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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.

Radiotherapy is a major part in the treatment of most common cancers, but many patients experience local recurrence with metastatic disease. In evaluating response biomarkers, we found that low doses of fractionated radiotherapy led to PD-L1 upregulation on tumor cells in a variety of syngeneic mouse models of cancer. Notably, fractionated radiotherapy delivered in combination with αPD-1 or αPD-L1 mAbs generated efficacious CD8+ T-cell responses that improved local tumor control, long-term survival, and protection against tumor rechallenge. These favorable outcomes were associated with induction of a tumor antigen–specific memory immune response. Mechanistic investigations showed that IFNγ produced by CD8+ T cells was responsible for mediating PD-L1 upregulation on tumor cells after delivery of fractionated radiotherapy. Scheduling of anti–PD-L1 mAb was important for therapeutic outcome, with concomitant but not sequential administration with fractionated radiotherapy required to improve survival. Taken together, our results reveal the mechanistic basis for an adaptive response by tumor cells that mediates resistance to fractionated radiotherapy and its treatment failure. With attention to scheduling, combination immunoradiotherapy with radiotherapy and PD-1/PD-L1 signaling blockade may offer an immediate strategy for clinical evaluation to improve treatment outcomes. Cancer Res; 74(19); 5458–68. ©2014 AACR.

Although topical TLR7 therapies such as imiquimod have proved successful in the treatment of dermatological malignancy, systemic delivery may be required for optimal immunotherapy of nondermatological tumors. We report that intravenous delivery of the novel small molecule TLR7 agonist, DSR-6434, leads to the induction of type 1 interferon and activation of T and B lymphocytes, NK and NKT cells. Our data demonstrate that systemic administration of DSR-6434 enhances the efficacy of ionizing radiation (IR) and leads to improved survival in mice bearing either CT26 or KHT tumors. Of the CT26 tumor-bearing mice that received combined therapy, 55% experienced complete tumor resolution. Our data reveal that these long-term surviving mice have a significantly greater frequency of tumor antigen specific CD8+ T cells when compared to age-matched tumor-naive cells. To evaluate therapeutic effects on spontaneous metastases, we showed that combination of DSR-6434 with local IR of the primary tumor significantly reduced metastatic burden in the lung, when compared to time-matched cohorts treated with IR alone. The data demonstrate that systemic administration of the novel TLR7 agonist DSR-6434 in combination with IR primes an antitumor CD8+ T-cell response leading to improved survival in syngeneic models of colorectal carcinoma and fibrosarcoma. Importantly, efficacy extends to sites outside of the field of irradiation, reducing metastatic load. Clinical evaluation of systemic TLR7 therapy in combination with IR for the treatment of solid malignancy is warranted. What's new? Recent evidence suggests that damage from ionizing radiation (IR) can render tumor cells immunogenic. Unfortunately, established tumors often suppress this anti-tumor immune response. Combination therapy with IR and immune-modulators such as Toll-like-receptor (TLR) family agonists may overcome this problem. In this proof-of-concept study, the authors examined one such small-molecule drug, called DSR-6434. They found that systemic administration of DSR-6434 can enhance the effectiveness of radiotherapy in mice, and that this occurs via the generation of tumor-specific immune responses. Easily delivered drugs that activate TLR-family molecules may thus offer a promising therapeutic approach.

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.

Although topical TLR7 therapies such as imiquimod have proved successful in the treatment of dermatological malignancy, systemic delivery may be required for optimal immunotherapy of nondermatological tumors. We report that intravenous delivery of the novel small molecule TLR7 agonist, DSR-6434, leads to the induction of type 1 interferon and activation of T and B lymphocytes, NK and NKT cells. Our data demonstrate that systemic administration of DSR-6434 enhances the efficacy of ionizing radiation (IR) and leads to improved survival in mice bearing either CT26 or KHT tumors. Of the CT26 tumor-bearing mice that received combined therapy, 55% experienced complete tumor resolution. Our data reveal that these long-term surviving mice have a significantly greater frequency of tumor antigen specific CD8+ T cells when compared to age-matched tumor-naïve cells. To evaluate therapeutic effects on spontaneous metastases, we showed that combination of DSR-6434 with local IR of the primary tumor significantly reduced metastatic burden in the lung, when compared to time-matched cohorts treated with IR alone. The data demonstrate that systemic administration of the novel TLR7 agonist DSR-6434 in combination with IR primes an antitumor CD8+ T-cell response leading to improved survival in syngeneic models of colorectal carcinoma and fibrosarcoma. Importantly, efficacy extends to sites outside of the field of irradiation, reducing metastatic load. Clinical evaluation of systemic TLR7 therapy in combination with IR for the treatment of solid malignancy is warranted. What's new? Recent evidence suggests that damage from ionizing radiation (IR) can render tumor cells immunogenic. Unfortunately, established tumors often suppress this anti-tumor immune response. Combination therapy with IR and immune-modulators such as Toll-like-receptor (TLR) family agonists may overcome this problem. In this proof-of-concept study, the authors examined one such small-molecule drug, called DSR-6434. They found that systemic administration of DSR-6434 can enhance the effectiveness of radiotherapy in mice, and that this occurs via the generation of tumor-specific immune responses. Easily delivered drugs that activate TLR-family molecules may thus offer a promising therapeutic approach.

Passive immunotherapy with monoclonal antibodies has improved outcome for patients with B-cell malignancies, although many still relapse and little progress has been made with T-cell malignancies. Novel treatment approaches are clearly required in this disease setting. There has been much recent interest in developing therapeutic approaches to enhance antitumor immune responses using novel immunomodulatory agents in combination with standard of care treatments. Here we report that intravenous administration of the Toll-like receptor 7 (TLR7) agonist, R848 in combination with radiation therapy (RT), leads to the longstanding clearance of tumor in T- and B-cell lymphoma bearing mice. In combination, TLR7/RT therapy leads to the expansion of tumor antigen-specific CD8+ T cells and improved survival. Furthermore, those mice that achieve long-term clearance of tumor after TLR7/RT therapy are protected from subsequent tumor rechallenge by the generation of a tumor-specific memory immune response. Our findings demonstrate the potential for enhancing the efficacy of conventional cytotoxic anticancer therapy through combination with a systemically administered TLR7 agonist to improve antitumor immune responses and provide durable remissions.