Fig 1: VV-ΔTK-4L expressed 4–1BBL on the tumor cell surface and showed a similar cytotoxicity to the VV-ΔTK control virus. A: Schematic diagram of the construction of the 4–1BBL recombinant oncolytic virus. VV-ΔTK-4L was generated by the homologous recombination of murine 4–1BBL into the tk locus of the VV genome. B–D: Murine pancreatic cancer cell line Pan02 (1 × 105 cells/well) in a 24-well/plate was infected with VV-ΔTK-4L or mock-infected with VV-ΔTK at different MOIs. The cell pellets were harvested 24 h post-infection to detect A34R and 4–1BBL expression using RT-qPCR (B, C) and cell-surface staining and flow cytometric assay (D). E–H: Tumor cells were plated at 1.0 × 104 cells/well (except B16-F10 cells, which were plated at 5 × 103 cells/well) in 96-well plates and infected with VV-ΔTK-4L or VV-ΔTK viruses the next day at different MOIs. Six replicate wells were set up for each sample. Cell viability was measured using an MTT assay at 48 h post-infection. MOI, multiplicity of infection; ns, not significant.
Fig 2: VV-ΔTK-4L administration produces 4–1BBL in tumors and is effective in therapeutic tumor models. A–C: C57BL/6 mice were intraperitoneally injected with 1 × 106 Pan02 cells per mouse and treated with PBS, or VV-ΔTK-4L or VV-ΔTK viruses at 2 × 108 PFU/mouse 9 days post tumor inoculation (n = 8/group). On day 14 post tumor inoculation, the mice were sacrificed (A). Tumor tissues (B) and spleens (C) were collected for quantitative assay for 4–1BBL expression by RT-qPCR. D–H: C57BL/6 mice were intraperitoneally injected with 1 × 106 Pan02 cells per mouse and treated with PBS, or VV-ΔTK-4L or VV-ΔTK viruses at 2 × 108 PFU/mouse 9 days post tumor inoculation (n = 10/group; D). Mouse survival was observed daily (E, F), and the other cohort of mice was enrolled to repeat this experiment (G, H). A log-rank (Mantel–Cox) test was used to analyze survival rates. A T-test was applied to compare 4–1BBL expression levels and survival periods among mice. *P < 0.05; **P < 0.01. ns: not significant.
Fig 3: VV-ΔTK-4L administration did not produce obvious hepatoxicity in therapeutic tumor models.A: Schematic diagram of the hepatotoxicity assay for α4–1BB mAb and VV-ΔTK-4L treatment in a pancreatic mouse model. B–E: C57BL/6 mice were intraperitoneally injected with 1 × 106 of Pan02 cells and treated with agonist α4–1BB mAb, rat IgG2a isotype control, PBS, VV-ΔTK-4L, or VV-ΔTK 9 days post tumor inoculation. The treated mice and healthy mice (n = 5/group) were sacrificed 21 days post tumor inoculation to harvest their livers, which were then photographed (B), weighed (C), and underwent blood collection to measure ALT (D) and AST (E) levels in sera. Liver tissues were collected to perform RT-qPCR to analyze 4–1BBL expression (F). *P < 0.05; **P < 0.01. ns: not significant.
Supplier Page from Sino Biological, Inc. for Mouse CD137L/4-1BBL/TNFSF9 Gene ORF cDNA clone in cloning vector