1. Volume 16, Issue 10, Pages 1665-1673 (October 2008)
Immunosuppression Enhances Oncolytic Adenovirus Replication and Antitumor Efficacy in the Syrian Hamster Model 
Maria A Thomas, Jacqueline F Spencer, Karoly Toth, John E Sagartz, Nancy J Phillips, William SM Wold 
Molecular Therapy 
Volume 16, Issue 10, Pages (October 2008)
DOI: /mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Suppression of all immune cell types was observed in animals receiving cyclophosphamide (CP). An antitumor efficacy study was conducted in which parallel groups of immunocompetent and immunosuppressed hamsters were treated with intratumoral injections of VRX-007, wild-type adenovirus serotype 5 (Ad5), or vehicle. In this study, the first dose of CP was given on day −4 and was followed by twice weekly CP dosing throughout the study. Intratumoral oncolytic Ad (or vehicle) injections were administered on days 0, 1, 2, 4–6. The tumor suppression data are shown in Figure 2. Blood was collected and hematological analysis was performed on days −4, 8, 20, and 34 to monitor immunosuppression. The counts of total white blood cells (a), neutrophils (b), lymphocytes (c), and monocytes (d) are shown. No basophils were observed. The data are shown as the mean of each treatment group, ±SEM, n = 6/group. The legend for all panels is shown in a.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Immunosuppression with cyclophosphamide (CP) resulted in significantly enhanced tumor control with oncolytic adenoviruses (Ads). Following randomization based on tumor volume, animals in treatment groups receiving CP were administered the first dose of CP on day −4. CP was administered twice weekly for the duration of the study. The immunosuppression data are shown in Figure 1. Animals received intratumoral injections of VRX-007, Ad5, or vehicle on days 0, 1, 2, 4–6 (n = 12 for each of six groups). The mean tumor volume on day 0 was 1,294 μl. Tumor growth was monitored by tumor measurement with digital calipers. Due to anemia in animals treated with CP, the study was terminated at 41 and 42 days. (a) Median tumor volumes (in microliters) are shown for each group for the duration of the study. Statistically significant differences were found between virus treatment alone (“VRX-007–” or “Ad5–”) and virus plus CP (“VRX CP” or “Ad5 + CP”), in which all P ≤ No significant difference was detected between “Vehicle–” and “Vehicle + CP” (P = 0.692). All groups that received intratumoral virus treatment showed statistically significant tumor suppression when compared with “Vehicle–” or “Vehicle + CP” (P ≤ for all such comparisons). (b) Individual tumor volumes (in microliters) at the last tumor measurement, on day 38, are shown. Each point represents one animal and the bar represents the mean volume for each treatment group.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Adenovirus (Ad)-neutralizing antibody formation was inhibited by cyclophosphamide (CP). Serum was obtained from half of the animals in the study (Figure 2) at day 22 and from all animals at study termination (days 41 and 42). The ability of these sera to neutralize adenovirus was quantitated in a cytopathic effect–based neutralization assay. The Ad-neutralizing antibody titer for each serum sample at (a) 22 days and at (b) 41–42 days is shown. Only a subset of vehicle-treated animals was tested. Serum samples that did not show inhibition of Ad infection at the lowest dilution tested (1:20) were considered to be negative.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Immunosuppression plus oncolytic adenovirus (Ad), VRX-007, resulted in striking changes in tumor histology and evidence of ongoing Ad infection by immunohistochemistry. At the termination of the previously described study (Figure 2), hematoxylin and eosin (H&E)-stained tumor sections were examined histologically. (a) An H&E-stained tumor section of a representative animal in the “Vehicle–” group is shown, original magnification of ×10. This tumor is composed of viable tumor cells (designated by “t”) and virtually no necrosis. The tumor architecture is organized, with some gland formation, and the tumor cells are densely packed. A portion of the original tumor capsule (indicated by “cap”) is visible in this image. (b) Significant changes in tumor histology were noted in immunocompetent animals treated with oncolytic Ad, such as mild to moderate disorganization of tumor architecture, islands of live tumor cells (t) surrounded by necrosis (nec), calcification (Ca++), and decreased density of remaining tumor cells. A representative tumor from the “VRX-007–” group is shown, original magnification of × 10. (c) The most dramatic effect on tumors was noted in hamsters that were both immunosuppressed and treated with oncolytic Ad. In these animals, tumor architecture was disorganized, with moderate to severe necrosis (nec) and calcification (Ca++), as evidence of previous necrosis. In approximately half of these animals, only a small rim of viable tumor cells (t) remained and the majority of the mass was actually necrotic debris, as represented by the tumor from the “VRX CP” group shown here, original magnification of ×10. (d) Immunohistochemistry was performed with an anti-hexon (Ad structural protein) antibody on tumor sections obtained at study termination. Upper panel, only immunosuppressed animals treated with oncolytic Ad demonstrated positive anti-hexon staining nearly 6 weeks after the initial virus injection, as represented by the tumor shown from the “VRX CP” group. Lower panel, no staining was observed with an isotype control antibody on an adjacent tumor section from the same specimen. No areas of positive anti-hexon staining were observed at study termination in the tumors of immunocompetent animals treated with oncolytic Ad (data not shown). CP, cyclophosphamide.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Immunosuppression allowed virus levels to remain elevated for the duration of the antitumor efficacy study, while virus was cleared in immunocompetent animals. The tumor and a portion of the liver from 6 of 12 animals in the “VRX-007–” and “VRX CP” groups in the antitumor efficacy study (Figure 2) were obtained at study termination for virus quantitation. Tumor and liver samples were harvested and processed for virus titration by a tissue culture infectious dose 50 (TCID50) infectious assay as well as a quantitative real-time PCR assay for adenovirus (Ad) genomes. The titers obtained from (a) tumor and (b) liver specimens are shown. Left panels, TCID50 infectious titration. Right panels, quantitative real-time PCR titration. Each point represents the titer from one animal and the bars indicate the median for each group. The threshold for the TCID50 assay is shown by the dashed horizontal line. Samples in which no infectious virus was detected are indicated by “–.” CP, cyclophosphamide.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Adenovirus (Ad) levels in the tumor, blood, and liver of hamsters following a single intratumoral injection were determined in an independent replication kinetics study. Animals received a single intratumoral injection of either VRX-007 (circle symbols) or wild-type Ad serotype 5 (Ad5) (triangle symbols). Parallel groups of immunocompetent (solid symbols) and immunosuppressed (open symbols) animals were evaluated at 3, 9, and 22 days after injection. At each time point, the tumor, blood, and liver were harvested and processed for virus quantitation by tissue culture infectious dose 50 (TCID50) infectious titration and quantitative real-time PCR titration. The titers obtained (a) per tumor, (b) per ml of blood, (c) and per liver samples are shown. Left panels, TCID50 infectious titration. Right panels, quantitative real-time PCR titration. Each point represents the titer from one animal and the bars indicate the median for each group. The threshold for each assay is shown by the dashed horizontal line. Samples in which infectious virus was detected but was below the threshold of the assay are indicated by “+”. Samples in which no infectious virus was detected are indicated by “–.”
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions