Volume 9, Issue 6, Pages 786-803 (June 2004) An E1B-19 kDa gene deletion mutant adenovirus demonstrates tumor necrosis factor- enhanced cancer selectivity and enhanced oncolytic potency  Ta-Chiang Liu, Gunnel Hallden, Yaohe Wang, Gabriel Brooks, Jennelle Francis, Nick Lemoine, David Kirn  Molecular Therapy  Volume 9, Issue 6, Pages 786-803 (June 2004) DOI: 10.1016/j.ymthe.2004.03.017 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Total viral replication in tumor cells is increased by E1B-19 kDa gene deletion. Cells were infected with 100 or 1000 ppc and harvested at indicated time points. Replication was assessed in the human tumor cells (A) PT45, (B) A2780, (C) A2780/CP, and (D) Hep3B; in murine tumor cells (E) CMT-167, (F) CMT-64, and (G) JC; and in human normal cells (H) NHBE. The resulting quantity of infectious virus was determined as described and values were normalized on an infectious unit produced per cell basis. Representative data from one experiment are shown here. Open bars, data from 48 h p.i.; black bars, data from 72 h p.i.; gray bars, data from 96 h p.i.; *level too low to be shown on the graph. Error bars represent standard errors between triplicates in one experiment. Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Virus release is increased by E1B-19 kDa gene deletion. Cells were infected as described, and culture media were collected and titered at indicated time points. Virus release was assessed in the human tumor cells (A) PT45, (B) A2780, and (C) A2780/CP; in murine tumor cells (D) CMT-167, (E) CMT-64, and (F) JC; and in human normal cells (G) NHBE. The resulting quantity of infectious virus was determined as described and values were normalized on an infectious unit produced per cell basis. Representative data from one experiment are shown here. Open bars, data from 48 h p.i.; black bars, data from 72 h p.i.; gray bars, data from 96 h p.i.; *level too low to be shown on the graph. Error bars represent standard errors between triplicates in one experiment. Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 EC50 value comparison between dl250 and Ad2wt; potency is increased by E1B-19 kDa gene deletion. The dose–response curves of Ad2wt, dl250, and inactivated dl312 in different cell lines were obtained by MTS assay as described. The EC50 values of each virus were then determined and compared in human cancer cells (A) PT45, (B) A2780, (C) A2780CP, and (D) Hep3B; in murine cancer cells (E) CMT-167, (F) CMT-64, and (G) JC; and in normal human cells (H) NHBE. Representative data from one experiment are shown here. Red curves, dl250; green curves, Ad2wt; blue curves, PUV-inactivated dl312. Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Effects of TNF-α on viral replication, cytopathic potency, and apoptosis induction. Viral replication, release, EC50, and apoptosis induction were measured with and without TNF treatment as described under Materials and Methods. Total viral replication was assessed in (A) PT45 cancer cells and (B) NHBE normal cells at 48, 72, and 96 h p.i., with/without TNF treatment. Viral release was assessed in (C) PT45 cancer cells and (D) NHBE normal cells at 48, 72, and 96 h p.i., with/without TNF treatment. Representative data from one experiment are shown here. Open bars, data from 48 h p.i.; black bars, data from 72 h p.i.; gray bars, data from 96 h p.i.; *TNF-α treatment; +level too low to be shown on the graph. Error bars represent standard errors between triplicates in one experiment. The percentage change in EC50 values of (E) Ad2wt and (F) dl250 after TNF treatment was studied. Error bars represent standard errors between different experiments (n = 4). In situ caspase-3 activation was determined in (G) NHBE and (H) PT45 cells as an indication of apoptosis induction. Cells were untreated or treated with viruses and/or TNF-α as described. At 24, 48, and 96 h p.i., cells were fixed and stained for active form caspase-3 as mentioned (n = 4). Open bars, data from 48 h p.i.; filled bars, data from 72 h p.i.; gray bars, data from 96 h p.i. Error bars represent standard errors between different experiments. Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Effects of TNF-α on viral replication, cytopathic potency, and apoptosis induction. Viral replication, release, EC50, and apoptosis induction were measured with and without TNF treatment as described under Materials and Methods. Total viral replication was assessed in (A) PT45 cancer cells and (B) NHBE normal cells at 48, 72, and 96 h p.i., with/without TNF treatment. Viral release was assessed in (C) PT45 cancer cells and (D) NHBE normal cells at 48, 72, and 96 h p.i., with/without TNF treatment. Representative data from one experiment are shown here. Open bars, data from 48 h p.i.; black bars, data from 72 h p.i.; gray bars, data from 96 h p.i.; *TNF-α treatment; +level too low to be shown on the graph. Error bars represent standard errors between triplicates in one experiment. The percentage change in EC50 values of (E) Ad2wt and (F) dl250 after TNF treatment was studied. Error bars represent standard errors between different experiments (n = 4). In situ caspase-3 activation was determined in (G) NHBE and (H) PT45 cells as an indication of apoptosis induction. Cells were untreated or treated with viruses and/or TNF-α as described. At 24, 48, and 96 h p.i., cells were fixed and stained for active form caspase-3 as mentioned (n = 4). Open bars, data from 48 h p.i.; filled bars, data from 72 h p.i.; gray bars, data from 96 h p.i. Error bars represent standard errors between different experiments. Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Adenovirus gene expression, replication, and infection-induced pathologic changes in normal liver tissue in vivo are inhibited by E1B-19 kDa deletion. Viruses were injected intravenously as described to infect the livers of immunocompetent mice. Viral gene expression, replication, pathology, and apoptosis induction by each virus were compared. (A) E1A expression (early viral gene). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (B) Representative examples of E1A staining from each treatment group (original magnification 10 × 20). (C) PFU (plaque-forming unit) recovery. Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (D) Cytopathic effect severity (scoring as under Materials and Methods). Open bars, 24 h postinjection; black bars, 48 h postinjection; gray bars, 120 h p.i. Error bars represent standard errors between different animals. *Undetectable. (E) Periportal inflammation severity (scoring as under Materials and Methods). Open bar, 24 h postinjection; filled bar, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (F) Representative examples of histopathology from each treatment group (original magnification 10 × 20). Filled arrows, classical cytopathic effect in livers after viral infection—eosinophilic cytoplasm and intranuclear inclusion body; open arrow, lymphocytic infiltration; N, necrotic area. (G) Active caspase-3 levels (scoring as under Materials and Methods). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (H) Representative examples of caspase-3 staining from each treatment group (original magnification 10 × 20). Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Adenovirus gene expression, replication, and infection-induced pathologic changes in normal liver tissue in vivo are inhibited by E1B-19 kDa deletion. Viruses were injected intravenously as described to infect the livers of immunocompetent mice. Viral gene expression, replication, pathology, and apoptosis induction by each virus were compared. (A) E1A expression (early viral gene). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (B) Representative examples of E1A staining from each treatment group (original magnification 10 × 20). (C) PFU (plaque-forming unit) recovery. Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (D) Cytopathic effect severity (scoring as under Materials and Methods). Open bars, 24 h postinjection; black bars, 48 h postinjection; gray bars, 120 h p.i. Error bars represent standard errors between different animals. *Undetectable. (E) Periportal inflammation severity (scoring as under Materials and Methods). Open bar, 24 h postinjection; filled bar, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (F) Representative examples of histopathology from each treatment group (original magnification 10 × 20). Filled arrows, classical cytopathic effect in livers after viral infection—eosinophilic cytoplasm and intranuclear inclusion body; open arrow, lymphocytic infiltration; N, necrotic area. (G) Active caspase-3 levels (scoring as under Materials and Methods). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (H) Representative examples of caspase-3 staining from each treatment group (original magnification 10 × 20). Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Adenovirus gene expression, replication, and infection-induced pathologic changes in normal liver tissue in vivo are inhibited by E1B-19 kDa deletion. Viruses were injected intravenously as described to infect the livers of immunocompetent mice. Viral gene expression, replication, pathology, and apoptosis induction by each virus were compared. (A) E1A expression (early viral gene). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (B) Representative examples of E1A staining from each treatment group (original magnification 10 × 20). (C) PFU (plaque-forming unit) recovery. Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (D) Cytopathic effect severity (scoring as under Materials and Methods). Open bars, 24 h postinjection; black bars, 48 h postinjection; gray bars, 120 h p.i. Error bars represent standard errors between different animals. *Undetectable. (E) Periportal inflammation severity (scoring as under Materials and Methods). Open bar, 24 h postinjection; filled bar, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (F) Representative examples of histopathology from each treatment group (original magnification 10 × 20). Filled arrows, classical cytopathic effect in livers after viral infection—eosinophilic cytoplasm and intranuclear inclusion body; open arrow, lymphocytic infiltration; N, necrotic area. (G) Active caspase-3 levels (scoring as under Materials and Methods). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (H) Representative examples of caspase-3 staining from each treatment group (original magnification 10 × 20). Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Adenovirus gene expression, replication, and infection-induced pathologic changes in normal liver tissue in vivo are inhibited by E1B-19 kDa deletion. Viruses were injected intravenously as described to infect the livers of immunocompetent mice. Viral gene expression, replication, pathology, and apoptosis induction by each virus were compared. (A) E1A expression (early viral gene). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (B) Representative examples of E1A staining from each treatment group (original magnification 10 × 20). (C) PFU (plaque-forming unit) recovery. Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (D) Cytopathic effect severity (scoring as under Materials and Methods). Open bars, 24 h postinjection; black bars, 48 h postinjection; gray bars, 120 h p.i. Error bars represent standard errors between different animals. *Undetectable. (E) Periportal inflammation severity (scoring as under Materials and Methods). Open bar, 24 h postinjection; filled bar, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (F) Representative examples of histopathology from each treatment group (original magnification 10 × 20). Filled arrows, classical cytopathic effect in livers after viral infection—eosinophilic cytoplasm and intranuclear inclusion body; open arrow, lymphocytic infiltration; N, necrotic area. (G) Active caspase-3 levels (scoring as under Materials and Methods). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (H) Representative examples of caspase-3 staining from each treatment group (original magnification 10 × 20). Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Adenovirus gene expression, replication, and infection-induced pathologic changes in normal liver tissue in vivo are inhibited by E1B-19 kDa deletion. Viruses were injected intravenously as described to infect the livers of immunocompetent mice. Viral gene expression, replication, pathology, and apoptosis induction by each virus were compared. (A) E1A expression (early viral gene). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (B) Representative examples of E1A staining from each treatment group (original magnification 10 × 20). (C) PFU (plaque-forming unit) recovery. Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (D) Cytopathic effect severity (scoring as under Materials and Methods). Open bars, 24 h postinjection; black bars, 48 h postinjection; gray bars, 120 h p.i. Error bars represent standard errors between different animals. *Undetectable. (E) Periportal inflammation severity (scoring as under Materials and Methods). Open bar, 24 h postinjection; filled bar, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (F) Representative examples of histopathology from each treatment group (original magnification 10 × 20). Filled arrows, classical cytopathic effect in livers after viral infection—eosinophilic cytoplasm and intranuclear inclusion body; open arrow, lymphocytic infiltration; N, necrotic area. (G) Active caspase-3 levels (scoring as under Materials and Methods). Open bars, 24 h postinjection; filled bars, 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. (H) Representative examples of caspase-3 staining from each treatment group (original magnification 10 × 20). Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Adenoviral gene expression and replication/persistence in replication-semipermissive murine tumor tissues in vivo. Viruses or PBS were injected on day 1 (109 particles) into JC tumors in immunocompetent mice. (A) E1A score (day 6 postinjection). Error bars represent standard errors between different animals. *Undetectable. (B) Plaque-forming unit (PFU) recovery data are shown on a per gram of tumor basis. Open bars, data from day 6; closed bars, day 10. Error bars represent standard errors between different animals. *Undetectable. Viruses (1 × 1010 particles) were injected on day 1 via tail vein into immunocompetent mice bearing CMT-167 tumors in the flank as described. (C) Hexon intensity score at various time points. Open bars, data from 24 h postinjection; filled bars, 48 h postinjection; gray bars, 120 h postinjection. Error bars represent standard errors between different animals. (D) PFU recovery data are shown on a per gram of tumor basis. Open bars, data from 24 h postinjection; filled bars, data from 48 h postinjection. Error bars represent standard errors between different animals. *Undetectable. Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 7 Antitumoral efficacy of E1B-19 kDa deletion mutant dl250 in vivo. PT45 human pancreatic cancer xenograft was implanted into C57BL/6 nu/nu mice as described. PBS or viruses (1 × 1010 particles) were injected intratumorally on days 1, 3, and 5. (A) Animal survival was monitored and compared. Both dl250 and Ad2wt treatment showed similar survival rates and both were higher than control group. (B) Tumor growth curve. Both dl250 and Ad2wt treatment groups showed significant delays in tumor growth compared with the PBS group. Molecular Therapy 2004 9, 786-803DOI: (10.1016/j.ymthe.2004.03.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions