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. 2016 Oct 31;113(46):E7240–E7249. doi: 10.1073/pnas.1608555113

Fig. 2.

Fig. 2.

Intratumoral SD-101 reverses tumor escape from anti–PD-1 therapy and leads to CD8+ T-cell and IFNAR-mediated tumor rejection. (A) Graphical representation of mean tumor volumes over time. CT26 tumor cells were injected s.c. in the flank of mice on day 0. Anti–PD-1 treatment started when tumor reached 5 mm (day 7). Mice were left untreated, or mice were treated with i.p. injections of anti–PD-1 administered on days 7, 10, 14, 17, 21, 25, 28, and 32. After four anti–PD-1 injections (day 19), mice started receiving intratumoral injections of 50 μg of SD-101 or CTRL-ODN administered on days 19, 21, 25, 28, 32, and 35. A separate group of mice with similar tumor size but no pretreatment with anti–PD-1 received SD-101 alone. (B) Long-term survival of mice in A. One representative experiment of four experiments, each performed with four to eight mice per group, is shown. (C and D) SD-101 increases response to anti–PD-L1 treatment. (C) Tumor volumes over time. (D) Overall survival. Experiment was performed with a similar schedule to that used in A but using an anti–PD-L1 blocking Ab with six mice per group. (E) Anti–PD-1 plus SD-101 combination therapy allows for rejection of contralateral tumors. Survival of mice bearing two s.c. tumors (right and left flank) that received systemic anti–PD-1 and IT SD-101 only in the left tumors. CT26 cells were injected s.c. on day 0 in the left flanks and on day 3 in the right flanks. Experiments were performed with a similar schedule to that used for the anti–PD-1 blocking antibody in A. Cumulative data from two independent experiments are shown. CTRL group, n = 18; SD-101 plus PD-1 group, n = 19. (F) CD8+ T cells but not CD4+ T cells are required for the efficacy of anti–PD-1 plus SD-101 combination treatment. Graph represents survival of mice upon depletion of CD8+ or CD4+ T cells. Experiment was performed with a similar schedule to that used in A. Depletion antibodies were started the day before the start of SD-101 treatment and given every 3 d until the end of SD-101 treatment; n = 7 per group. (G) SD-101 treatment restores IFN production in anti–PD-1-treated tumors. Anti–PD-1 treatment was administered on days 5, 9, 12, 14, 19, and 22. After four anti–PD-1 injections (day 14), SD-101 or CTRL-ODN was started and administered on days 16, 19, and 22. TAQMAN analysis was performed on whole tumors harvested 4 d after the last treatment. (G, Upper) Graph shows the tumor size at time of excision. (Lower) Graphs show TAQMAN analysis. Cumulative data from two independent experiments are shown. **P ≤ 0.01; ***P ≤ 0.001. (H) Blockade of IFNAR impairs tumor rejection in response to anti–PD-1 plus SD-101 combination treatment. Experiments were performed with a similar schedule to that used in A. IFNAR blocking Ab or anti-IgG control were started 1 d before the first SD-101 treatment and given every 3 d for the duration of SD-101 treatment.