Stromal cell inhibition of anti-CD20 antibody mediated killing of B-cell malignancies.

Introduction: The glycoengineered type II anti-CD20 monoclonal antibody obinutuzumab has been licensed for treatment in follicular non-Hodgkin lymphoma and B-CLL following clinical trials demonstrating superior outcomes to standard of care treatment. However, ultimately many patients still relapse, highlighting the need to understand the mechanisms behind treatment failure to improve patient care. Resistance to chemotherapy is often caused by the ability of malignant B-cells to migrate to the bone marrow and home into the stromal layer. Therefore, this study aimed to investigate whether stromal cells were also able to inhibit type II anti-CD20 antibody mechanisms of action, contributing to resistance to therapy. Methods: A stromal-tumor co-culture was established in vitro between Raji or Daudi B-cell tumor cells and M210B4 stromal cells in 24 well plates. Results: Contact with stromal cells was able to protect tumor cells from obinutuzumab mediated programmed cell death (PCD), antibody dependent cellular phagocytosis and antibody dependent cellular cytotoxicity. Furthermore, such protection required direct contact between stroma and tumor cells. Stromal cells appeared to interfere with obinutuzumab mediated B-cell homotypic adhesion through inhibiting and reversing actin remodelling, potentially as a result of stromal-tumor cell contact leading to downregulation of CD20 on the surface of tumor cells. Further evidence for the potential role of CD20 downregulation comes through the reduction in surface CD20 expression and inhibition of obinutuzumab mediated PCD when tumor cells are treated with Ibrutinib in the presence of stromal cells. The proteomic analysis of tumor cells after contact with stromal cells led to the identification of a number of altered pathways including those involved in cell adhesion and the actin cytoskeleton and remodeling. Discussion: This work demonstrates that contact between tumor cells and stromal cells leads to inhibition of Obinutuzumab effector functions and has important implications for future therapies to improve outcomes to anti-CD20 antibodies. A deeper understanding of how anti-CD20 antibodies interact with stromal cells could prove a useful tool to define better strategies to target the micro-environment and ultimately improve patient outcomes in B-cell malignancies.

Reprogramming the immunosuppressive tumor microenvironment results in successful clearance of tumors resistant to radiation therapy and anti-PD-1/PD-L1.

Despite breakthroughs in immune checkpoint inhibitors (ICI), the majority of tumors, including those poorly infiltrated by CD8+ T cells or heavily infiltrated by immunosuppressive immune effector cells, are unlikely to result in clinically meaningful tumor responses. Radiation therapy (RT) has been combined with ICI to potentially overcome this resistance and improve response rates but reported clinical trial results have thus far been disappointing. Novel approaches are required to overcome this resistance and reprogram the immunosuppressive tumor microenvironment (TME) and address this major unmet clinical need. Using diverse preclinical tumor models of prostate and bladder cancer, including an autochthonous prostate tumor (Pten-/-/trp53-/-) that respond poorly to radiation therapy (RT) and anti-PD-L1 combinations, the key drivers of this resistance within the TME were profiled and used to develop rationalized combination therapies that simultaneously enhance activation of anti-cancer T cell responses and reprogram the immunosuppressive TME. The addition of anti-CD40mAb to RT resulted in an increase in IFN-y signaling, activation of Th-1 pathways with an increased infiltration of CD8+ T-cells and regulatory T-cells with associated activation of the CTLA-4 signaling pathway in the TME. Anti-CTLA-4mAb in combination with RT further reprogrammed the immunosuppressive TME, resulting in durable, long-term tumor control. Our data provide novel insights into the underlying mechanisms of the immunosuppressive TME that result in resistance to RT and anti-PD-1 inhibitors and inform therapeutic approaches to reprogramming the immune contexture in the TME to potentially improve tumor responses and clinical outcomes.

The CD40 agonist HERA-CD40L results in enhanced activation of antigen presenting cells, promoting an anti-tumor effect alone and in combination with radiotherapy.

Introduction: The ability to modulate and enhance the anti-tumor immune responses is critical in developing novel therapies in cancer. The Tumor Necrosis Factor (TNF) Receptor Super Family (TNFRSF) are potentially excellent targets for modulation which result in specific anti-tumor immune responses. CD40 is a member of the TNFRSF and several clinical therapies are under development. CD40 signaling plays a pivotal role in regulating the immune system from B cell responses to myeloid cell driven activation of T cells. The CD40 signaling axis is well characterized and here we compare next generation HERA-Ligands to conventional monoclonal antibody based immune modulation for the treatment of cancer. Methods & results: HERA-CD40L is a novel molecule that targets CD40 mediated signal transduction and demonstrates a clear mode of action in generating an activated receptor complex via recruitment of TRAFs, cIAP1, and HOIP, leading to TRAF2 phosphorylation and ultimately resulting in the enhanced activation of key inflammatory/survival pathway and transcription factors such asNFkB, AKT, p38, ERK1/2, JNK, and STAT1 in dendritic cells. Furthermore, HERA-CD40L demonstrated a strong modulation of the tumor microenvironment (TME) via the increase in intratumoral CD8+ T cells and the functional switch from pro-tumor macrophages (TAMs) to anti-tumor macrophages that together results in a significant reduction of tumor growth in a CT26 mouse model. Furthermore, radiotherapy which may have an immunosuppressive modulation of the TME, was shown to have an immunostimulatory effect in combination with HERA-CD40L. Radiotherapy in combination with HERA-CD40L treatment resulted in an increase in detected intratumoral CD4+/8+ T cells compared to RT alone and, additionally, the repolarization of TAMs was also observed, resulting in an inhibition of tumor growth in a TRAMP-C1 mouse model. Discussion: Taken together, HERA-CD40L resulted in activating signal transduction mechanisms in dendritic cells, resulting in an increase in intratumoral T cells and manipulation of the TME to be pro-inflammatory, repolarizing M2 macrophages to M1, enhancing tumor control.

Updates on radiotherapy-immunotherapy combinations: Proceedings of 6th annual ImmunoRad conference.

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference.

The suitability of micronuclei as markers of relative biological effect.

Micronucleus (MN) formation is routinely used as a biodosimeter for radiation exposures and has historically been used as a measure of DNA damage in cells. Strongly correlating with dose, MN are also suggested to indicate radiation quality, differentiating between particle and photon irradiation. The "gold standard" for measuring MN formation is Fenech's cytokinesis-block micronucleus (CBMN) cytome assay, which uses the cytokinesis blocking agent cytochalasin-B. Here, we present a comprehensive analysis of the literature investigating MN induction trends in vitro, collating 193 publications, with 2476 data points. Data were collected from original studies that used the CBMN assay to quantify MN in response to ionizing radiation in vitro. Overall, the meta-analysis showed that individual studies mostly have a linear increase of MN with dose [85% of MN per cell (MNPC) datasets and 89% of percentage containing MN (PCMN) datasets had an R2 greater than 0.90]. However, there is high variation between studies, resulting in a low R2 when data are combined (0.47 for MNPC datasets and 0.60 for PCMN datasets). Particle type, species, cell type, and cytochalasin-B concentration were suggested to influence MN frequency. However, variation in the data meant that the effects could not be strongly correlated with the experimental parameters investigated. There is less variation between studies when comparing the PCMN rather than the number of MNPC. Deviation from CBMN protocol specified timings did not have a large effect on MN induction. However, further analysis showed less variation between studies following Fenech's protocol closely, which provided more reliable results. By limiting the cell type and species as well as only selecting studies following the Fenech protocol, R2 was increased to 0.64 for both measures. We therefore determine that due to variation between studies, MN are currently a poor predictor of radiation-induced DNA damage and make recommendations for futures studies assessing MN to improve consistency between datasets.

Radiotherapy transiently reduces the sensitivity of cancer cells to lymphocyte cytotoxicity.

The impact of radiotherapy on the interaction between immune cells and cancer cells is important not least because radiotherapy can be used alongside immunotherapy as a cancer treatment. Unexpectedly, we found that X-ray irradiation of cancer cells induced significant resistance to natural killer (NK) cell killing. This was true across a wide variety of cancer-cell types as well as for antibody-dependent cellular cytotoxicity. Resistance appeared 72 h postirradiation and persisted for 2 wk. Resistance could also occur independently of radiotherapy through pharmacologically induced cell-cycle arrest. Crucially, multiple steps in NK-cell engagement, synapse assembly, and activation were unaffected by target cell irradiation. Instead, radiotherapy caused profound resistance to perforin-induced calcium flux and lysis. Resistance also occurred to a structurally similar bacterial toxin, streptolysin O. Radiotherapy did not affect the binding of pore-forming proteins at the cell surface or membrane repair. Rather, irradiation instigated a defect in functional pore formation, consistent with phosphatidylserine-mediated perforin inhibition. In vivo, radiotherapy also led to a significant reduction in NK cell-mediated clearance of cancer cells. Radiotherapy-induced resistance to perforin also constrained chimeric antigen receptor T-cell cytotoxicity. Together, these data establish a treatment-induced resistance to lymphocyte cytotoxicity that is important to consider in the design of radiotherapy-immunotherapy protocols.

Immunomodulation by radiotherapy in tumour control and normal tissue toxicity.

Radiotherapy (RT) is a highly effective anticancer treatment that is delivered to more than half of all patients with cancer. In addition to the well-documented direct cytotoxic effects, RT can have immunomodulatory effects on the tumour and surrounding tissues. These effects are thought to underlie the so-called abscopal responses, whereby RT generates systemic antitumour immunity outside the irradiated tumour. The full scope of these immune changes remains unclear but is likely to involve multiple components, such as immune cells, the extracellular matrix, endothelial and epithelial cells and a myriad of chemokines and cytokines, including transforming growth factor-ß (TGFß). In normal tissues exposed to RT during cancer therapy, acute immune changes may ultimately lead to chronic inflammation and RT-induced toxicity and organ dysfunction, which limits the quality of life of survivors of cancer. Here we discuss the emerging understanding of RT-induced immune effects with particular focus on the lungs and gut and the potential immune crosstalk that occurs between these tissues.

Radiotherapy-Immunotherapy Combination: How Will We Bridge the Gap Between Pre-Clinical Promise and Effective Clinical Delivery?

Radiotherapy (RT) is highly effective at directly killing tumor cells and plays an important part in cancer treatments being delivered to around 50% of all cancer patients. The additional immunomodulatory properties of RT have been investigated, and if exploited effectively, have the potential to further improve the efficacy of RT and cancer outcomes. The initial results of combining RT with immunomodulatory agents have generated promising data in pre-clinical studies, which has in turn led to a large number of RT and immunotherapy clinical trials. The overarching aim of these combinations is to enhance anti-tumor immune responses and improve responses rates and patient outcomes. In order to maximize this undoubted opportunity, there remain a number of important questions that need to be addressed, including: (i) the optimal RT dose and fractionation schedule; (ii) the optimal RT target volume; (iii) the optimal immuno-oncology (IO) agent(s) to partner with RT; (iv) the optimal site(s)/route(s) of administration of IO agents; and finally, the optimal RT schedule. In this review, we will summarize progress to date and identify current gaps in knowledge that need to be addressed in order to facilitate effective clinical translation of RT and IO agent combinations.

Reprogramming the tumour microenvironment by radiotherapy: implications for radiotherapy and immunotherapy combinations.

Radiotherapy (RT) is a highly effective anti-cancer therapy delivered to around 50-60% of patients. It is part of therapy for around 40% of cancer patients who are cured of their disease. Until recently, the focus of this anti-tumour efficacy has been on the direct tumour cytotoxicity and RT-induced DNA damage. Recently, the immunomodulatory effects of RT on the tumour microenvironment have increasingly been recognized. There is now intense interest in potentially using RT to induce an anti-tumour immune response, which has led to rethinking into how the efficacy of RT could be further enhanced. Following the breakthrough of immune check point inhibitors (ICIs), a new era of immuno-oncology (IO) agents has emerged and established immunotherapy as a routine part of cancer treatment. Despite ICI improving outcomes in many cancer types, overall durable responses occur in only a minority of patients. The immunostimulatory effects of RT make combinations with ICI attractive to potentially amplify anti-tumour immunity resulting in increased tumour responses and improved outcomes. In contrast, tumours with profoundly immunosuppressive tumour microenvironments, dominated by myeloid-derived cell populations, remain a greater clinical challenge and RT may potentially further enhance the immunosuppression. To harness the full potential of RT and IO agent combinations, further insights are required to enhance our understanding of the role these immunosuppressive myeloid populations play, how RT influences these populations and how they may be therapeutically manipulated in combination with RT to improve outcomes further. These are exciting times with increasing numbers of IO targets being discovered and IO agents undergoing clinical evaluation. Multidisciplinary research collaborations will be required to establish the optimal parameters for delivering RT (target volume, dose and fractionation) in combination with IO agents, including scheduling to achieve maximal therapeutic efficacy.

Investigating Radiotherapy Response in a Novel Syngeneic Model of Prostate Cancer.

The prostate cancer (PCa) field lacks clinically relevant, syngeneic mouse models which retain the tumour microenvironment observed in PCa patients. This study establishes a cell line from prostate tumour tissue derived from the Pten-/-/trp53-/- mouse, termed DVL3 which when subcutaneously implanted in immunocompetent C57BL/6 mice, forms tumours with distinct glandular morphology, strong cytokeratin 8 and androgen receptor expression, recapitulating high-risk localised human PCa. Compared to the commonly used TRAMP C1 model, generated with SV40 large T-antigen, DVL3 tumours are immunologically cold, with a lower proportion of CD8+ T-cells, and high proportion of immunosuppressive myeloid derived suppressor cells (MDSCs), thus resembling high-risk PCa. Furthermore, DVL3 tumours are responsive to fractionated RT, a standard treatment for localised and metastatic PCa, compared to the TRAMP C1 model. RNA-sequencing of irradiated DVL3 tumours identified upregulation of type-1 interferon and STING pathways, as well as transcripts associated with MDSCs. Upregulation of STING expression in tumour epithelium and the recruitment of MDSCs following irradiation was confirmed by immunohistochemistry. The DVL3 syngeneic model represents substantial progress in preclinical PCa modelling, displaying pathological, micro-environmental and treatment responses observed in molecular high-risk disease. Our study supports using this model for development and validation of treatments targeting PCa, especially novel immune therapeutic agents.

Toll-Like Receptor Agonists and Radiation Therapy Combinations: An Untapped Opportunity to Induce Anticancer Immunity and Improve Tumor control.

The premise that therapies targeting immune checkpoints can enhance radiation therapy (RT)-induced antitumor immunity is being explored rigorously in the preclinical setting, and early clinical trials testing this hypothesis are beginning to report. Although such approaches might prove efficacious in certain settings, it is likely that many tumor types, particularly those that have a deeply immune-suppressed microenvironment with little or no T cell infiltration, will require alternative approaches. Thus, there is now considerable drive to develop novel immune modulatory therapies that target other areas of the cancer immunity cycle. Toll-like receptors (TLRs) are expressed on sentinel immune cells and play a key role in the host defense against invading pathogens. Innate sensing via TLR-mediated detection of pathogen-derived molecular patterns can lead to maturation of antigen-presenting cells and downstream activation of adaptive immunity. After demonstrating promising efficacy in preclinical studies, drugs that stimulate TLR have been approved for use clinically, albeit to a limited extent. There is a growing body of preclinical evidence that novel agonists targeting TLR3, TLR7/8, or TLR9 in combination with RT might lead to enhanced antitumor immunity. Mechanistic studies have revealed that TLR agonists enhance dendritic cell-mediated T cell priming after RT, in some cases leading to the generation of systemic antitumor immunity and immune memory. In this report, we describe results from preclinical studies that advocate the strategy of combining RT with TLR agonists, discuss reported mechanisms of action, and explore the exciting opportunities of how this approach may be successfully translated into clinical practice.

Diffusion model comparison identifies distinct tumor sub-regions and tracks treatment response.

PURPOSE: MRI biomarkers of tumor response to treatment are typically obtained from parameters derived from a model applied to pre-treatment and post-treatment data. However, as tumors are spatially and temporally heterogeneous, different models may be necessary in different tumor regions, and model suitability may change over time. This work evaluates how the suitability of two diffusion-weighted (DW) MRI models varies spatially within tumors at the voxel level and in response to radiotherapy, potentially allowing inference of qualitatively different tumor microenvironments. METHODS: DW-MRI data were acquired in CT26 subcutaneous allografts before and after radiotherapy. Restricted and time-independent diffusion models were compared, with regions well-described by the former hypothesized to reflect cellular tissue, and those well-described by the latter expected to reflect necrosis or oedema. Technical and biological validation of the percentage of tissue described by the restricted diffusion microstructural model (termed %MM) was performed through simulations and histological comparison. RESULTS: Spatial and radiotherapy-related variation in model suitability was observed. %MM decreased from a mean of 64% at baseline to 44% 6 days post-radiotherapy in the treated group. %MM correlated negatively with the percentage of necrosis from histology, but overestimated it due to noise. Within MM regions, microstructural parameters were sensitive to radiotherapy-induced changes. CONCLUSIONS: There is spatial and radiotherapy-related variation in different models' suitability for describing diffusion in tumor tissue, suggesting the presence of different and changing tumor sub-regions. The biological and technical validation of the proposed %MM cancer imaging biomarker suggests it correlates with, but overestimates, the percentage of necrosis.

Evaluation of apoptosis imaging biomarkers in a genetic model of cell death.

PURPOSE: We have previously developed the caspase-based radiotracer, 18F-ICMT-11, for PET imaging to monitor treatment response. We further validated 18F-ICMT-11 specificity in a murine melanoma death-switch tumour model with conditional activation of caspase-3 induced by doxycycline. METHODS: Caspase-3/7 activity and cellular uptake of 18F-ICMT-11, 18F-ML-10 and 18F-FDG were assessed in B16ova and B16ovaRevC3 cells after death-switch induction. Death-switch induction was confirmed in vivo in xenograft tumours, and 18F-ICMT-11 and 18F-ML-10 biodistribution was assessed by ex vivo gamma counting of select tissues. PET imaging was performed with 18F-ICMT-11, 18F-ML-10 and 18F-FDG. Caspase-3 activation was confirmed by immunohistochemistry. RESULTS: Significantly increased caspase-3/7 activity was observed only in B16ovaRevC3 cells after death-switch induction, accompanied by significantly increased 18F-ICMT-11 (p < 0.001) and 18F-ML-10 (p < 0.05) and decreased 18F-FDG (p < 0.001) uptake compared with controls. B16ova and B16ovaRevC3 tumours had similar growth in vivo; however, B16ovaRevC3 growth was significantly reduced with death-switch induction (p < 0.01). Biodistribution studies showed significantly increased 18F-ICMT-11 tumour uptake following death-switch induction (p < 0.01), but not for 18F-ML-10. Tumour uptake of 18F-ICMT-11 was higher than that of 18F-ML-10 after death-switch induction. PET imaging studies showed that 18F-ICMT-11 can be used to detect apoptosis after death-switch induction, which was accompanied by significantly increased expression of cleaved caspase-3. 18F-FDG signal decreased in tumours after death-switch induction. CONCLUSIONS: We demonstrate that 18F-ICMT-11 can be used to detect caspase-3 activation in a death-switch tumour model, independent of the confounding effects of cancer therapeutics, thus confirming its specificity and supporting the development of this radiotracer for clinical use to monitor tumour apoptosis and therapy response.

The anti-PD-1 era - an opportunity to enhance radiotherapy for patients with bladder cancer.

An urgent need exists to improve the outcomes of patients with muscle-invasive bladder cancer (MIBC), and especially of those with metastatic disease. Treatments that enhance antitumour immune responses - such as immune-checkpoint inhibition - provide an opportunity to do this. Despite initial success, durable response rates in patients with advanced-stage MIBC treated with novel inhibitory antibodies targeting programmed cell death protein 1 (PD-1) or its endogenous ligand programmed cell death 1 ligand 1 (PD-L1) remain low. Radiotherapy is part of the management of bladder cancer in many patients. Evidence that radiotherapy has immunogenic properties is now available, but radiotherapy-induced immune responses are often negated by immunosuppression within the tumour microenvironment. Anti-PD-1 or anti-PD-L1 antibodies might enhance radiotherapy-induced antitumour immunity. This effect has been demonstrated in preclinical models of bladder cancer, and clinical trials involving this approach are currently recruiting. Combination treatment strategies provide an exciting opportunity for urological oncologists to not only improve the chances of cure in patients undergoing radical treatment for MIBC, but also to increase long-term response rates in those with metastatic disease.

Fractionated Radiation Therapy Stimulates Antitumor Immunity Mediated by Both Resident and Infiltrating Polyclonal T-cell Populations when Combined with PD-1 Blockade.

Purpose: Radiotherapy is a highly effective anticancer treatment forming part of the standard of care for the majority of patients, but local and distal disease recurrence remains a major cause of mortality. Radiotherapy is known to enhance tumor immunogenicity; however, the contribution and mechanisms of radiotherapy-induced immune responses are unknown.Experimental Design: The impact of low-dose fractionated radiotherapy (5 × 2 Gy) alone and in combination with αPD-1 mAb on the tumor microenvironment was evaluated by flow cytometry and next-generation sequencing of the T-cell receptor (TCR) repertoire. A dual-tumor model was used, with fractionated radiotherapy delivered to a single tumor site to enable evaluation of the local and systemic response to treatment and ability to induce abscopal responses outside the radiation field.Results: We show that fractionated radiotherapy leads to T-cell infiltration at the irradiated site; however, the TCR landscape remains dominated by polyclonal expansion of preexisting T-cell clones. Adaptive resistance via the PD-1/PD-L1 pathway restricts the generation of systemic anticancer immunity following radiotherapy, which can be overcome through combination with αPD-1 mAb leading to improved local and distal tumor control. Moreover, we show that effective clearance of tumor following combination therapy is dependent on both T cells resident in the tumor at the time of radiotherapy and infiltrating T cells.Conclusions: These data provide evidence that radiotherapy can enhance T-cell trafficking to locally treated tumor sites and augment preexisting anticancer T-cell responses with the capacity to mediate regression of out-of-field tumor lesions when delivered in combination with αPD-1 mAb therapy. Clin Cancer Res; 23(18); 5514-26. ©2017 AACR.

The influence of radiation in the context of developing combination immunotherapies in cancer.

In addition to tumouricidal activity, radiotherapy is now recognized to display potent immunostimulatory properties that can contribute to the generation of anti-cancer immune responses. Treatment with radiation can induce a variety of pro-immunogenic and phenotypic changes in malignant cells, and recalibrate the immune contexture of the tumour microenvironment, leading to enhanced activation of the innate immune system, and priming of tumour-specific T-cell immunity. The immune-dependent effects of radiotherapy provide a sound rationale for the development of combination strategies, whereby the immunomodulatory properties of radiation can be exploited to augment the activity of immunotherapeutic agents. Encouraged by the recent success of breakthrough therapies such as immune checkpoint blockade, and a wealth of experimental data demonstrating the efficacy of radiotherapy and immunotherapy combinations, the clinical potential of this approach is now being explored in numerous trials. Successful translation will require careful consideration of the most suitable dose and fractionation of radiation, choice of immunotherapy and optimal sequencing and scheduling regimen. Immunological control of cancer is now becoming a clinical reality. There is considerable optimism that the development of effective radiotherapy and immunotherapy combinations with the capacity to induce durable, systemic immunity will further enhance patient outcome and transform the future management of cancer.

Stereotactic ablative radiotherapy and immunotherapy combinations: turning the future into systemic therapy?

Radiotherapy (RT) is effective at cytoreducing tumours and until relatively recently the focus in radiobiology has been on the direct effects of RT on the tumour. Increasingly, however, the effect of RT on the tumour vasculature, tumour stroma and immune system are recognized as important to the overall outcome. RT is known to lead to the induction of immunogenic cell death (ICD), which can generate tumour-specific immunity. However, systemic immunity leading to "abscopal effects" resulting in tumour shrinkage outside of the RT treatment field is rare, which is thought to be caused by the immunosuppressive nature of the tumour microenvironment. Recent advances in understanding the nature of this immunosuppression and therapeutics targeting immune checkpoints such as programmed death 1 has led to durable clinical responses in a range of cancer types including malignant melanoma and non-small-cell lung cancer. The effects of RT dose and fraction on the generation of ICD and systemic immunity are largely unknown and are currently under investigation. Stereotactic ablative radiotherapy (SABR) provides an opportunity to deliver single or hypofractionated large doses of RT and potentially increase the amount of ICD and the generation of systemic immunity. Here, we review the interplay of RT and the tumour microenvironment and the rationale for combining SABR with immunomodulatory agents to generate systemic immunity and improve outcomes.

Antitumor Efficacy of Radiation plus Immunotherapy Depends upon Dendritic Cell Activation of Effector CD8+ T Cells.

Tumor cells dying after cytotoxic therapy are a potential source of antigen for T-cell priming. Antigen-presenting cells (APC) can cross-present MHC I-restricted peptides after the uptake of dying cells. Depending on the nature of the surrounding environmental signals, APCs then orchestrate a spectrum of responses ranging from immune activation to inhibition. Previously, we had demonstrated that combining radiation with either agonistic monoclonal antibody (mAb) to CD40 or a systemically administered TLR7 agonist could enhance CD8 T-cell-dependent protection against syngeneic murine lymphoma models. However, it remains unknown how individual APC populations affect this antitumor immune response. Using APC depletion models, we now show that dendritic cells (DC), but not macrophages or B cells, were responsible for the generation of long-term immunologic protection following combination therapy with radiotherapy and either agonistic CD40 mAb or systemic TLR7 agonist therapy. Novel immunotherapeutic approaches that augment antigen uptake and presentation by DCs may further enhance the generation of therapeutic antitumor immune responses, leading to improved outcomes after radiotherapy. Cancer Immunol Res; 4(7); 621-30. ©2016 AACR.

Intravenous administration of the selective toll-like receptor 7 agonist DSR-29133 leads to anti-tumor efficacy in murine solid tumor models which can be potentiated by combination with fractionated radiotherapy.

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.

Immuno-regulatory antibodies for the treatment of cancer.

INTRODUCTION: After years of limited success, progress of anti-cancer immuno-therapeutics has been considerable over the past decade. Key to this progress has been the application of new biological insights around the importance and nature of immune checkpoints that are able to reverse down-regulation of anti-tumor immunity. AREAS COVERED: An overview of the preclinical and recent clinical trial data on key immuno-regulatory agents currently in development, including antibody targeting of cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death receptor 1 (PD-1) on T-lymphocytes and its principal ligand (PD-L1) on tumor cells as well as immune agonists (e.g., anti-CD40). EXPERT OPINION: Durable long-term responses in some patients with advanced melanoma, initially with ipilimumab (anti-CTLA-4) and more recently antibodies targeting either PD-1 or PD-L1 in patients with melanoma and renal cancer, non-small-cell lung, bladder and head and neck cancers with less toxicity, have provided real optimism that immunotherapeutic approaches can improve outcomes in a wide range of cancer. The manageable tolerability of PD-1-pathway blockers and their unique mechanism of action are encouraging combination approaches. Current efforts focus on registration trials of single agents plus combinations in many different tumor types and treatment settings and identifying and developing predictive biomarkers of immunological response.