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BackgroundImmune responses against tumors are subject to negative feedback regulation. Immune checkpoint inhibitors (ICIs) blocking Programmed cell death protein 1 (PD-1), a receptor expressed on T cells, or its ligand PD-L1 have significantly improved the treatment of cancer, in particular malignant melanoma. Nevertheless, responses and durability are variables, suggesting that additional critical negative feedback mechanisms exist and need to be targeted to improve therapeutic efficacy.MethodsWe used different syngeneic melanoma mouse models and performed PD-1 blockade to identify novel mechanisms of negative immune regulation. Genetic gain-of-function and loss-of-function approaches as well as small molecule inhibitor applications were used for target validation in our melanoma models. We analyzed mouse melanoma tissues from treated and untreated mice by RNA-seq, immunofluorescence and flow cytometry to detect changes in pathway activities and immune cell composition of the tumor microenvironment. We analyzed tissue sections of patients with melanoma by immunohistochemistry as well as publicly available single-cell RNA-seq data and correlated target expression with clinical responses to ICIs.ResultsHere, we identified 11-beta-hydroxysteroid dehydrogenase-1 (HSD11B1), an enzyme that converts inert glucocorticoids into active forms in tissues, as negative feedback mechanism in response to T cell immunotherapies. Glucocorticoids are potent suppressors of immune responses. HSD11B1 was expressed in different cellular compartments of melanomas, most notably myeloid cells but also T cells and melanoma cells. Enforced expression of HSD11B1 in mouse melanomas limited the efficacy of PD-1 blockade, whereas small molecule HSD11B1 inhibitors improved responses in a CD8+T cell-dependent manner. Mechanistically, HSD11B1 inhibition in combination with PD-1 blockade augmented the production of interferon-γ by T cells. Interferon pathway activation correlated with sensitivity to PD-1 blockade linked to anti-proliferative effects on melanoma cells. Furthermore, high levels of HSD11B1, predominantly expressed by tumor-associated macrophages, were associated with poor responses to ICI therapy in two independent cohorts of patients with advanced melanomas analyzed by different methods (scRNA-seq, immunohistochemistry).ConclusionAs HSD11B1 inhibitors are in the focus of drug development for metabolic diseases, our data suggest a drug repurposing strategy combining HSD11B1 inhibitors with ICIs to improve melanoma immunotherapy. Furthermore, our work also delineated potential caveats emphasizing the need for careful patient stratification.

2050 Background: Pre-clinical studies consistently demonstrate that inhibition of the CXCL12/CXCR4/CXCR7 axis abrogates recruitment of pro-vasculogenic bone marrow-derived cells after radiotherapy (RT) of glioblastoma (GBM) and promotes T cell exclusion from the tumor microenvironment (TME). The German multicenter phase 1/2 trial GLORIA (NCT04121455) assesses safety of RT plus escalating dose levels (DL) of the CXCL12-neutralizing RNA-Spiegelmer Olaptesed pegol (OLA; NOX-A12) in patients with chemotherapy-resistant GBM. Methods: Until now, GLORIA enrolled 10 patients newly diagnosed with incompletely resected (n = 8) or biopsied (n = 2) GBM with ECOG≤2, age ≥18 and without MGMT promoter hypermethylation. All patients receive standard RT (60 Gy in 30 fractions or 40.05 Gy in 15 fractions) and continuous (24/7) i.v. infusions of either 200 mg (DL1; n = 3), 400 mg (DL2; n = 3) or 600 mg (DL3; n = 4) per week of OLA for 26 weeks. The primary endpoint (EP) is safety as per incidence of treatment-related adverse events (AE). Secondary EPs include radiographic response as per mRANO criteria, dynamic susceptibility contrast perfusion (DSC) and the fraction of highly-perfused tumor (FTBhigh) as well as the apparent diffusion coefficient (ADC). Target lesions (TL) and non-target lesions (NTL, i.e. in-field satellite lesions) are analyzed separately. Tumor tissue is assessed by high-plex immunofluorescence imaging (co-detection by indexing; CODEX). Matched reference cohorts serve as controls for MRI (n = 14) and CODEX (n = 8) data. Results: Combination of RT and OLA was well-tolerated and safe. Of all G ≥ 2 AEs (n = 77), 3 (4%) were deemed to be solely OLA-related, including 1 grade 3 AE at DL3. There were no dose limiting toxicities and no treatment-related deaths. In total, eight of the nine patients (89%) with TLs at baseline showed a TL response during OLA therapy, with four (40%) reaching partial remission (PR) as per radiologic mRANO criteria (n = 2 at DL1 and n = 2 at DL3). All three patients treated at DL1 and all four of DL3 reached PR of one or more NTLs. In three cases (n = 2 at DL1; n = 1 at DL3), at least one NTL completely disappeared. Under OLA, radiographic responses of NTL were best at the highest DL (DL1 +49.5/DL2 +488.3/DL3 -59%), as was the increase in diffusion (mean ADC increase +46.4/+28.2/+56.7%) and the decrease in FTBhigh (mean -33.5/-32.8/-47.7%). Matched pre-/post-surgery CODEX of a confirmed pseudoprogression revealed intralesional clusters of proliferating cytotoxic T cells. Analysis of tissue from a non-responding patient showed T-cell encapsulation by M2-polarized macrophages in an immune-cell enriched TME. Additional follow-up is ongoing. Conclusions: Interim data from the ongoing GLORIA trial demonstrates safety of RT plus OLA and suggests promising clinical efficacy of a new class of drugs targeting CXCL12 in GBM. Clinical trial information: NCT04121455.

BACKGROUND We recently reported favorable safety, promising clinical efficacy and immunohistochemical indicators of response after radiotherapy (RT) plus escalating doses of the CXCL12-neutralizing RNA-Spiegelmer olaptesed pegol (NOX-A12) for glioblastoma in the German multicenter phase 1/2 GLORIA trial (NCT04121455). Here, we report outcomes after RT plus dual inhibition of vasculogenesis (NOX-A12) and angiogenesis (bevacizumab). METHODS After establishing safety in the monotherapy arm, we enrolled six patients with incompletely resected GBM, ECOG ≤ 2, age ≥ 18 and without MGMT promoter hypermethylation into a pre-planned expansion arm. Patients received standard RT (60 Gy in 30 fractions), continuous i.v. infusions of NOX-A12 (600 mg/week) and i.v. infusions of bevacizumab (10 mg/kg q2w). The primary endpoint was safety. Secondary endpoints included radiographic response, perfusion/diffusion imaging and neurologic performance. RESULTS Dual treatment was well-tolerated and safe. Of all G ≥ 2 AEs (n = 37), two G2 events (5.4%) were deemed related to NOX-A12. There were no dose-limiting toxicities and no treatment-related deaths. Longitudinal NANO assessment revealed stable neurologic functioning in all patients. Five out of six patients achieved partial responses (PRs) as per mRANO in week 9. All PRs remained durable at a median follow up of 5.6 months (range 3.6 to 9.3 months). No progression occurred. The mean best response was -65.9% (-13.3% to -99.9%) for target lesion sums and -92.1% (-76.2% to -100%) for non-target lesion (NTL) sums. In all three patients with NTL at least one lesion disappeared. The mean best change from baseline of the highly perfused-tumor fraction was -84.5% (-51.9% to -100%) and the mean best change of the apparent diffusion coefficient was 20.1% (-24.5% to 59.1%). CONCLUSION Interim data of the ongoing trial confirm the previously established safety profile of NOX-A12 and suggest improved efficacy of dual inhibition of post-radiogenic angio- and vasculogenesis by the addition of bevacizumab.

There is increasing evidence for a link between inflammation and thrombosis. Following tissue injury, vascular endothelium becomes activated, losing its antithrombotic properties whereas inflammatory mediators build up a prothrombotic environment. Platelets are the first elements to be activated following endothelial damage; they participate in physiological haemostasis, but also in inflammatory and thrombotic events occurring in an injured tissue. While physiological haemostasis develops rapidly to prevent excessive blood loss in the endothelium activated by inflammation, hypoxia or by altered blood flow, thrombosis develops slowly. Activated platelets release the content of their granules, including ATP and ADP released from their dense granules. Ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39 dephosphorylates ATP to ADP and to AMP, which in turn, is hydrolysed to adenosine by ecto-5′-nucleotidase (CD73). NTPDase1/CD39 has emerged has an important molecule in the vasculature and on platelet surfaces; it limits thrombotic events and contributes to maintain the antithrombotic properties of endothelium. The aim of the present review is to provide an overview of platelets as cellular elements interfacing haemostasis and inflammation, with a particular focus on the emerging role of NTPDase1/CD39 in controlling both processes.

BackgroundCD73 is an ectonucleotidase producing the immunosuppressor mediator adenosine. Elevated levels of circulating CD73 in patients with cancer have been associated with disease progression and poor response to immunotherapy. Immunosuppressive pathways associated with exosomes can affect T-cell function and the therapeutic efficacy of anti-programmed cell-death protein 1 (anti-PD-1) therapy. Here, we conducted a retrospective pilot study to evaluate levels of exosomal CD73 before and early during treatment with anti-PD-1 agents in patients with melanoma and its potential contribution to affect T-cell functions and to influence the clinical outcomes of anti-PD-1 monotherapy.MethodsExosomes were isolated by mini size exclusion chromatography from serum of patients with melanoma (n=41) receiving nivolumab or pembrolizumab monotherapy. Expression of CD73 and programmed death-ligand 1 (PD-L1) were evaluated on exosomes enriched for CD63 by on-bead flow cytometry. The CD73 AMPase activity was evaluated by mass spectrometry, also in the presence of selective inhibitors of CD73. Interferon (IFN)-γ production and granzyme B expression were measured in CD3/28 activated T cells incubated with exosomes in presence of the CD73 substrate AMP. Levels of CD73 and PD-L1 on exosomes were correlated with therapy response. Exosomes isolated from healthy subjects were used as control.ResultsIsolated exosomes carried CD73 on their surface, which is enzymatically active in producing adenosine. Incubation of exosomes with CD3/28 activated T cells in the presence of AMP resulted in a significant reduction of IFN-γ release, which was reversed by the CD73 inhibitor APCP or by the selective A2A adenosine receptor antagonist ZM241385. Expression levels of exosomal CD73 from serum of patients with melanoma were not significantly different from those in healthy subjects. Early on-treatment, expression levels of both CD73 and PD-L1 on exosomes isolated from patients receiving pembrolizumab or nivolumab monotherapy were significantly increased compared with baseline. Early during therapy exosomal PD-L1 increased in responders, while exosomal CD73 resulted significantly increased in non-responders.ConclusionsCD73 expressed on exosomes from serum of patients with melanoma produces adenosine and contributes to suppress T-cell functions. Early on-treatment, elevated expression levels of exosomal CD73 might affect the response to anti-PD-1 agents in patients with melanoma who failed to respond to therapy.

Adenosine, deriving from ATP released by dying cancer cells and then degradated in the tumor environment by CD39/CD73 enzyme axis, is linked to the generation of an immunosuppressed niche favoring the onset of neoplasia. Signals delivered by extracellular adenosine are detected and transduced by G-protein-coupled cell surface receptors, classified into four subtypes: A1, A2A, A2B, and A3. A critical role of this nucleoside is emerging in the modulation of several immune and nonimmune cells defining the tumor microenvironment, providing novel insights about the development of novel therapeutic strategies aimed at undermining the immune-privileged sites where cancer cells grow and proliferate.

Adenosine, deriving from ATP released by dying cancer cells and then degradated in the tumor environment by CD39/CD73 enzyme axis, is linked to the generation of an immunosuppressed niche favoring the onset of neoplasia. Signals delivered by extracellular adenosine are detected and transduced by G-protein-coupled cell surface receptors, classified into four subtypes: A

BackgroundInhibitors of immune checkpoint programmed cell death protein 1 (PD-1) receptor on T cells have shown remarkable clinical outcomes in metastatic melanoma. However, most patients are resistant to therapy. Production of extracellular adenosine, via CD73-mediated catabolism of AMP, contributes to suppress T-cell-mediated responses against cancer. In this study, we analyzed the expression and activity of soluble CD73 in sera of patients with melanoma undergoing anti-PD-1± cytotoxic T-lymphocyte-associated antigen 4 therapy.MethodsSoluble CD73 expression and activity were retrospectively analyzed in serum of a total of 546 patients with melanoma from different centers before starting treatment (baseline) with anti-PD-1 agents, nivolumab or pembrolizumab, and compared with those of 96 healthy subjects. The CD73 activity was correlated with therapy response and survival of patients.ResultsPatients with melanoma show significantly higher CD73 activity and expression than those observed in healthy donors (pConclusionOur data indicate the relevance of serum CD73 in patients with advanced melanoma receiving anti-PD-1 therapy and support further investigation on targeting CD73 in combination with anti-PD-1 antibodies.

Background PD-1 blocking agents, such as nivolumab, have demonstrated clear anti-tumor effects and clinical benefits in a subset of patients with advanced malignancies. Nonetheless, more efforts are needed to identify reliable biomarkers for outcome, to correctly select patients who will benefit from anti-PD-1 treatment. The aim of this study was to investigate the role of peripheral CD8+T cells expressing CD73, involved in the generation of the immune suppressive molecule adenosine, in predicting outcome after nivolumab treatment in advanced melanoma patients. Methods PBMCs from 100 melanoma patients treated with nivolumab were collected at National Cancer Institute “G. Pascale” of Naples. Frequencies of CD8+ lymphocytes phenotypes were assessed by flow cytometry at baseline before nivolumab treatment, along with clinical characteristics and blood count parameters. Healthy controls (n = 20) were also analysed. Percentages of baseline T cells expressing PD-1 and CD73 were correlated with outcome after nivolumab treatment. Results Melanoma patients presented a lower frequency of total circulating CD8+ lymphocytes than control subjects (p = 0.008). Patients with low baseline percentage of circulating CD8+PD-1+CD73+ lymphocytes (vs 6.9 months, p = 0.001). Patients (39%) with clinical benefit from nivolumab therapy presented a significantly lower frequency of circulating CD8+PD-1+CD73+ lymphocytes than patients who progressed to nivolumab treatment (p = 0.02). Conclusions Our observations suggest that baseline CD73 expression on circulating CD8+PD-1+ lymphocytes appear a promising biomarker of response to anti-PD-1 treatment in melanoma patients. Further investigations are needed for validation and for clarifying its role as prognostic or predictive marker.

CD73 is an ectonucleotidase able to catabolize 5'-adenosine monophosphate (AMP) into adenosine at the extracellular level. Extracellular adenosine plays a critical role in regulating many processes under physiological and pathological conditions. In the context of cancer, the expression and activity of CD73, either in tissue and in biological fluids, is increased leading to high levels of adenosine that potently suppress T-cell mediated responses, promoting tumor progression through stimulation of adenosine receptors. Compelling evidence indicates that elevated levels of CD73-generating adenosine limit the efficacy of cancer immunotherapy. Inhibitors of ectonucleotidases and antagonists of adenosine receptors have emerged as new therapeutic tools to improve anti-tumor immune response and potentially synergize with currently used immunotherapeutic agents. Measurement of CD73 levels in serum of cancer patients is a promising approach that, although it needs to be validated, may help to select patients who will benefit from adenosine-targeting agents and predict response to immunotherapy. Here, we describe a simple and fast method to evaluate the AMPase activity of CD73 in peripheral blood that may also be applied to other biological fluids.

CD73 is an ectonucleotidase able to catabolize 5'-adenosine monophosphate (AMP) into adenosine at the extracellular level. Extracellular adenosine plays a critical role in regulating many processes under physiological and pathological conditions. In the context of cancer, the expression and activity of CD73, either in tissue and in biological fluids, is increased leading to high levels of adenosine that potently suppress T-cell mediated responses, promoting tumor progression through stimulation of adenosine receptors. Compelling evidence indicates that elevated levels of CD73-generating adenosine limit the efficacy of cancer immunotherapy. Inhibitors of ectonucleotidases and antagonists of adenosine receptors have emerged as new therapeutic tools to improve anti-tumor immune response and potentially synergize with currently used immunotherapeutic agents. Measurement of CD73 levels in serum of cancer patients is a promising approach that, although it needs to be validated, may help to select patients who will benefit from adenosine-targeting agents and predict response to immunotherapy. Here, we describe a simple and fast method to evaluate the AMPase activity of CD73 in peripheral blood that may also be applied to other biological fluids.