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Supplemental figure 1: Gating strategy for determining tumor cell PD-L1 expression. Supplemental figure 2: Impact of low-dose fractionated RT on PD-L1 expression by CD11b+GR1Hi cells. Supplemental figure 3: Dual blockade of PD-1 and PD-L1 does not further enhance efficacy of RT combination. Supplemental figure 4: Blockade of PD-1 / PD-L1 in combination with local RT is well tolerated in mice. Supplemental figure 5: PD-1 / PD-L1 blockade does not directly sensitise tumor cells to RT. Supplemental figure 6: Scheduling of RT and anti-PD-L1 is critical for anti-tumor activity. Supplemental figure 7: Only acute administration of anti-PD-L1 mAb may be required to achieve efficacy when delivered in combination with RT.

Strategies to augment anti-cancer immune responses have recently demonstrated therapeutic utility. To date clinical success has been achieved through targeting co-inhibitory checkpoints such as CTLA-4, PD-1, and PD-L1. However, approaches that target co-activatory pathways are also being actively being developed. Here we report that the novel TLR7-selective agonist DSR-29133 is well tolerated in mice and leads to acute immune activation. Administration of DSR-29133 leads to the induction of IFNα/γ, IP-10, TNFα, IL-1Ra and IL-12p70, and to a reduction in tumor burden in syngeneic models of renal cancer (Renca), metastatic osteosarcoma (LM8) and colorectal cancer (CT26). Moreover, we show that the efficacy of DSR-29133 was significantly improved when administered in combination with low-dose fractionated radiotherapy (RT). Effective combination therapy required weekly administration of DSR-29133 commencing on day 1 of a fractionated RT treatment cycle, whereas no enhancement of radiation response was observed when DSR-29133 was administered at the end of the fractionated RT cycle. Combined therapy resulted in curative responses in a high proportion of mice bearing established CT26 tumors which was dependent on the activity of CD8+ T-cells but independent of CD4+ T-cells and NK/NKT cells. Moreover, long-term surviving mice originally treated with DSR-29133 and RT were protected by a tumor-specific memory immune response which could prevent tumor growth upon rechallenge. These results demonstrate that DSR-29133 is a potent selective TLR7 agonist that when administered intravenously can induce anti-tumor immune responses that can be further enhanced through combination with low-dose fractionated RT.