Volume 16, Issue 2, Pages 244-251 (February 2008) Virotherapy of Ovarian Cancer With Polymer-cloaked Adenovirus Retargeted to the Epidermal Growth Factor Receptor  Joanne Morrison, Simon S Briggs, Nicola Green, Kerry Fisher, Vladimir Subr, Karel Ulbrich, Sean Kehoe, Leonard W Seymour  Molecular Therapy  Volume 16, Issue 2, Pages 244-251 (February 2008) DOI: 10.1038/sj.mt.6300363 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Poly-(2-hydroxypropyl)methacrylamide (pHPMA) production and physical properties of pHPMA-adenovirus. (a) Diagrammatic representation of mEGF-pHPMA synthesis. For further details see methods. (b) Photon correlation spectroscopy measurement of virus particle size, before and following pHPMA or mEGF-pPHMA coating. Mean unmodified adenovirus = 118 nm; pHPMA-adenovirus and mEGF-pHPMA-adenovirus coated at 10 mg/ml = ∼135 nm. Ad, unmodified adenovirus; p-Ad, pHPMA-adenovirus; mEGF-p-Ad, mEGF-pHPMA-adenovirus; mEGF, murine epidermal growth factor. Molecular Therapy 2008 16, 244-251DOI: (10.1038/sj.mt.6300363) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Biological activity of epidermal growth factor (EGF) following conjugation with poly-(2-hydroxypropyl)methacrylamide (pHPMA). (a) EGF receptor (EGFR) negative (K562) and positive (A431) cells were incubated with biotinylated mEGF-pPHMA (solid histogram) and a biotinylated pHPMA control (broken line, open histogram) followed by labeling with extravidin phycoerythrin (PE) and analysis by flow cytometry (cells plus extravidin-PE shown with solid line, open histogram). Percentage of positive cells following incubation with mEGF-pHPMA and extravidin-PE are indicated. (b) Western blot of mEGF-pHPMA and murine EGF (mEGF) stimulation of EGFR autophosphorylation (Y1092). A431 cells were serum starved and stimulated for 30 minutes with mEGF or mEGF-pHPMA. Cell lysates were run on a 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis, blotted onto nitrocellulose paper, and developed with antibodies against phosphorylated EGFR. Concentrations of mEGF-pHPMA relate to the mEGF content only. (c) mEGF (top row) and mEGF-pHPMA (bottom row) were added to serum-starved A431 cells and EGFR labeled (green) in cells incubated for 0, 20, and 60 minutes to demonstrate the effect of mEGF and mEGF-pHPMA on EGFR internalization. Labeling of mEGF-pHPMA with Texas red (red) in the right hand panels demonstrates co-localization of EGFR and mEGF-pHPMA following internalization (yellow). Scale bars represent 10 μm. These data are representative of at least three independent experiments. Molecular Therapy 2008 16, 244-251DOI: (10.1038/sj.mt.6300363) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Epidermal growth factor receptor (EGFR) status of cell lines used. Murine A9 fibroblast cells were stably transfected with a human EGFR containing plasmid, clones isolated and screened for EGFR expression. (a) Flow cytometry for EGFR status of murine A9 fibroblast cells transfected with human EGFR: A549 cells (positive control); parental A9 cells (negative control) and A9-EGFR clone. Broken line histograms represent isotype control antibody labeling and solid line histograms represent anti-EGFR antibody labeling. Percentage of positive cells following incubation with anti-EGFR antibody indicated for each cell line. Data are representative of repeated measurements over a 12-month period. (b) poly-(2-hydroxypropyl)methacrylamide (pHPMA) and mEGF-pHPMA (10 mg/ml) were used to coat AdCMV-LucΔE1 and transduction efficiency measured by a luciferase assay in the EGFR-positive, coxsackie-adenovirus receptor (CAR)-negative A9 cell line (right panel) (P = 0.0018), and EGFR-negative, CAR-negative parental A9 cells (left panel). (c) murine EGF (mEGF) binding to EGFR was blocked in A9-EGFR cells by preincubating cells for 30 minutes with an anti-EGFR antibody (GR01L). No anti-EGFR blockage (unfilled bars); preincubation with anti-EGFR antibody (black bars); (***P < 0.001). Data in b and c are representative of three independent experiments performed in triplicate. Bars represent mean + SD. Ad, unmodified adenovirus; p-Ad, pHPMA-adenovirus; mEGF-p-Ad, mEGF-pHPMA-adenovirus. Molecular Therapy 2008 16, 244-251DOI: (10.1038/sj.mt.6300363) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 In vitro transduction efficiency of mEGF-pHPMA modified adenovirus (a) mEGF-pHPMA-adenovirus transduction of a range of epidermal growth factor (EGFR)-positive cell lines. Unmodified AdCMV-LucΔE1 (unfilled bars); poly-(2-hydroxypropyl)methacrylamide (pHPMA)-adenovirus (hashed bars); mEGF-pHPMA-adenovirus (black bars).For all cell lines the level of transgene expression following transduction with pHPMA-adenovirus was compared to mEGF-pHPMA-adenovirus (***P < 0.001). (b) Coxsackie-adenovirus receptor (CAR) and EGFR status of each cell line was assessed by flow cytometry (data not shown) and EGFR levels were formally quantified using a commercial enzyme-linked immunosorbent assay, which gave a similar pattern of results. (c) Levels of luciferase gene expression following transduction with unmodified AdCMV-Luc ΔE1 and mEGF-pHPMA-adenovirus were compared with cellular EGFR levels. (d) Specific activity of mEGF-pHPMA-adenovirus in EGFR-positive cells (luciferase activity per virus particle, as measured by quantitative polymerase chain reaction (QPCR)) was assessed by transduction of A431 cells with AdCMV-LucΔE1. After 24 hours, cell lysate was analyzed for luciferase activity and DNA extracted for viral particle estimation by QPCR. Data are representative of at least two independent experiments performed in at least triplicate. Bars represent + SD. Ad, unmodified adenovirus; p-Ad, pHPMA-adenovirus; mEGF-p-Ad, mEGF-pHPMA-adenovirus. (e, f) Cytotoxicity analysis of mEGF-pHPMA-Ad5WT. pHPMA and mEGF-pHPMA (10 mg/ml) were used to coat Ad5WT. Known concentrations of adenovirus in plaque forming units (pfu) were incubated with A2780 cells (e) and SKOV-3 cells (f). After 6 days an MTS assay was performed to assess cell survival. Bars represent + SD. Dashed line with open squares, unmodified Ad5WT; dotted line with crosses, pHPMA-Ad5WT; solid line with filled diamonds, mEGF-pHPMA-Ad5WT. mEGF, murine EGF; RLU, relative light units. Molecular Therapy 2008 16, 244-251DOI: (10.1038/sj.mt.6300363) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 In vivo efficacy of mEGF-pHPMA retargeted wild-type Ad5. Female nude mice implanted with SKOV-luc cells intraperitoneally and treated with 3 doses of Ad5WT (5 × 1010 virus particles of unmodified Ad5WT [solid line, open inverted triangles); pHPMA-Ad5WT (dashed line, pink triangles); or mEGF-pHPMA-Ad5WT (dashed line, blue diamonds)] or a phosphate-buffered saline (PBS) control (solid line, closed squares). (a) Kaplan–Meier survival curves. (b) Bioluminescence measurements of tumor load 16 days after tumor inoculation. Representative merged luminescence images for each group are shown. Bars represent median group values and points are individual mouse luminescence. (**P < 0.01; ***P < 0.001; ns, not significant (P > 0.05)) (c) Adhesion scores for each group; mEGF-pHPMA-adenovirus treated mice had significantly fewer adhesions than mice treated with unmodified adenovirus by Chi-squared analysis (P = 0.00039). Bars represent the percentage of each group: no adhesions (open bars); mild adhesions (hashed bars); moderate adhesions (dotted bars); severe adhesions (black bars). (**P < 0.01, ***P < 0.001) (d) Representative images of mice from each group are shown. Ad, unmodified Ad5WT; p-Ad, pHPMA-Ad5WT; mEGF-p-Ad, mEGF-pHPMA-Ad5WT. Data are consistent with data from a randomized and blinded tumor load pilot study (data not shown), which were used in an a priori power calculation for this survival experiment. mEGF, murine epidermal growth factor; ns, not significant; pHPMA, N-(2-hydroxypropyl)methacrylamide. Molecular Therapy 2008 16, 244-251DOI: (10.1038/sj.mt.6300363) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions