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The prognosis for patients with pancreatic adenocarcinoma (PDAC) remains extremely poor. PDAC is resistant to both conventional therapies and emerging immunotherapies (IOT), apart from tumors with mismatch repair deficiency. This may be explained in part by its low tumor mutational burden (TMB) but also by its immunosuppressive tumor microenvironment (TME). It has been suggested that CD73, a member of the adenosine pathway, expressed on cancer cells contributes to immune escape and resistance to cytotoxic/radiotherapy treatment. The adenosine pathway converts the immune activator ATP, released by dying cells during cell turnover or after treatment, to extracellular Adenosine (eAdo), which is immunosuppressive. Using syngeneic, in vivo models by s.c. implantation of KPC-derived cell lines (courtesy of Ben Stanger, UPenn) with differential immune infiltration and response to IOT [resistant (IOTResi) or responsive (IOTResp)], we showed by flow cytometry that the adenosine pathway is enriched in the tumor-infiltrating immune cells (in particular myeloid populations) which co-express CD39 and CD73, enabling the formation of eAdo. Mass Spec Imaging (MSI) revealed that adenosine distribution is heterogeneous in the tumors with high concentrations in the hypoxic margins that surround necrotic areas. Subpopulations of myeloid cells infiltrating the lesions are a target for eAdo, expressing high levels of adenosine receptor Adora2a. We discovered that pro-tumorigenic M2 macrophages have the highest expression of the receptor and significantly higher in the IOTResi model. Blocking the in vivo formation and function of eAdo in IOTResi tumors, using a combination of anti-CD73 antibody (2C5, murine IgG1-Fc) and an inhibitor of Adora2a (AZD4635) reduced the presence of eAdo, slowed tumor growth and reduced the lung metastatic burden. The combination remodeled the TME, reducing the infiltration by M2 macrophages, particularly those that are PD-L1 positive and diminished the frequency of infiltrating Tregs. Bulk RNAseq analysis demonstrated a profound dependency of the TME on the presence of eAdo. Genes related to cytokine/chemokine signaling, immunosuppression/inflammation, hypoxia, metastasis and collagen production are strongly downregulated following administration of anti-CD73Ab/Adora2ai. In addition, blocking the adenosine pathway improved the efficacy of combinations of cytotoxics (gemcitabine/ATR inhibitor) and immunotherapy (aCD40/anti-PDL1Ab). The formation of eAdo appears to be a factor in the development of the immunosuppressive TME in PDAC, contributing to its resistance to conventional and novel therapies. Therefore, inhibition of the adenosine pathway using a CD73Ab and an Adora2ai may represent a strategy to modulate the PDAC stroma and improve therapy response in patients with PDAC. Citation Format: Vincenzo Graziano, Andreas Dannhorn, Kate Williamson, Heather Hulme, Hannah Buckley, Sheng Y. Lee, Sabita Islam, James E. Thaventhiran, Richard Goodwin, Rebecca Brais, Simon J. Dovedi, Alwin Schuller, Jim Eyles, Duncan I. Jodrell. Reshaping the myeloid-dependent pro-tumorigenic microenvironment in PDAC by targeting the extracellular adenosine pathway: A therapeutic opportunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6103.

The catabolism of ATP into immunosuppressive adenosine contributes to the dysfunction of tumor infiltrating leukocytes (TIL). CD73 is an ectonucleotidase which catabolizes conversion of AMP to adenosine and is expressed on a range of immune cells including B cells. Oleclumab (MEDI9447) is a monoclonal antibody specific for human CD73 and is currently in clinical development for the treatment of cancer. B cells constitute a significant proportion of human TIL; however, their importance to Immuno-Oncology (IO) treatments remains unclear. Recent publications demonstrate that B cells sustain inflammation and predict response to immune checkpoint blockade in human melanoma, their presence within the tumor microenvironment is correlated with improved prognosis in several human tumor types, and CD73 expression has been shown to vary on different human B cell subtypes. Further investigations into the role of CD73 in control of human B cell function are therefore warranted. We show that blockade of CD73 by oleclumab and other monoclonal antibodies on peripheral blood B cells from healthy human donors upregulates expression of CD69, CD83 and CD86, and induces secretion of IL-6, MIP-1α and MIP-1β. Activation is mediated by antibodies that block and internalize CD73 and is reduced by pharmacological inhibition of BTK, thus highlighting involvement of the canonical B-cell receptor signaling pathway. Furthermore, our analyses highlight a differential effect of CD73 blockade on individual B-cell populations, with the most robust increases in activation marker expression being observed on naïve subsets that retain expression of IgD. Our research has identified that oleclumab activates human peripheral blood B cells. Given the renewed interest in B-cell biology in IO, this is an area we believe warrants further clinical investigation. Citation Format: James Hair, Fabien Garcon, Michelle Hsueh, Laura Dallaway, Elena Bibikova, Maria Letizia Giardino Torchia, Gordon Moody, Alwin Schuller, Simon J. Dovedi, Zachary A. Cooper, Kris Sachsenmeier, Rakesh Kumar, Jim Eyles, Robert W. Wilkinson. Activation of B cells by CD73 blocking antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1695.

CD39 has recently been identified as critically differentiating tumour-reactive from bystander tumour-infiltrating leukocyte (TIL) CD8+ T cells. CD39 is an ectonucleotidase that degrades immunostimulatory ATP to ADP and AMP. CD73 then further metabolises AMP to generate immunosuppressive adenosine in the tumor microenvironment. To facilitate our understanding of CD39 as a therapeutic target we established a detailed receptor co-expression profile for CD39 with other immuno-oncology (IO)-relevant targets on TIL T/NK cells. TIL samples from three diseases (NSCLC, Renal cancer [RC] and Melanoma) were profiled, via the creation and use of a single large flow cytometry panel, combined with computational analysis. Unbiased clustering identified the expected major T and NK cell populations, however one of the most striking findings was that CD39 was highly differentially expressed across the different cell subsets. Almost all TREG cells were CD39 positive, whereas CD8+ T cells and NK cells were highly variable in their degree of expression of CD39. CD4+CD8+ T cells predominantly did not express cell surface CD39 and only very few CD4+ TCONV cells expressed CD39. The co-expression profile for TREG, TCONV and NK cells is presented, but we have also defined a detailed phenotype of the critical tumour-specific CD39+CD8+ T cell population. Our analyses show that CD39+CD8+ T cells express more markers associated with antigen-experienced T cells versus CD39-CD8+ T cells; a significantly greater proportion of CD39+CD8+ T cells expressed TIM3, CTLA-4, PD-1, TIGIT, CD137 and GITR versus CD39-CD8+ T cells. Significantly fewer CD39+CD8+ T cells than CD39-CD8+ T cells expressed CD28, a receptor known to be downregulated on antigen-experienced cytotoxic CD8+ T cells. These data reveal the detailed co-expression patterns of key IO-relevant receptors across TIL T and NK cell subsets and are consistent with recent reports identifying CD39 as a key marker differentiating the critical tumor-specific, antigen-experienced tumor infiltrating CD8+ T cells. Citation Format: Jim Hair, Nadia Luheshi, Simon J. Dovedi, Robert W. Wilkinson. Characterisation of co-expression of CD39 with other IO-relevant markers on major T/NK cell TIL subsets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2152.

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.

Immuno-oncology research relies heavily on murine syngeneic tumor models. However, whilst the median age for a cancer diagnosis is 65 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that aging has been shown to have a significant impact on the immune response. Using aged mice bearing CT26 tumors, we analysed how aging impacts the immune composition of the tumor, spleen and tumor-draining lymph nodes by flow cytometry. We found many age-related changes between aged (60-72 weeks old) and young (6-8 weeks old) mice, such as a reduction in the naïve T cell population and a decreased CD8/Treg ratio in aged animals. Profiling of co-inhibitory and co-stimulatory receptor expression levels on immune cells in aged versus young mice also identified altered expression profiles in both the periphery and tumor. We hypothesised that these differences may contribute to impaired anti-cancer immune responses in aged mice. To investigate this, we compared the anti-tumor efficacy of immune checkpoint blockade (PD-L1 and CTLA-4) and T-cell costimulation (OX-40) in aged versus young mice. Our data demonstrate that aged mice retained their capacity to generate effective anti-tumor immune responses, albeit often attenuated when compared to the responses observed in young mice. These differences highlight the potential importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models. Citation Format: Suzanne I. Sitnikova, Michelle Morrow, Viia Valge-Archer, Robert W. Wilkinson, Simon J. Dovedi, Matthew J. Robinson. Age-induced changes in anti-tumor immunity alter the tumor immune infiltrate and reduce response to immuno-oncology treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4987.

Despite the recent breakthrough of checkpoint blockade antibodies targeting T cells to combat cancer, it has become apparent that the majority of patients are not fully responsive. Engaging innate immune cells is a promising way to fully exploit the anti-cancer potential of the immune system. Our strategy is to stimulate TOLL-like receptors (TLRs) with MEDI9197, a potent TLR7 and TLR8 agonist, which stimulates the release of interferon-alpha (IFN-α) from plasmacytoid dendritic cells (pDCs) and interleukin-12 (IL-12) and other cytokines from myeloid DCs. While several compounds including MEDI9197, CpG and STING agonists can induce the production of IFN-α, we show that only MEDI9197 is able to induce high levels of interferon-gamma (IFN-γ) and IL-12p70 in human PBMCs, a hallmark of TLR 7/8 agonists. We also demonstrate in vitro that MEDI9197 upregulates activation markers on both innate and adaptive human immune cells, repolarizes macrophages and skews immunity from a Th2 towards a Th1 phenotype in a PHA assay, thereby increasing the functional activity of NK and cytotoxic CD8 cells. MEDI9197 is an imidazoquinoline with a unique lipid tail and formulation designed for intratumoral (IT) dosing and retention at the site of injection to minimize unwanted systemic exposure. Mass spectrometry imaging of MEDI9197 in mouse tumors and whole-body autoradiography in rats confirm local retention of MEDI9197, with < 5% of the initial IT dose detectable in the rat serum. We show in the B16-OVA mouse syngeneic tumor model that a single IT dose increases the percentage of tumor infiltrating lymphocytes (TILs), enhances activation of T cells, and leads to a qualitative shift from single to dual cytokine production (IFN-γ and Tumor necrosis factor alpha, TNF-α). Gene expression analysis further reveals increased expression of activatory innate and adaptive immune gene signatures consistent with increased inflammation. As our mouse flow cytometry studies show elevated levels of PD-1 and PD-L1 on TILs, the combination of MEDI9197 with anti-PD-L1 mAb was also evaluated and resulted in enhanced efficacy compared to MEDI9197 alone. We further demonstrate in vitro that combining MEDI9197 with durvalumab (anti-PD-L1) augments cytokine production (e.g. IL-2, IFN-γ) in a co-culture of human DCs and allogenic T cells. These data showcase MEDI9197 as a potent modulator of the innate and adaptive immune system in the fight against cancer and support the rationale for evaluating MEDI9197 in combination with checkpoint blockade and costimulatory agonists in the clinic. MEDI9197 is currently being evaluated in human clinical trials for safety and efficacy (NCT02556463). Citation Format: Stefanie R. Mullins, John Vasilakos, Katharina Deschler, Iwen Grigsby, Song Ren, Matthew J. Elder, Simon J. Dovedi, Andrew J. Leishman, Patricia Ryan, Zachary Cooper, James Elvecrog, Ronald Herbst, Rakesh Kumar, Mark Tomai, Robert W. Wilkinson. Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 711.

Studies have demonstrated that the clinical benefit of PD-1 blockade can be further improved by combination with an αCTLA-4 mAb in some indications. However, this increased activity is commensurate with significant immune related adverse events (irAE's). Therefore, novel approaches are required to uncouple toxicity from anti-tumour efficacy and realise the full potential of this combination. MEDI5752 is a monovalent bispecific human IgG1 monoclonal antibody (mAb) with an engineered fragment crystallisable (Fc) domain to reduce Fc effector function, that specifically binds two clinically validated negative T cell regulators; PD-1 (programmed cell death 1) and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). MEDI5752 has been designed to suppress the PD-1 pathway and provide modulated CTLA-4 inhibition to uncouple CTLA-4 dependent peripheral toxicity from tumour efficacy. PD-1 expression is a defining feature of tumour infiltrating lymphocytes (TILs). We show that MEDI5752 can saturate CTLA-4 on PD-1+ cells at orders of magnitude lower concentrations than required to saturate CTLA-4 on PD-1- cells. Moreover, our data demonstrate that monovalent targeting of CTLA-4 with MEDI5752 is significantly less potent (15 fold) than bivalent targeting with a parental αCTLA-4 mAb in reporter assays. In contrast, the switch to monovalent targeting of PD-1 has limited effect on potency (within 3-fold compared to a parental αPD-1 mAb) in a PD-1/L1 reporter assay. Together these data demonstrate the potential for MEDI5752 to inhibit CTLA-4 on TILs whilst sparing peripheral T cell populations and reducing toxicity. Furthermore, profiling of MEDI5752 in a range of primary T cell activation assays reveals equivalent activity to a combination of parental PD-1 and CTLA-4 antibodies. MEDI5752 is rapidly internalised upon target binding with kinetics similar to the parental αCTLA-4 mAb reflecting the rapid recycling of this receptor. However, in contrast to an αCTLA-4 mAb (or an αPD-1 mAb), MEDI5752, by tethering CTLA-4 to PD-1, leads to the internalisation and subsequent degradation of PD-1. This novel mechanism of action further differentiates MEDI5752 from a combination of mAb's targeting PD-1 and CTLA-4. MEDI5752 is a novel monovalent bispecific which may provide an improved therapeutic index when compared to a combination of bivalent αPD-1 and αCTLA-4 mAb's, and could provide benefit in cancer indications. Citation Format: Simon J. Dovedi, Yariv Mazor, Matthew Elder, Sumati Hasani, Bo Wang, Suzanne Mosely, Desmond Jones, Anna Hansen, Chuning Yang, Yanli Wu, Ikbel Achour, Nick Durham, Gareth Browne, Thomas Murray, James Hair, Michelle Morrow, Godfrey Rainey, Maria Jure Kunkel, John Gooya, Daniel Freeman, Ronald Herbst, Robert Wilkinson. MEDI5752: A novel bispecific antibody that preferentially targets CTLA-4 on PD-1 expressing T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2776.

CD137 is a costimulatory molecule associated with tumour infiltrating lymphocytes and cytotoxic T lymphocytes in particular. Murine anti-CD137 monoclonal antibodies (mAbs) have shown potent anti-tumour effects in syngeneic tumour models, which can be further enhanced by combination with checkpoint inhibitor (CPI) mAbs to PD-1, or CTLA-4 and OX40. Two anti-CD137 monoclonal antibodies (mAbs), Urelumab and Utomilumab, are in development for both haematological and solid cancers, and studies include combinations with other IO therapies such as anti-PD-1, anti-PD-L1 and anti-OX40 mabs. We studied the impact of combining anti-CD137 with CPIs anti-PD-L1, anti-CTLA4, and IO agonist GITRL fusion protein (GITRL-FP) and anti-OX40 on pharmacodynamic responses, tumour growth and survival in mouse syngeneic models. In a responsive model, CT26, combining anti-CD137 with anti-PD-L1 had a greater impact on tumour growth inhibition, which correlated with an enhanced proliferation of CD8 T cells, as determined Ki67 expression using flow cytometry. In MC-38, a model that is less sensitive to both monotherapies, the combination was synergistic and resulted in enhanced tumour growth inhibition. Next, we compared the anti-CTLA4, anti-OX40 and GITRL-FP as monotherapies and in combination with anti-CD137 in the CT26 model. As before, anti-CD137 alone induced CD8 T cell proliferation, whereas anti-OX40 and GITRL-FP stimulated expansion of CD8 and CD4 T cells. Interestingly, in all combinations anti-CD137 appeared to enhance both CD8 and CD4 T cell proliferation. Anti-CTLA4 and GITR-FP had a greater impact on tumour growth inhibition when combined with anti-CD137. In summary, in vivo models indicate that anti-CD137 mAbs are a promising therapy in cancer, but the most benefit may be obtained in combination with other IO therapies. Citation Format: Matthew J. Robinson, Ines Osma-Garcia, Jane Coates-Ulrichisen, Amanda Watkins, Suzanne Mosely, Chrisopher Lloyd, Geoff Williams, Michelle Morrow, Simon Dovedi, Ronald Herbst, Robert Wilkinson. An anti-CD137 antibody can show greater efficacy in syngeneic mouse tumor models when combined with other immuno-oncology (IO) therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3765.

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is a co-stimulatory receptor, and programmed cell death protein 1 (PD-1) is an important co-inhibitory receptor involved in controlling T-cell activation and function. Preclinical studies using murine cross-reactive molecules demonstrate that the combination of an αPD-1 monoclonal antibody (mAb) with a GITR-ligand (GITRL) fusion protein can elicit synergistic antitumor activity and long-term survival benefit in syngeneic mouse tumor models. Here we describe the activity of two bispecific fusion proteins, each comprising two GITR ligand trimers fused to the Fc region of an αPD1 mAb, in an IgG1 (MEDI5771) or IgG4P (MEDI3387) format. We show that these bispecific molecules are able to concurrently engage both targets and are effective in cell-based PD-1 and GITR reporter assays, and in a range of human primary T-cell assays. Furthermore, the PD-1/GITRL FP bispecific constructs elicit T-cell responses in vitro that are at least equivalent to a combination of a) the parental molecules and b) MEDI1873 (a GITR agonist currently undergoing clinical evaluation) and an αPD-1 mAb. Moreover, using surrogate molecules we demonstrate that IV administration leads to dose-dependent pharmacodynamic responses on CD4+ and CD8+ T cells for both isotypes of the bispecific molecule. These data demonstrate that the antitumor activity for these novel molecules is equivalent to the combination of GITRL-FP and PD-1 mAb in the B16 model. In summary, the simultaneous dual targeting of PD-1 and GITR pathways mediated by these two novel bispecific molecules induces robust T-cell activity that is at least equivalent to a combination of molecules targeting these pathways, thereby demonstrating strong potential as a novel anticancer therapy. Citation Format: Simon J. Dovedi, Jonathan Seaman, Ines Osma-Garcia, Katharina Deschler, Edmund Poon, Ronald Herbst, Robert Wilkinson. Novel PD-1/GITRL bispecific fusion protein delivering concurrent T cell co-stimulation and checkpoint pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3820.

Recent successes in immuno-oncology have focussed primarily on the T-cell synapse, targeting co-activatory /inhibitory pathways to modulate T cell function. However, strategies that target tumor-resident myeloid cells may be required to fully exploit the therapeutic potential of the anti-cancer immune response. TOLL-like receptors (TLRs) are expressed on a broad range of myeloid cells and function to recognise highly evolutionarily conserved pathogen-associated molecular patterns. Signalling through TLRs leads to the activation of antigen-presenting cells (APCs) and to expression of inflammatory cytokines. However, whilst topical application of TLR agonists have proved successful in the treatment of dermatological tumors, systemic administration have proved to be poorly tolerated. MEDI9197 (formerly 3M-052), is a novel lipophilic TLR7/8 agonist designed with a lipid tail to facilitate retention at the site of injection, limiting systemic exposure. These properties make MEDI9197 ideal for intratumoral (IT) administration. We have demonstrated that MEDI9197 is a potent TLR7 and TLR8 agonist and induces pro-inflammatory cytokines through activation of a diverse range of myeloid and lymphoid cells. In mice bearing established tumors, IT injection of MEDI9197 induces a local inflammatory response, characterized by upregulation of genes associated with the activation of innate and adaptive immunity in the injected tumor and tumor draining lymph nodes. Moreover, treatment leads to an increase in the frequency of tumor infiltrating lymphocytes (TILs), such as CD8+ cytotoxic T cells and increased expression of activation markers, such as CD69. Cellular depletion studies reveal that CD8+ T cells are required for therapeutic activity. Furthermore, using in vitro co-cultures we demonstrate that MEDI9197 is able to enhance cytotoxic activity of NK cell and T cells. Importantly, in models that respond poorly to mAbs targeting either PD-L1 or CTLA-4, combination with MEDI9197 significantly improved anti-tumor activity when compared to either monotherapy alone. These preclinical data demonstrate that MEDI9197 can modulate both the myeloid and lymphoid immune compartments to mediate anti-tumor activity and combines productively with immune checkpoint blockade. MEDI9197 is currently being evaluated as a monotherapy for safety and efficacy in human clinical trials (NCT02556463). Citation Format: Stefanie R. Mullins, Katharina Vogel, John Vasilakos, Iwen Grigsby, Simon Dovedi, Ryan Patricia, Zachary Cooper, Ronald Herbst, Rakesh Kumar, Mark Tomai, Robert W. Wilkinson. Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment and holds potential for combination with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4697. doi:10.1158/1538-7445.AM2017-4697

Radiation therapy (RT) is administered to around 50% of all cancer patients making it one of the most important cancer treatments. In addition to the direct cytoreductive effect of RT there is increasing evidence that radiation-induces immunogenic tumor cell death. Despite the immunogenicity of RT-induced tumor cell death, RT delivered to tumours in the clinic rarely generates therapeutic systemic anti-cancer immune responses or ‘abscopal effects’. Here we show that tumor infiltrating CD8+ cytotoxic T lymphocytes (CTL) have increased expression of PD-1 following RT in vivo. Moreover, our data demonstrate that treatment of established syngeneic tumors with RT leads to upregulation of tumor cell expression of PD-L1 in vivo but not when cells are irradiated in vitro. Using depleting antibodies we determined that the depletion of CD8+ T cells but not CD4+ T cells or NK cells could abrogate this RT-induced increase in tumor cell expression of PD-L1 in vivo. Furthermore, silencing of IFNγR1 using ShRNA confirmed that this process was dependent on CD8+ T cell production of IFNγ suggesting an adaptive upregulation of PD-L1 following RT occurs in response to CTL activation. This novel finding suggests that the immunogenicity of RT may be limited via the PD-L1/PD-1 signalling axis and may contribute to treatment failure. We next sought to determine whether blockade of the PD-1/PD-L1 signalling axis could enhance the therapeutic response to RT. Our study demonstrates that administration of either an anti-PD-1 or anti-PD-L1 mAb in combination with RT leads to substantially improved survival when compared to either monotherapy alone with approximately 60% of treated mice undergoing a complete response. In addition, our data reveal that combination therapy generates long-term immunological memory in mice that have undergone complete response protecting against tumor rechallenge. This is the first report to our knowledge that demonstrates adaptive resistance through the upregulation of tumour cell PD-L1 expression in response to an RT-induced CTL response. This study demonstrates the potential for enhancing the efficacy of conventional RT through blockade of the PD-1/PD-L1 axis and warrants clinical evaluation. Citation Format: Simon J. Dovedi, Graznya Lipowska-Bhalla, Eleanor Cheadle, Edmund Poon, Michelle Morrow, Ross Stewart, Robert Wilkinson, Jamie Honeychurch, Timothy Illidge. The antitumor immune response generated by radiation therapy may be limited by tumor cell adaptive resistance and can be circumvented by PD-L1 blockade. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5034. doi:10.1158/1538-7445.AM2014-5034

Introduction: Systemic administration of the small molecule Toll-like receptor (TLR)-7 agonist DSR-6434 leads to potent activation of innate immunity and to the generation of anti-tumor immune responses. However, clinical responses with systemically administered TLR7 agonists have been underwhelming, in part because activation of TLRs with small-molecule agonists can induce TLR tolerance; defined as a state of hyporesponsiveness to subsequent agonism. This study is undertaken to identify conditions to overcome TLR tolerance. Experimental procedures: To confirm the anti-tumor effect of DSR-6434, mice were inoculated with the mouse renal cell carcinoma cell line, Renca, at day 0, and then administrated DSR-6434 weekly from day 1, or twice weekly from day 6. To examine tolerance, wild-type or IFN-α/β receptor knockout (IFN-AR KO) mice were intravenously administrated DSR-6434 at intervals of 3, 7 or 10 days. Plasma samples were taken 2 hours after the second administration of DSR-6434 and IFN-α levels were measured. Splenocytes were isolated 24 hours following either single or sequential i.v. doses. To measure lymphocyte activation, CD69 expression was assessed and splenocyte-mediated cytotoxicity against YAC-1 target cells was determined. Bone-marrow derived pDC (BM-pDC) were also treated with DSR-6434 for 5 or 48 hours. Expression of TLR7 signaling-related genes was determined by real-time quantitative RT-PCR. Results: Weekly administration of DSR-6434 significantly reduced tumor burden when compared to vehicle-treated mice. By contrast, pre-inoculation and 2qw administration of DSR-6434 completely abolished anti-tumor activity. IFN-α induction was completely impaired following the second administration of DSR-6434 after 3 days, partially impaired after 7 days, and fully functional when the dosing interval was extended to 10 days. Sequential dosing of DSR-6434 reduced the frequency of activated splenocytes (defined as CD69+) and the level of cytotoxicity, compared with a single administration of DSR-6434. TLR7 tolerance was also observed in IFN-AR KO mice, suggesting this effect was independent of IFN signaling. Interestingly, TLR7 down-regulation was only observed after DSR-6434 dosing, while the expression of other TLR7-signaling related genes was unaltered. Conclusion: The dosing schedule of systemically administered TLR7 agonists significantly affect TLR tolerance and antitumor activity, offering a potential solution to overcome TLR tolerance . Furthermore, TLR7 expression on BM-pDC cells may serve as a useful biomarker of TLR7 tolerance and aid in the optimization of dosing schedules involving systemically administered TLR7 agonists. Citation Format: Erina Koga-Yamakawa, Masashi Murata, Simon J. Dovedi, Robert W. Wilkinson, Hiroki Umehara, Eiji Sugaru, Yuko Hirose, Hideyuki Harada, David T. Robinson, Philip J. Jewsbury, Setsuko Yamamoto, Chiang J. Li. Enhancement of antitumor activity of DSP-6434, a novel TLR7 agonist through reduction of TLR tolerance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2567. doi:10.1158/1538-7445.AM2014-2567