Volume 16, Issue 12, Pages 1919-1926 (December 2008) Potent Systemic Anticancer Activity of Adenovirally Expressed EGFR-Selective TRAIL Fusion Protein  Edwin Bremer, Gooitzen M van Dam, Marco de Bruyn, Manon van Riezen, Marike Dijkstra, Gera Kamps, Wijnand Helfrich, Hidde Haisma  Molecular Therapy  Volume 16, Issue 12, Pages 1919-1926 (December 2008) DOI: 10.1038/mt.2008.203 Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 1 Schematic representation of the mechanism of action of scFv425:sTRAIL. Binding of scFv425:sTRAIL to the epidermal growth factor receptor (EGFR), via antibody fragment scFv425, inhibits mitogenic signaling of the active EGFR dimer. Consequently, the cell is sensitized to induction of apoptosis by TRAIL. In addition, scFv425-mediated binding immobilizes soluble scFv425:sTRAIL on the cell surface of EGFR-positive tumor cells and converts soluble scFv425:sTRAIL into a membrane-bound form. This membrane-bound from of TRAIL can efficiently cross-link and activate the agonistic TRAIL receptors TRAIL-R1 and TRAIL-R2, resulting in the induction of apoptosis. TM, transmembrane. Molecular Therapy 2008 16, 1919-1926DOI: (10.1038/mt.2008.203) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 2 EGFR-restricted induction of apoptosis by adenovirally produced scFv425:sTRAIL. (a) Cell death induced by Ad-scFv425:sTRAIL shown by MTS assay. RC21.luc cells were infected Ad-scFv425:sTRAIL or Ad-Luciferase at varying multiplicity of infections (MOIs). At 6 days after infection, loss of viability was quantified by MTS assay. (b) RC21 cells were infected with Ad-scFv425:sTRAIL or Ad-Luciferase (MOI 100), after which cell viability was determined at various time points. (c) RC21.luc cells were infected with Ad-scFv425:sTRAIL or Ad-Luciferase (MOI 100), after which DNA was isolated at day 6 of infection and evaluated for apoptotic DNA fragmentation. (d) Supernatant produced by Ad-scFv425:sTRAIL–infected RC21.luc cells was used to treat noninfected RC21.luc cells in the presence or absence of TRAIL-neutralizing mAb 2E5 or EGFR-blocking mAb 425. (e) RC21.luc cells were seeded in a trans-well system, after which cells in the lower compartment were treated with Ad-scFv425:sTRAIL at varying MOI or Ad-Luciferase at an MOI of 100. After 6 days, induction of apoptosis in both infected and noninfected RC21.luc target cells was analyzed by MTS assay. Values are representatives of three independent experiments. Molecular Therapy 2008 16, 1919-1926DOI: (10.1038/mt.2008.203) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 3 Efficient production and secretion of scFv425:sTRAIL upon Ad-scFv425:sTRAIL infection of nude mice. (a) Mice (n = 3) were infected with control adenovirus Ad-Luciferase and were imaged for bioluminescence 3 days after infection. A representative picture of bioluminescence (ph/sec/cm2/sr) after Ad-Luciferase infection is given. (b) Mice were infected with saline or low and high dose of Ad-scFv425:sTRAIL (109 and 1010 vp, respectively). After 3 days, concentration of scFv425:sTRAIL in plasma was determined by TRAIL ELISA. (c) RC21.luc cells were treated with plasma obtained from mice treated with saline, Ad-Luciferase, Ad-scFv425:sTRAIL (109 vp), or Ad-scFv425:sTRAIL (1010 vp). After 16 hours, induction of apoptosis was assessed by Δψ. (d) RC21.luc cells were treated with plasma obtained from mice infected with high-dose Ad-scFv425:sTRAIL in the presence or absence of TRAIL-neutralizing mAb 2E5 or EGFR-blocking mAb 425. Apoptosis was assessed by Δψ. Molecular Therapy 2008 16, 1919-1926DOI: (10.1038/mt.2008.203) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 4 No detectable liver damage after infection with Ad-scFv425:sTRAIL. (a) A representative picture of an isolated liver after Ad-scFv425:sTRAIL (1010 vp) infection. (b) Isolated livers of saline, low- and high-dose Ad-scFv425:sTRAIL-treated mice were analyzed for caspase-3/-7 activity. As positive control for liver caspase-3–7 activation, mice were treated with Flag-FasL secondarily cross-linked with anti-Flag mAb M2. Values are represented as relative light units per mg of liver. Normal liver histology, as determined by hematoxylin and eosin staining upon (c) saline treatment, (d) Ad-scFv425:sTRAIL (109 vp)-treated and (e) in Ad-scFv425:sTRAIL (1010 vp)-treated mice. Original magnification ×200. (f) Primary human hepatocytes (PHHs) were infected with Ad-scFv425:sTRAIL (MOI 100), Ad-Luciferase (MOI 100), or treated with killer-FasL (5 µg/ml). Viability at 3 days after infection was measured using MTS assay. (g) PHHs were treated for 16 hours with plasma obtained from mice infected with Ad-scFv425:sTRAIL (1010 vp), yielding a concentration of 5 µg/ml, saline control, or killer-FasL (5 µg/ml). Apoptosis was assessed by Δψ. Molecular Therapy 2008 16, 1919-1926DOI: (10.1038/mt.2008.203) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 5 Ad-scFv425:sTRAIL infection eradicates established RC21.luc xenografts in nude mice. Nude mice with established IP RC21.luc xenografts were treated with (a) saline, (b) Ad-scFv425:sTRAIL (109 vp), (c) Ad-scFv425:sTRAIL (1010 vp). Tumor size was monitored real-time using bioluminescence imaging. The luminescence image was merged with the reference image to create an overlay image that enables anatomical localization. Images were displayed and quantified in log radiance (photons/s/cm2/sr), enabling absolute comparison between bioluminescent images across different treatment groups. Mice treated with saline and low-dose Ad-scFv425:sTRAIL were killed after 32 days. Mice treated with high dose of Ad-scFv425:sTRAIL were killed at day 64. The two panels in a, b and c show the variability in tumor size within each treatment group at the start of treatment. Molecular Therapy 2008 16, 1919-1926DOI: (10.1038/mt.2008.203) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 6 Ad-scFv425:sTRAIL infection eradicates established RC21.luc xenografts in nude mice. (a) Graph of tumor size in mice treated with saline, Ad-scFv425:sTRAIL (109 vp), and Ad-scFv425:sTRAIL (1010 vp). Tumor size was monitored using bioluminescence imaging and is represented as relative size compared with tumor size at start of treatment. (b). Concentration of plasma scFv425:sTRAIL in mice treated with Ad-scFv425:sTRAIL (109 vp). (c) Concentration of plasma scFv425:sTRAIL in mice treated with Ad-scFv425:sTRAIL (1010 vp) (d) Concentration of scFv425:sTRAIL in plasma and IP fluid in mice treated with Ad-scFv425:sTRAIL (109vp) at day 64 after infection. In b–d, TRAIL concentrations were determined using TRAIL ELISA. Molecular Therapy 2008 16, 1919-1926DOI: (10.1038/mt.2008.203) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions