Fig. 4.

Combining vaccinia virus expressing IL-2 with immune checkpoint therapy. B6 mice were inoculated with 5 × 10⁵ MC38-luc cells and treated with PBS, vvDD, vvDD-IL-2-FG, or vvDD-IL-2-RG at 2 × 10⁸ PFU per mouse 9 days post-tumour inoculation. Tumour-bearing mice were sacrificed 5 days post-treatment and primary tumours were collected and analysed using RT-qPCR to determine the expressions of PD-1 (a), PD-L1 (b), and CTLA-4 (c) in the tumour microenvironment. Five mice were used for each treatment group in one experiment and data are combined from three independent experiments. In a separate experiment, B6 mice were i.p. inoculated with 5 × 10⁵ MC38-luc cells and treated with vvDD/vvDD-IL-2-RG or PBS 9 days post-tumour inoculation. Anti-PD-1 Ab (200 µg per injection), anti-PD-L1 Ab (200 µg per injection), or anti-CTLA-4 Ab (100 µg per injection) were i.p. injected into mice as scheduled (nine mice per group) (d), and a log-rank (Mantel-Cox) test was used to compare survival rates (e, f). In some experiments, to measure the abscopal effect, B6 mice were also s.c. inoculated with 5 × 10⁵ MC38-luc 4 days after i.p. tumour inoculation, and treated with i.p. vvDD-IL-2-RG alone or combined with α-PD-1/PD-L1 Ab as scheduled (ten mice per group) (g) to monitor s.c. tumour growth (h, i). *P<0.05; **P<0.01; ***P<0.001; and ****P<0.0001. ns: not significant