The Enhanced Tumor Specificity of TG6002, an Armed Oncolytic Vaccinia Virus Deleted in Two Genes Involved in Nucleotide Metabolism  Johann Foloppe, Juliette Kempf, Nicolas Futin, Jacqueline Kintz, Pascale Cordier, Christelle Pichon, Annie Findeli, Fabien Vorburger, Eric Quemeneur, Philippe Erbs  Molecular Therapy - Oncolytics  Volume 14, Pages 1-14 (September 2019) DOI: 10.1016/j.omto.2019.03.005 Copyright © 2019 The Author(s) Terms and Conditions

Figure 1 Oncolytic Activity and In Vitro Replication of Copenhagen-Based VACVs (A) Oncolytic effect of ΔJ2R/FCU1 VACV and TG6002 on a panel of human tumor cells. Cells were infected at MOI 10−4, 10−3, or 10−2 with the indicated vectors, and cell viability was measured 5 days later by trypan blue exclusion. The results are presented as a mean of triplicate experiments ± SD. (B) Virus production of the different VACVs in primary normal cells and tumor cells. Normal human hepatocytes and tumor human Hep G2 hepatocarcinoma cells were infected by VACVwt, ΔJ2R/FCU1 VACV, and TG6002 at MOI 10−4 (102 PFU/well) or 10−2 (104 PFU/well). Viruses produced after 48 h were titered by plaque assay. The results are presented as a mean of triplicate experiments ± SD. The asterisks indicate a significant difference between groups (p < 0.05). (C) Ratio of virus yield in Hep G2 cells versus human hepatocytes 48 h after infection. Values are represented as the mean of three individual determinations. (D) Amplification factor of the different VACVs in Phenion full-thickness human skin model. 3D Phenion full-thickness human skin models were infected by VACVwt, ΔJ2R/FCU1 VACV, and TG6002 at 1 × 105 PFU. Seven days after infection, 3D skin and supernatant were collected and sonicated, and viral titers were determined by plaque assay. Results are expressed as viral fold increased (corresponding to output/input ratio). Values are represented as means ± SD. The asterisks indicate a significant difference between groups (p < 0.05). Molecular Therapy - Oncolytics 2019 14, 1-14DOI: (10.1016/j.omto.2019.03.005) Copyright © 2019 The Author(s) Terms and Conditions

Figure 2 Effects of siRNA-Mediated Knockdown of Cellular RRM1 and TK1 on Replication of VACVs (A) Western blot detection of RRM1 (left) and TK1 (right) protein expression in LoVo cells 24, 48, and 72 h after transfection with specific siRNA and non-targeting control siRNA (siRNAc). Molecular weight standards are shown (M). The western blots are representative of three different experiments. (B) Virus production in RRM1 and TK1-knockdown LoVo cells following infection with the indicated VACVs at MOI 10−3 for 48 h before performing plaque assays. The data represent the average of three independent experiments and are shown as the mean ± SD. *p < 0.05 compared to cells transfected with the non-targeting siRNAc. Molecular Therapy - Oncolytics 2019 14, 1-14DOI: (10.1016/j.omto.2019.03.005) Copyright © 2019 The Author(s) Terms and Conditions

Figure 3 In Vitro Evaluation of FCU1 Expression (A) Specific CDase and UPRTase activities. LoVo cells were infected at MOI 10−2 with the indicated vectors. After 16 h, enzymatic activities were measured as described in Materials and Methods. CDase and UPRTase activities are expressed as the number of nanomoles 5-FC deaminated per minute per milligram of protein and the number of nanomoles 5-FU phosphorylated per minute per milligram of protein, respectively. Each value represents the average of three independent experiments ± SD. (B) Combination of oncolytic and prodrug activation cytotoxicity. LoVo cells were infected at MOI 10−3 with the indicated vectors. After 48 h, 5-FC was added in a range of concentrations, and cell survival was determined 3 days later, as described in the Materials and Methods section. Results were standardized against values for wells lacking virus and drug, which represented 100% viability. Values are represented as means ± SD of three individual determinations. Molecular Therapy - Oncolytics 2019 14, 1-14DOI: (10.1016/j.omto.2019.03.005) Copyright © 2019 The Author(s) Terms and Conditions

Figure 4 Biodistribution and Pathogenicity of Single- and Double-Deleted VACVs (A) Comparison of biodistribution of ΔJ2R/FCU1 VACV and TG6002 in tumor and normal tissues. Female nude mice bearing established subcutaneous LoVo tumors were infected intravenously with 1 × 106 PFU of ΔJ2R/FCU1 VACV or TG6002. On day 14 after virus administration, tumors and normal tissues were harvested and homogenized, and viral titers were determined by a standard plaque assay. Results are expressed in PFU per milligram tissue. Values are presented as means ± SD of three animals. (B) Survival of nude mice treated with 1 × 108 PFU of ΔJ2R/FCU1 VACV or TG6002 by one intravenous injection (n = 12/group). TG6002-infected mice had significantly prolonged survival (p < 0.01) compared with ΔJ2R/FCU1 VACV. (C) Survival of immunocompetent mice treated with 1 × 108 PFU of ΔJ2R/FCU1 VACV or TG6002 in one intravenous injection (n = 12/group). TG6002-infected mice had significantly prolonged survival (p < 0.001) compared with ΔJ2R/FCU1 VACV. (D) Mean number of pocks on tails after systemic injection of VACVs. Healthy nude mice were treated with one intravenous injection of ΔJ2R/FCU1 VACV and TG6002 at 1 × 106 PFU or 1 × 107 PFU. Pocks on tails were counted at days 9 and 34 after injection. Values represent the means ± SD of 12 animals. Molecular Therapy - Oncolytics 2019 14, 1-14DOI: (10.1016/j.omto.2019.03.005) Copyright © 2019 The Author(s) Terms and Conditions

Figure 5 In Vivo Antitumor Activity of VACVs (A) Mean tumor volume after systemic treatment with 1 × 107 PFU of ΔJ2R/FCU1 VACV or TG6002 plus 5-FC administration. Mice bearing LoVo subcutaneous xenografts were treated with one intravenous administration of vehicle, ΔJ2R/FCU1 VACV or TG6002 (indicated by a vertical arrow). Seven days after virus injection, the animals were then treated twice daily with per os administration of water or 5-FC (200 mg/kg per day) for 3 weeks (indicated by a horizontal arrow). The data represent the mean of 12 animals. (B) Antitumor activity of TG6002 in multiple human tumor models. OE19 esophagus cancer cells, SW780 bladder cancer cells, Hep G2 hepatocarcinoma cells, Hs 746T stomach cancer cells, CAL33 head and neck cancer cells, and HCT 116 colorectal carcinoma cells were implanted subcutaneously in mice. At the day indicated by the vertical arrow, the mice were treated with one intravenous administration of vehicle (open diamonds, vehicle + water; solid diamonds, vehicle + 5-FC) or with one intravenous administration of TG6002 at the dose indicated in Materials and Methods (open red squares, TG6002 + water; solid red squares, TG6002 + 5-FC). Seven days after virus injection, the animals were treated twice daily with per os administrations of water or 5-FC (200 mg/kg per day) for 3 weeks (indicated by a horizontal arrow). The data represent the mean ± SD of 12 animals. Molecular Therapy - Oncolytics 2019 14, 1-14DOI: (10.1016/j.omto.2019.03.005) Copyright © 2019 The Author(s) Terms and Conditions

Figure 6 Antitumor Activity of Multiple Cycles of TG6002/5-FC Mean tumor volume after multiple cycles of TG6002/5-FC. Mice bearing LoVo subcutaneous xenografts were treated with one or two cycles of TG6002/5-FC. In the single cycle of treatment, at day 10 (indicated by vertical arrow), mice were treated with one intravenous administration of vehicle (open diamonds, vehicle + water; solid diamonds, vehicle + 5-FC) or with one intravenous administration of TG6002 at 1 × 107 PFU (open red squares, TG6002 + water; closed red squares, TG6002 + 5-FC). Seven days after viral injection, 5-FC (200 mg/kg per day) was administered for 3 weeks (indicated by a horizontal arrow). In the multiple cycles of TG6002/5-FC treatment, at days 10 and 31 (indicated by vertical arrows), mice were treated with one intravenous administration of TG6002 at 1 × 107 PFU (open green squares, TG6002 + water; closed green squares, TG6002 + 5-FC). Seven days after each viral injection, 5-FC (200 mg/kg per day) was administered for 2 weeks (indicated by horizontal arrows). The data represent the mean ± SD of 12 animals. Molecular Therapy - Oncolytics 2019 14, 1-14DOI: (10.1016/j.omto.2019.03.005) Copyright © 2019 The Author(s) Terms and Conditions

Figure 7 Mouse Pharmacokinetic Studies Concentrations of 5-FC and 5-FU in plasma and tumor tissue samples. TG6002 was injected intravenously at 1 × 106 PFU in nude mice bearing subcutaneous human Hep G2 tumors. Before virus injection (day 0) and at 7, 14, 21, 28, and 60 days after viral injection, blood and tumor tissues were collected 1 h after administration of 5-FC (per os at 100 mg/kg) and the concentrations of 5-FC and 5-FU were measured in plasma (A) or in tumors (B) as described in Materials and Methods. Samples were obtained from groups of three mice at each sample time. Concentrations determined in samples from individual animals are indicated as 5-FC (green triangle) and 5-FU (red downward triangle). Symbols drawn through the x axis denote samples with concentrations below the limit of quantitation. Horizontal bars represent mean concentrations. Molecular Therapy - Oncolytics 2019 14, 1-14DOI: (10.1016/j.omto.2019.03.005) Copyright © 2019 The Author(s) Terms and Conditions