Volume 18, Issue 4, Pages 852-860 (April 2010) A Phase I Trial of Repeated Intrapleural Adenoviral-mediated Interferon-β Gene Transfer for Mesothelioma and Metastatic Pleural Effusions  Daniel H Sterman, Adri Recio, Andrew R Haas, Anil Vachani, Sharyn I Katz, Colin T Gillespie, Guanjun Cheng, Jing Sun, Edmund Moon, Luana Pereira, Xinzhong Wang, Daniel F Heitjan, Leslie Litzky, Carl H June, Robert H Vonderheide, Richard G Carroll, Steven M Albelda  Molecular Therapy  Volume 18, Issue 4, Pages 852-860 (April 2010) DOI: 10.1038/mt.2009.309 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Pleural gene transfer and serum adenoviral neutralizing antibody (Nab) data. (a,b) Levels of IFN-β protein (ng/ml) in pleural fluid samples removed via the tunneled pleural catheter (PleurX) (y axis) were measured by enzyme-linked immunosorbent assay and plotted versus the time after Ad.IFN-β instillation (arrows). Day 1 shows levels of preadministration. a shows data from patients given a second dose on day 14. b shows data from patients given a second dose on day 7. (c) Adenovirus neutralizing antibodies. c plots the inverse titer of Nab versus time (in weeks) after the first Ad.IFN-β instillation. Virtually every patient rapidly developed high titers (>1:1,000) of anti-Ad Nab within 1 week. (d,e) Relationship between Nab titers and gene transfer. d plots the relationship between the pleural fluid IFN-β level (in ng/ml on the y axis) versus the serum adenovirus neutralizing antibody titer (expressed as 1/titer on the x axis). A value of 10 signifies a titer of 1:10 or less. e plots the percentage of patients who showed detectable levels of IFN-β after gene transfer versus their Nab titers at the time of the instillation of Ad.IFN-β. Data from both the first and second viral instillation are graphed. Molecular Therapy 2010 18, 852-860DOI: (10.1038/mt.2009.309) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Antitumor immune responses. Antibody responses against tumor antigens were visualized on immunoblots using patient serum (diluted 1:1,500) before and after gene transfer. (a) Response of mesothelioma patient 213 to purified mesothelin (meso) and SV40 large T-antigen (SV40). Purified mesothelin and SV40 protein were run on SDS-PAGE gels, transferred to nitrocellulose, and immunoblotted with diluted pre- and 6-week postgene transfer serum. Blots were stripped and blotted with commercial anti-SV40 Tag and antimesothelin antibodies to show equal loading of SV40 Tag and mesothelin (data not shown). (b) Immunologic response of patient 214 to an autologous breast cancer cell extract. Extracts from a breast cancer cell line established from this patient's own pleural fluid were run on an SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted with diluted pre- and postgene transfer serum. Molecular Therapy 2010 18, 852-860DOI: (10.1038/mt.2009.309) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Clinical responses. (a) Waterfall plot of tumor responses determined by CT scan using modified RECIST criteria. The plot shows the change in tumor size comparing pretreatment to 2 months postvector measurements for each patient. By modified RECIST criteria, >30% reduction in tumor size defines a partial response, >25% increase in size defines progressive disease, and change in tumor size from <30% reduction to <25% increase is defined as stable disease. Data from patient 203 (outside films were not available for quantification) and patients 211 and 219 (whose tumor was not measurable) are not included. (b) CT scans and FDG-PET scans from patient 218 prevector instillation (left panels), 3 months after vector instillation (middle panels), and 6 months post-therapy (right panels) show a mixed response. PET scan identifies a right apical lesion (solid white arrows) that does not change over time (upper two rows) and a basilar lesion (dotted white arrow) pretherapy that demonstrated decreased FDG uptake at the 3- and 6-month time point. The patient received no other treatment. (c) A Kaplan–Meier plot of survival of the 17 patients with malignant pleural mesothelioma treated with Ad.IFN-β in our original one dose trial5 and the current study. The median survival (arrow) is 22 months. Three patients remain alive at 42, 39, and 18 months after vector administration (as of October 2009). CT, computed tomography; FDG, fluorodeoxyglucose; PET, positron emission tomography; RECIST, Response Evaluation Criteria in Solid Tumors. Molecular Therapy 2010 18, 852-860DOI: (10.1038/mt.2009.309) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions