Volume 8, Issue 3, Pages 449-458 (September 2003) Enhanced therapeutic efficacy for ovarian cancer with a serotype 3 receptor-targeted oncolytic adenovirus  Anna Kanerva, Kurt R Zinn, Tandra R Chaudhuri, John T Lam, Kaori Suzuki, Taco G Uil, Tanja Hakkarainen, Gerd J Bauerschmitz, Minghui Wang, Bin Liu, Zhihong Cao, Ronald D Alvarez, David T Curiel, Akseli Hemminki  Molecular Therapy  Volume 8, Issue 3, Pages 449-458 (September 2003) DOI: 10.1016/S1525-0016(03)00200-4 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Structure of Ad5/3-Δ24. Ad5/3-Δ24 has a 24-bp deletion in constant region 2 (CR2) of the E1A gene, corresponding to the region utilized for Rb protein binding. This results in an agent replication competent and oncolytic in cells defective in the Rb/p16 pathway, such as ovarian cancer cells. The fiber is modified to incorporate the serotype 3 knob, while retaining the Ad5 shaft and tail. Molecular Therapy 2003 8, 449-458DOI: (10.1016/S1525-0016(03)00200-4) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Ad5/3-Δ24 displays increased cell killing of ovarian cancer cells. Cells were infected with 0, 0.1, 1, or 10 vp/cell of Ad5/3-Δ24, Ad5-Δ24E3 (non-fiber-modified isogenic control virus), or AdCMVHSV-TK (E1-deleted control virus). On day 6 (SKOV3.ip1), day 13 (OV-4), day 6 (OV-3), day 10 (Hey), or day 10 (ES-2) cell viability was measured with the MTS assay. The OD490 values of uninfected cells were set as 100%. Data are expressed as means ± SD of quadruplicate experiments. With all cell lines, oncolysis was significantly improved with Ad5/3-Δ24 compared to Ad5-Δ24E3 or AdCMVHSV-TK (all P values < 0.004). Molecular Therapy 2003 8, 449-458DOI: (10.1016/S1525-0016(03)00200-4) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Ad5/3-Δ24 replicates in three-dimensional human ovarian primary cancer cell spheroids. Purified, unpassaged cancer cells were allowed to form spheroids, which were infected with 1000 vp/surface cell of Ad5/3-Δ24, Ad5-Δ24E3 (non-fiber-modified isogenic control virus), or AdCMVHSV-TK (E1-deleted control virus). Spheroids and growth medium were harvested at indicated time points, and virus copy number was measured with quantitative PCR. (A–D) The increase in virus copy number with Ad5/3-Δ24 and Ad5-Δ24E3 in patient samples 1–4, respectively. (E–H) Cumulative values from patients 1–4 allow comparison of total virus production and replication kinetics. The background values (uninfected spheroids) were subtracted from the data at each time point. Molecular Therapy 2003 8, 449-458DOI: (10.1016/S1525-0016(03)00200-4) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Therapeutic effect of Ad5/3-Δ24 in an animal model of peritoneally disseminated ovarian cancer. SKOV3.ip1 cells were injected ip into SCID mice and advanced carcinomatosis was allowed to develop for 10 days. (A) The mice received a single ip injection of 3 × 107 vp of Ad5/3-Δ24, Ad5-Δ24E3, AdCMVHSV-TK or no virus. (B) Intraperitoneal injection with 1 × 108 vp daily on 3 consecutive days (days 10, 11, and 12). The median survival was not reached with Ad5/3-Δ24 in either experiment by day 135. In both A and B, the overall survival of mice treated with Ad5/3-Δ24 was statistically significant in comparison to other groups (P < 0.0001). Molecular Therapy 2003 8, 449-458DOI: (10.1016/S1525-0016(03)00200-4) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Noninvasive assessment of Ad5/3-Δ24 efficacy in vivo. SKOV3-luc cells were injected ip into SCID mice (day 0) and advanced carcinomatosis was allowed to develop. The mice received a single ip injection of 3 × 107 vp of Ad5/3-Δ24 or no virus on day 8. Bioluminescence was imaged weekly starting on day 7. Location and magnitude of light captured by optical CCD imaging after ip injection of d-luciferin on day 21, Ad5/3-Δ24 treated (A), no virus (B). A pseudocolor image representing light intensity (blue, least intense, and red, most intense) and gray-scale reference images were superimposed. (C) Signal from the entire abdominal region of each mouse were quantified, and the mean photon counts are shown. SKOV3-luc ovarian cancer cells express firefly luciferase, which reacts with d-luciferin in a bioluminescence reaction. Therefore, reduction in signal indicates killing of cancer cells by Ad5/3-Δ24. Bars indicate SD. *Imaging performed after removal of the abdominal wall. Molecular Therapy 2003 8, 449-458DOI: (10.1016/S1525-0016(03)00200-4) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 The oncolytic efficacy of Ad5/3-Δ24 is comparable or superior to that of Ad5-Δ24RGD. (A) SKOV3.ip1 monolayers were infected with 0, 1, or 100 vp/cell of Ad5/3-Δ24 or Ad5-Δ24RGD (isogenic control virus with an RGD-4C capsid modification). Cell viability was measured with MTS assay on days 5, 9, and 13. The OD490 values of uninfected cells were set as 100% at each time point. Data are expressed as means ± SD of quadruplicate experiments. Oncolysis was significantly improved with Ad5/3-Δ24 compared to Ad5-Δ24RGD (on day 13, 100 vp/cell, P = 0.2652; all other P < 0.005). (B) Human primary ovarian adenocarcinoma cells were purified and cultured as spheroids, and CRAd infections were performed. Cell killing was analyzed with MTS assay. With patient sample A, Ad5/3-Δ24 displayed faster oncolysis (P = 0.0020 on day 8). Molecular Therapy 2003 8, 449-458DOI: (10.1016/S1525-0016(03)00200-4) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions