Volume 15, Issue 6, Pages 1121-1128 (June 2007) Carrier Cell–mediated Delivery of a Replication-competent Adenovirus for Cancer Gene Therapy  Katsuyuki Hamada, Junzo Desaki, Kou Nakagawa, Ting Zhang, Toshiro Shirakawa, Akinobu Gotoh, Masatoshi Tagawa  Molecular Therapy  Volume 15, Issue 6, Pages 1121-1128 (June 2007) DOI: 10.1038/sj.mt.6300128 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Viral production and cytotoxicity of AdE3-IAI.3B-infected cells. (a) Viral production of AdE3-IAI.3B or AdE3 in cell lysates (cell) and supernatants (supt). (b) Cytotoxicity of AdE3-IAI.3B-infected cells against ovarian cancer PA-1 cells was determined by cell count assay. Values in a and b are the means ± SD for five replicates. PFU, plaque-forming unit; IC50, 50% growth inhibitory concentration. Molecular Therapy 2007 15, 1121-1128DOI: (10.1038/sj.mt.6300128) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Anti-tumor effects of AdE3-IAI.3B alone and AdE3-IAI.3B-infected A549, 293, and SW626 carrier cells on subcutaneous PA-1 (a) and RMG-1 (b) tumor growth in nude mice. Mice were intra-tumorally injected with medium alone, AdE3-IAI.3B alone, or AdE3-IAI.3B-infected A549, 293, and SW626 carrier cells on days 0, 1, 2, 3, 4, and 5. Values in a and b are the means ± SD for ten female nude mice. Molecular Therapy 2007 15, 1121-1128DOI: (10.1038/sj.mt.6300128) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Effects of immunization with Ad-β-gal or UV-inactivated Ad-β-gal on survival of OVHM tumor–bearing mice that received AdE3-IAI.3B- and/or AxCAmGM-CSF-infected A549 and/or 293 carrier cells. (a) Mice were intra-tumorally injected with medium alone, AdE3-IAI.3B alone, or AdE3-IAI.3B-infected A549 carrier cells with or without immunization on days 0, 1, 2, 3, 4, and 5. (b) After one (×1), two (×2), or three (×3) immunizations with Ad-β-gal, mice were treated with AdE3-IAI.3B-infected A549 carrier cells on days 0, 1, 2, 3, 4, and 5. (c) After immunization with Ad-β-gal or UV-inactivated Ad-β-gal, mice were treated with A549 carrier cells infected with AdE3-IAI.3B and/or AxCAmGM-CSF on days 0, 1, and 2. (d) After immunization with Ad-β-gal or UV-inactivated Ad-β-gal, mice were treated with A549 and/or 293 carrier cells co-infected with AdE3-IAI.3B and AxCAmGM-CSF on day 0. Values in a, b, c, and d are the means ± SD for ten female (C57BL/6 × C3H/He) F1 mice. Molecular Therapy 2007 15, 1121-1128DOI: (10.1038/sj.mt.6300128) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Carrier cells circumvent adenoviral humoral immune responses and induce tumor-specific immunity. (a) Time course of change in anti-adenovirus antibody titer after subcutaneous injection of Ad-β-gal (Ad) or UV-inactivated Ad-β-gal (UV) in syngeneic mice. Serum samples were collected 3, 6, 9, and 12 weeks (wk) after a single injection (×1) of Ad-β-gal (black column) or UV-inactivated Ad-β-gal(gray column). Serum samples were also collected 3 weeks after the first (×1), second (×2), and third injections (×3) of Ad-β-gal, and 6 weeks after the third injection (×3) of Ad-β-gal (white column). AdE3-IAI.3B-infectedHT-3, SW626, A549, and 293 cells inhibited growth of PA-1 cells in medium with (Ab (+)) or without (Ab (−)) anti-adenovirus antibody, but AdE3-IAI.3B did so only in medium without anti-adenovirus antibody (Ab (−)) (b). (c) A549 carrier cells with or without three freeze-and-thaw cycles after infection with AdE3-IAI.3B for 24 hours were incubated in anti-adenovirus antibody–containing medium for 10 days with separation from adherent target PA-1 cells by Millicell filter chambers (0.4 μm, 12 μm). (d) Splenic lymphocytes were isolated from tumor-bearing syngeneic mice after injection of phosphate-buffered saline, Ad-β-gal, and UV-inactivated Ad-β-gal(UV-Ad-β-gal), and from mice with complete tumor regression after injection of A549 carrier cells infected with AdE3-IAI.3B or with AdE3-IAI.3B and AxCAmGM-CSF after immunization with Ad-β-gal. OVHM cells infected with Ad-β-gal(Ad-OVHM) or uninfectedcells (OVHM) were used as target cells for the assay of cytotoxic T-lymphocyte (CTL) activity. Values in a, b, c, and d are the means ± SD for five replicates. Molecular Therapy 2007 15, 1121-1128DOI: (10.1038/sj.mt.6300128) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Electron micrographs of A549 carrier cells and of A549 and PA-1 target cells. (a) Control A549 cells generated microvilli and short cytoplasmic processes (arrows). A549 carrier cells were infected with AdE3-IAI.3B for 24 hours, harvested, and then cultured with target A549 cells for 24 hours. A549 carrier cells generated viral particle–containing blebs (arrow) (b) and delivered viral particle–containing cell fragment vesicles (arrows) (c). (d) Nuclei and intracellular organelles of A549 carrier cells still contained viral particles after necrosis. Viral particle–containing cell fragments (arrow) were captured by cytoplasmic processes of target A549 cells (e) and directly infected cytoplasmic process of target A549 cell with viruses (arrow) (f). (g) A viral particle–containing cell fragment (arrows) engulfed by a target A549 cell. (h) Control PA-1 target cells had cytoplasmic processes (arrow). AdE3-IAI.3B-infected A549 carrier cells were harvested and cultured with target PA-1 cells for 24 hours. (i) A viral particle–containing cell fragment (arrow) engulfed by a target PA-1 cell. C and T, carrier and target cells, respectively. The scale bar in the lower right corner of each figure indicates 1 μm. Molecular Therapy 2007 15, 1121-1128DOI: (10.1038/sj.mt.6300128) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions