.23 In this article we have demonstrated successful retargeting of MV by combining single-chain antibody targeted entry via EGFR (as well as the EGFRvIII mutant variant) with ablation of entry via the MV natural receptors CD46 and SLAM. The EGFRretargeted virus resulted in EGFR/EGFRvIII-specific oncolytic activity against primary glioma lines that was comparable to the antitumor activity of the unmodified MV-GFP strain both in vitro and in vivo. A significant problem in the preclinical assessment of novel therapeutic approaches in gliomas is the difficulty involved in developing clinically relevant animal models.24 Established GBM cell lines frequently fail to preserve molecular characteristics of GBM, including invasiveness and preservation of EGFR amplification and mutation.25 We have recently developed cell lines derived from glioblastoma patients that are subsequently maintained as heterotopic xenografts through serial passaging in the flank of nude mice.17 These GBM lines preserve the genetic alterations, including EGFR gene amplification and mutation, and the invasive nature of human tumors after orthotopic implantation. For the in vivo experiments in this study, we used an orthotopic animal model deriving from such a line: the GBM12 model, which maintains EGFR amplification in vivo. Furthermore, we stably transduced these primary glioma lines with the luciferase gene before implantation and we employed a bioluminescence imaging system (IVIS 200, Xenogen, Caliper Life Sciences, Hopkinton, MA) to follow tumor growth in vivo.26–28 EGFR–retargeted MV strains increased survival and induced sustained tumor regression in both in vivo models that was comparable to the therapeutic effect of the unmodified strain, indicating that EGFR MV retargeting does not decrease antitumor potency. Epidermal growth factor receptor–targeted therapeutics including EGFR tyrosine kinase inhibitors and monoclonal antibodies recognizing EGFR are an area of active investigation in www.moleculartherapy.org vol. 15 no. 4, april 2007



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viral retargeting and represents a novel addition to the armamentarium of therapeutic agents attacking gliomas through EGFR/ EGFRvIII-targeted cytotoxicity. The results from our pilot toxicity experiment using Ifnarko CD46 Ge mice, a very sensitive model of measles neurotoxicity, are reassuring. Nevertheless, primate toxicology studies would likely be required before clinical translation

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glioma treatment.29 Efforts to retarget DNA or RNA viruses against EGFR-expressing cells have mostly employed bispecific antibodies or soluble adapter proteins30–32 that are not covalently linked to the vector particle, the latter representing a potential obstacle if clinical translation is contemplated. Our approach, using display of the EGFR single-chain antibody at the C terminus of H, results in true

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Figure 4 Cytopathic effect of MV-GFP-HAA-scEGFR in the primary glioblastoma cell lines GBM8, GBM12, GBM14, GBM39, and GBM6. (a) The EGFR-retargeted measles virus (MV) strain transduces efficiently all EGFR- or EGFRvIII-expressing cell lines (GBM8, GBM12, GBM39, GBM6), as indicated by GFP positivity, which is comparable to the unmodified strain MV-GFP. There was no transduction of the EGFR/EGFRvIII-negative cell line GBM14. (b) Cell viability was determined by trypan blue exclusion. MV-GFP-HAA-scEGFR resulted in significant cytopathic effect in all EGFR- and/or EGFRvIII-positive cell lines, but not in the negative line GBM14.

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Figure 5 One-step viral growth curves in primary glioma lines expressing the EGFR or EGFRvIII receptor demonstrate comparable replication to MVGFP in receptor-positive cells (a, b), but no replication of the MV-GFP-HAA-scEGFR virus in receptor-negative cells (c).

of this technology because of similarities in EGFR distribution between humans and non-human primates that a rodent model cannot simulate and the degree of receptor homology.33–35 In summary, we have demonstrated that EGFR-retargeted MV strains enter tumor cells selectively via EGFR or EGFRvIII and have significant antitumor activity in vitro and in vivo that is comparable to the unmodified strain MV-GFP. They therefore represent promising candidates for clinical translation.

MATERIALS AND Methods Cell culture. Vero (African green monkey kidney) and CHO cells were

purchased from American Type Culture Collection (ATCC, Manassas,VA). All cell lines were grown at 37°C in media recommended by American Type Culture Collection in a humidified atmosphere of 5% CO2. CHO

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transfectants stably expressing CD46, SLAM, EGFR, and EGFRvIII were generated as previously described.7,16 Receptor expression was confirmed by fluorescence-activated cell sorting analysis. Primary glioblastoma lines GBM6, GBM8, GBM12, GBM14, and GBM39 were generated from Mayo Clinic glioblastoma patients and maintained as subcutaneous xenografts, as previously described.17 To establish short-term cultures, we excised the xenografts and placed them in culture dishes, where the tissue was initially minced with a scalpel and then mechanically disrupted to create a cell suspension. Following short-term culture of the tumor cells (3–7 days) in Dulbecco’s modified Eagle’s medium containing 2.5% fetal bovine serum, 1× penicillin/streptomycin, cells were either used for orthotopic implantation or further maintained in culture in Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, 1× penicillin/streptomycin. Construction of MV-GFP-HAA-scEGFR. The MV-GFP-HAA-scEGFR virus

containing a single CD46-ablating mutation (Y481A) and SLAMwww.moleculartherapy.org vol. 15 no. 4, april 2007



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Figure 6 Assessment of antitumor activity of MV-GFP-HAA-scEGFR in vivo. (a) Representative bioluminescent images from animals bearing orthotopic GBM12 xenografts. There is bioluminescence signal stability in animals treated with the retargeted MV-GFP-HAA-scEGFR virus or the unmodified MV-GFP virus but significant tumor growth in the animals treated with inactive virus. (b) Assessment according to photons released by orthotopic GBM12 shows significant decrease in tumor volume in MV-GFP- or MV-GFP-HAA-scEGFR-treated animals, as compared to animals treated with the inactive virus. (c) Treatment with the epidermal growth factor receptor (EGFR)–retargeted strain MV-GFP-HAA-scEGFR resulted in significant prolongation of survival of mice bearing GBM12 xenografts and comparable antitumor effect to the unmodified strain MV-GFP.

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Days Figure 7 Survival of Ifnarko CD46 mice after central nervous system administration of MV-GFP or MV-GFP-HAA-scEGFR. In contrast to the lethal neurotoxicity observed by day 7 in all MV-GFP-treated mice, mice treated with the epidermal growth factor receptor (EGFR) retargeted virus survived without toxicity.

a blating mutation (R533A) was rescued by employing the pseudoreceptor STAR system of Nakamura et al.7 A six-histidine tag at the C terminus of H allows rescue and propagation of the virus in Vero-HIS cells transduced with a pDisplace (Invitrogen, Carlsbad, CA) plasmid to express a membrane-bound single-chain antibody that recognizes the six-histidine peptide.36 The MV-GFP virus was rescued as previously described37 and propagated in Vero cells. Virus stocks were prepared by infecting the appropriate Vero cell line with MV at a multiplicity of infection of 0.02 and incubating at 32 °C, 5% CO2. Virus was harvested by three freezethaw cycles from cellular substrate and resuspended in Opti-MEM (Life Technologies, Carlsbad, CA) after the third serial passage. Titers were determined by TCID50 according to the Kärber method on Vero-HIS or Vero cells.7 Western immunoblotting for EGFR and EGFRvIII. GBM6, GBM8, GBM12,

GBM14, and GBM39 xenograft tumors were mechanically dispersed in lysis buffer, and protein samples were subsequently sonicated and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gels were transferred to nitrocellulose (Bio-Rad, Hercules, CA) and blocked overnight at 4 °C in 1× casein in Tween-TBS (Vector, Burlingame, CA). The blot was incubated with rabbit anti-EGF receptor (Cell Signaling Technology, Danvers, MA) (1:1,000 in Tween-TBS, 0.5% nonfat dry milk) at room temperature for 60 minutes, then washed for 60 minutes in Tween-TBS and incubated with goat anti-rabbit IgG (Pierce, Rockford, IL) (1:1,000 in Tween-TBS containing 0.5% nonfat dry milk). After a final 2-hour wash, the blot was developed with SuperSignal West Femto Chemiluminescent Substrate (Pierce, Rockford, IL). Beta-actin was detected in parallel with a monoclonal anti–beta-actin antibody (Sigma, St. Louis, MO) followed by filter incubation with SuperSignal West Femto Chemiluminescent Substrate. Assessment of cytopathic effect in vitro. Cells were plated in 6-well plates

at a density of either 105 cells/well (CHO, CHO-CD46, CHO-SLAM, CHOEGFR, CHO-EGFRvIII) or 5 × 105 cells/well (GBM6, GBM8, GBM 12, GBM14, GBM39). Twenty-four hours after seeding, the cells were infected with MV-GFP or MV-GFP-HAA-scEGFR at different multiplicities of infection (0.1–1.0) in 1 ml of Opti-MEM for 2 hours at 37 °C. At the end of the incubation period, the virus was removed and the cells were maintained in their standard medium. Uninfected cells were used as controls. The number of viable cells in each well was counted

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using a hemocytometer at days 3, 5, 7, 9, and 11 after infection. Viable cells were identified using trypan blue exclusion. The percentage of surviving cells was calculated by dividing the number of viable cells in the infected well by the number of viable cells in the uninfected control well corresponding to the same time point. Presence of infection was confirmed using fluorescent microscopy at corresponding time points. Assessment of viral replication in glioma cell lines. Glioma lines (GBM6,

GBM8, GBM12, GBM14, and GBM39) were plated in 6-well plates at a density of 5 × 105 cells/well. The cells were infected as described above at a multiplicity of infection of 1.0 and harvested on days 1, 2, 3, 4, 5, and 7 after infection. The virus was released with two cycles of freeze-thawing. The viral titer was determined by 50% end point dilution assay as described above on Vero or Vero-HIS cells in 96-well plates. Generation of primary glioblastoma multiforme lines expressing firefly luciferase (Fluc). HIV-based lentiviral vectors expressing the firefly lucif-

erase gene were generated by transient transfection of 293T cells with plasmids encoding the vesicular stomatitis virus G envelope, gag-pol genes and pHRSIN-fluc with FuGENE 6 (Roche, Indianapolis, IN), using the calcium phosphate method. Forty-eight hours after transfection, conditioning medium containing the viral vectors was harvested, filtered using a 0.45 µm filter and frozen at −80°C until use. Primary GBM12 tumors were excised from mice and grown as previously described in T150 flasks using Dulbecco’s modified Eagle’s medium with 2.5% fetal bovine serum, 1× penicillin/streptomycin. When the cells reached 60% confluency, they were transduced using 2 ml of the supernatant and expression of Fluc was confirmed by measuring cellular luciferase activity (IVIS 200; Xenogen, Alameda, CA). Animal experiments. All animal experiments were approved by the Mayo

Clinic Institutional Animal Care and Use Committee. Orthotopic Gbm39 tumor model. We implanted 3 × 105 GBM12 Flucexpressing cells resuspended in phosphate-buffered saline orthotopically into the right caudate nucleus of 5-week old BALB/c nude mice using the small animal stereotactic frame (ASI Instruments, Warren, MI) and a 26-gauge Hamilton syringe, as described previously.2 Treatment was initiated 7 days after tumor cell implantation. The virus was administered intratumorally using the same coordinates as for tumor cell implantation. Treatment was repeated three times a week over a 3-week period for a total dose of 9 × 105 TCID50. The following three groups were included www.moleculartherapy.org vol. 15 no. 4, april 2007


(8–10 animals each): MV-GFP, UV-inactivated MV-GFP, and MV-GFPHAA-scEGFR. One mouse per group was euthanized 4 days after completion of treatment; tumor was fixed in paraformaldehyde and embedded in paraffin for histologic analysis. Mice were observed daily and were euthanized when they developed neurologic deficits or more than 10% weight loss was observed. In vivo bioluminescence imaging. Animals received intraperitoneal injection of 3 mg d-luciferin (Gold Biotechnology, St. Louis, MO) with xylazine/ketamine anesthesia. Images were acquired 10 minutes after luciferin administration using the Xenogen Ivis 200 System (Caliper Life Sciences, Hopkinton, MA). The Ivis 100 cooled CCD camera system was used for emitted light acquisition. Luciferase activity was analyzed using Living Image Software (version 2.5; Xenogen), according to the manufacturer’s instructions. Assessment of MV-GFP and MV-GFP-HAA-sc-EGFR toxicity in an Ifnarko CD46 Ge mouse model. Mouse cells normally do not express the MV CD46 or SLAM receptors; therefore, the animal models we used to assess efficacy do not allow adequate evaluation of the toxicity of MVGFP or the retargeted strain. For this purpose we performed a pilot toxicity study using the measles-susceptible transgenic mouse model Ifnarko CD46 Ge. The model was kindly provided by Dr. Roberto Cattaneo (Mayo Clinic) and was developed by insertion of a yeast artificial chromosome containing the human CD46 receptor and knockout of the interferon-αβ receptor.15 CD46 expression in the brain of CD46 Ge mice, as examined by western immunoblotting, has been shown to be comparable to CD46 expression in human brain.38 Injection of MV into the central nervous system of these mice causes lethal encephalitis.18,19 For this pilot toxicity study we employed the same parameters for stereotactic injection of the virus as for the efficacy studies described above. The animals were injected with a total dose of 2 × 105 TCID50 of either MV-GFP or MVGFP-HAA-scEGFR. The mice were followed on a daily basis, and were euthanized if they developed neurologic toxicity or weight loss of more than 10% of body weight. The experiment was terminated after a 4-week observation period. Statistical analysis. To assess animal survival, we generated Kaplan– Meir survival curves and compared them using the log-rank test. A Pvalue of