Volume 14, Issue 3, Pages 361-370 (September 2006) Systemic Armed Oncolytic and Immunologic Therapy for Cancer with JX-594, a Targeted Poxvirus Expressing GM-CSF  J.H. Kim, J.Y. Oh, B.H. Park, D.E. Lee, J.S. Kim, H.E. Park, M.S. Roh, J.E. Je, J.H. Yoon, S.H. Thorne, D. Kirn, T.H. Hwang  Molecular Therapy  Volume 14, Issue 3, Pages 361-370 (September 2006) DOI: 10.1016/j.ymthe.2006.05.008 Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 1 (A) Genetic construct for JX-594 (reproduced from Mastrangelo et al. [13]). (B) Replicative burst (plaque-forming units per well) of wild-type and thymidine kinase (TK)-deleted Wyeth vaccinia viruses in cancer cells (MCF-7, Panc-1) and in normal MRC-5 fibroblasts (proliferating or nonproliferating). (C) Mean (±SE) GM-CSF concentrations in the infected cell supernatant 24 h after infection with JX-594 at an m.o.i. of 0.1. Molecular Therapy 2006 14, 361-370DOI: (10.1016/j.ymthe.2006.05.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 2 VX2 rabbit tumor model development for use with JX-594. (A) Replicative burst over time (pfu per cell) of JX-594 (m.o.i. 0.1) in vitro in VX2 rabbit cells versus murine (TIB-75) and human (A2780) carcinoma cells. (B) Mean (±SE) VX2 primary tumor size within the liver over time as measured by serial CT scanning (n = 18). (C) The mean (±SE) number of detectable lung metastases per animal (n = 18) over time. (D) Lung CT scans of animals at baseline and after 8 weeks. Multiple opaque lung nodules are visible on week 8. Molecular Therapy 2006 14, 361-370DOI: (10.1016/j.ymthe.2006.05.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 3 (A) Infectious units (pfu/mg tissue) per biopsy sample of JX-594 from rabbit VX2 tumors or peritumoral normal liver following intratumoral or intrahepatic injection on day 4 postinjection (n = 3 animals per group; 5 biopsy samples per animal). (B) Infectious units (pfu/mg; ±SE) of JX-594 in tumors vs normal lung, liver, heart, ovaries, kidneys, spleen, brain, bone marrow, skeletal muscle, and colon over time following i.v. administration in liver tumor-bearing mice (107 pfu; TIB-75 tumor) or rabbits (109 pfu; VX2 tumor) (n = 3 per group per time point) (NB: viral titers in skeletal muscle, heart, kidney, spleen, brain, bone marrow, liver, and colon all below level of detection). (C) hGM-CSF concentrations over time following intratumoral or intravenous injection in rabbits with or without VX2 tumors. (D) Mean immunohistochemistry (IHC) score (±SE) for CD4 and CD8 staining for JX-594- and PBS-injected tumors (day 8). *P < 0.01 vs PBS-injected tumors (n = 5 tumors per group; three random fields). Molecular Therapy 2006 14, 361-370DOI: (10.1016/j.ymthe.2006.05.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 4 (A) Antitumoral efficacy against primary liver tumors: bar graph for mean tumor volume (±SE) on week 7 after treatment, for JX-594-treated vs PBS-injected controls in VX2 model (IT, intratumoral; IV, intravenous). (B) Antitumoral efficacy against lung metastases: bar graph for % of rabbits with detectable lung metastases on week 6 or 7 after treatment, for JX-594-treated vs PBS-injected controls in VX2 model. (C) Mean animal weights (±SE) over time for control and treated groups. (D) Kaplan–Meier survival curve for JX-594-treated (n = 6 per group) vs PBS-treated control animals (n = 20; pooled from two experiments). (E) Dose response of i.v. JX-594 against metastases: bar graph for mean tumor volume (±SE) on week 7 after treatment for PBS-injected controls vs two dose levels of JX-594 (108 and 109 pfu) i.v. or i.t. Molecular Therapy 2006 14, 361-370DOI: (10.1016/j.ymthe.2006.05.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 5 (A) Methods used for development, treatment, and follow-up of NNM-induced rat tumor model. (B) Intraoperative photos of NNM-induced cirrhosis and primary hepatocellular carcinoma in liver of rat on week 18 of study. (C) Growth curves (mean ± SE) of intravenous JX-594-treated vs PBS-injected control animals over time in rat carcinoma model (n = 6 per group). Molecular Therapy 2006 14, 361-370DOI: (10.1016/j.ymthe.2006.05.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

Supplementary Fig. S1 Immunohistochemistry for CD4 and CD8(+) infiltration in PBS and JX-594-injected tumors on day 8 after injection. Molecular Therapy 2006 14, 361-370DOI: (10.1016/j.ymthe.2006.05.008) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions