Volume 6, Issue 3, Pages 342-348 (September 2002) Impact of Preimmunization on Adenoviral Vector Expression and Toxicity in a Subcutaneous Mouse Cancer Model  Maria T. Vlachaki, Andres Hernandez-Garcia, Michael Ittmann, Madhu Chhikara, Laura K. Aguilar, Xiaohong Zhu, Bin S. The, E.Brian Butler, Shiao Woo, Timothy C. Thompson, Hugo Barrera-Saldana, Estuardo Aguilar-Cordova  Molecular Therapy  Volume 6, Issue 3, Pages 342-348 (September 2002) DOI: 10.1006/mthe.2002.0669 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 Total anti-AdV antibodies evaluated by ELISA. Significantly higher circulating antibody titers were observed in preimmunized compared with naïve animals at all time points (P < 0.011). Antibody titers peaked 1 week after intratumoral AdV-luc administration in both groups (P < 0.009). Molecular Therapy 2002 6, 342-348DOI: (10.1006/mthe.2002.0669) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Tumor and peripheral organ luciferase activity in naïve and immunized animals. Luciferase levels are expressed as relative light units per g of tissue (RLU/g). Animals (five per group) were immunized with 1 × 1011 vp 14 days before intratumor inoculation. Preimmunization lowered luciferase expression by 714- to 2392-fold (P < 0.001) in peripheral organs, but only by 24-fold (P = 0.004) in tumor. Molecular Therapy 2002 6, 342-348DOI: (10.1006/mthe.2002.0669) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 AdV-luc expression in tumor and liver as a function of inoculum dose. Animals were injected with the doses indicated and tissues were analyzed 3 days post-intratumoral injection. Bars denote luciferase activity in escalating doses of naïve (first two groups) and immunized (next five groups) animals. Molecular Therapy 2002 6, 342-348DOI: (10.1006/mthe.2002.0669) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 AdV-luc expression and tumor to liver gene expression ratio as a function of time. Animals were injected with 2 × 1011 vp/tumor and tissues were analyzed at the times noted. The left axis indicates the level of luciferase activity in naïve and immunized animals. The right axis indicates the tumor to liver gene expression ratio of naïve (open circles) and immunized (closed squares) animals. Intratumoral luciferase levels peaked on day 2 after AdV-luc administration and decreased significantly on day 7, both in naïve and preimmunized animals. In naïve animals, intratumoral luciferase levels were significantly higher at all time points (P < 0.004). Molecular Therapy 2002 6, 342-348DOI: (10.1006/mthe.2002.0669) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Histologic analysis after intratumor adenoviral injection in naïve and preimmunized animals. Tissues for histologic evaluation were harvested on day 3 after intratumoral AdV-luc administration of 6 × 1011 vp. (A) Normal control liver showing portal area. (B) Similar area from injected naïve animal. Mixed inflammatory infiltrates around central vein (C) and portal area (D) from injected immunized animals are shown. Note large infiltrate designated by arrowheads. (E) Graphic presentation of peripheral organ pathological toxicity classification, from 0 (normal histology) to 4 (extensive inflammation with areas of necrosis). Significantly higher scores for hepatic toxicity were noted in all preimmunized animals (P < 0.001), which consistently presented with histologic evidence of inflammation, lymphocytic infiltration and necrosis. Molecular Therapy 2002 6, 342-348DOI: (10.1006/mthe.2002.0669) Copyright © 2002 American Society for Gene Therapy Terms and Conditions