Adenosine-mediated Immunosuppression Research Articles

Adenosine Uptake through the Nucleoside Transporter ENT1 Suppresses Antitumor Immunity and T Cell Pyrimidine Synthesis.

Immunosuppression by adenosine is an important cancer immune checkpoint. Extracellular adenosine signals through specific receptors and can be transported across the cell membrane through nucleoside transporters. While adenosine receptors are well-known to regulate tumor immunity, the impact of adenosine transporters remains unexplored. In this study, we investigated the effect on tumor immunity of equilibrative nucleoside transporter-1 (ENT1), the major regulator of extracellular adenosine concentrations. Blocking or deleting host ENT1 significantly enhanced CD8+ T cell-dependent antitumor responses. Tumors inoculated into ENT1-deficient mice showed increased infiltration of effector CD8+ T cells with an enhanced cytotoxic transcriptomic profile and significant upregulation of granzyme B, IFN-γ, IL-2, TNF-α, and CXCL10. ENT1-deficiency was further associated with decreased tumor-infiltrating T regulatory cells and CD206high macrophages and suppressed CCL17 production. ENT1-deficiency notably potentiated the therapeutic activity of PD-1 blockade. T cells upregulated ENT1 upon activation, and blocking ENT1 enhanced their function when co-cultured with cognate antigen/HLA-matched melanoma cells. Mechanistically, ENT1-mediated adenosine uptake inhibited the activity of phosphoribosyl pyrophosphate synthetase (PRPS) in activated T cells, thereby suppressing production of uridine 5'-monophosphate (UMP) and its derivatives required for DNA and RNA synthesis. In summary, this study identified ENT1-mediated adenosine uptake as an important mechanism of adenosine-mediated immunosuppression and pyrimidine starvation that can be targeted to enhance antitumor T cell responses.

Abstract C007: Quemliclustat (CD73 Inhibitor) reduces adenosine-regulated NR4A gene expression and increases mPDAC inflammation in patients from the ARC-8 trial

Abstract Background: Metastatic pancreatic ductal adenocarcinomas (mPDACs) express high levels of the adenosine-generating enzyme CD73 and therapeutically represent a significant unmet medical need, with median patient overall survival (OS) of <1 year. The small-molecule quemliclustat (quemli; AB680) was designed to inhibit soluble and cell-bound CD73 enzymatic activity, thereby providing the capability of reversing adenosine-mediated anti-tumor immune suppression. Quemli, in combination with gemcitabine/nab-paclitaxel (G/nP) ± anti-PD-1 antibody zimberelimab (zim), demonstrated encouraging clinical activity in the first line (1L) mPDAC study ARC-8 (NCT04104672), with median OS ranging from 14.6-19.4 months. Methods and Results: We began by identifying surrogates for tumor adenosine levels, given that it is not possible to measure adenosine levels directly in clinical samples. Cell culture experiments indicated that the stable adenosine analogue NECA can drive upregulation of the NR4A family of transcription factors (NR4A1, NR4A2, and NR4A3) in cancer cells as well as cancer-associated stromal cells (fibroblasts). Treatment with quemli reversed NR4A gene family upregulation, supporting a model in which high baseline levels of NR4A expression in tumors may be reflective of tumors with higher levels of adenosine. Next, we investigated quemli’s mechanism of action (MoA) by transcriptomic assessment of 37 matched pairs of pre- compared to post-treated mPDACs from ARC-8 patients dosed with 100mg of quemli. Treatment with quemli-containing regimens led to a significant reduction in tumor expression levels of the NR4A gene family. Decreased NR4A family tumor expression after treatment was accompanied by a significant increase in tumor inflammation, as measured by a T-cell activation gene signature. Analysis of a published transcriptional dataset from the nivolumab + G/nP control arm of the PRINCE trial in front-line mPDAC (NCT03214250) indicated that high levels of tumor NR4A1, 2, and 3 expression portend significantly worse clinical outcomes in patients treated with this IO/chemotherapy regimen, highlighting a potential opportunity for quemli to provide clinical benefit by reversing adenosine-mediated immunosuppression in mPDAC. Conclusion:Quemli + G/nP treatment was associated with a reduction in tumor expression levels of adenosine-regulated NR4A gene family and was accompanied by an increase in tumor inflammation. Given the adenosine-modulating MoA, patients with mPDACs expressing high levels of NR4A in baseline tumor tissue, which portended poor prognosis in 1L mPDACs treated with G/np-containing regimen from the PRINCE study, may benefit from treatment with quemli. A phase 3 trial (PRISM-1) of G/nP +/- quemli in the 1L mPDAC setting is being planned. Citation Format: Ji Yun Kim, Ning Wang, Ben Weeder, Enzo Stagnaro, Karim Mrouj, Jose Aquino, Xian He, Eileen M O’Reilly, Shubham Pant, Gulam A Manji, Zev A Wainberg, Joon Rhee, Brandon Beagle, Daniel DiRenzo, Matthew J Walters, Angelo Kaplan, Sean Cho, Omar Kabbarah. Quemliclustat (CD73 Inhibitor) reduces adenosine-regulated NR4A gene expression and increases mPDAC inflammation in patients from the ARC-8 trial [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C007.

Dual A2A/A2B Adenosine Receptor Antagonist M1069 Counteracts Immunosuppressive Mechanisms of Adenosine and Reduces Tumor Growth In Vivo.

While A2A adenosine receptor (AR) was considered as a major contributor to adenosine-mediated immunosuppression, A2B, having the lowest affinity to adenosine, has also emerged as a potential contributor to tumor promotion. Therefore, in adenosine-rich tumor microenvironment (TME), where A2B could be complementary and/or compensatory to A2A, simultaneous targeting of A2A and A2B ARs can provide higher potential for cancer immunotherapy. We developed M1069-a highly selective dual antagonist of the A2A and A2B AR. In assays with primary human and murine immune cells, M1069 rescued IL2 production from T cells (A2A dependent) and inhibited VEGF production by myeloid cells (A2B dependent) in adenosine-high settings. M1069 also demonstrated superior suppression of the secretion of protumorigenic cytokines CXCL1, CXCL5, and rescue of IL12 secretion from adenosine-differentiated dendritic cells compared to an A2A-selective antagonist (A2Ai). In a one-way mixed lymphocyte reaction (MLR) assay, adenosine-differentiated human and murine dendritic cells treated with M1069 demonstrated superior T-cell stimulatory activity compared to dendritic cells differentiated in presence of A2Ai. In vivo, M1069 decreased tumor growth as a monotherapy and enhanced antitumor activity of bintrafusp alfa (BA) or cisplatin in syngeneic adenosinehi/CD73hi 4T1 breast tumor model, but not in the CD73 knockout 4T1 tumor model or in adenosinelow/CD73low MC38 murine colon carcinoma model. In summary, our dual A2A/A2B AR antagonist M1069 may counteract immune-suppressive mechanisms of high concentrations of adenosine in vitro and in vivo and enhance the antitumor activity of other agents, including BA and cisplatin.

Interference of ATP-Adenosine Axis by Engineered Biohybrid for Amplifying Immunogenic Cell Death-Mediated Antitumor Immunotherapy.

Immunogenic cell death (ICD) often results in the production and accumulation of adenosine (ADO), a byproduct that negatively impacts the therapeutic effect as well as facilitates tumor development and metastasis. Here, an innovative strategy is elaborately developed to effectively activate ICD while avoiding the generation of immunosuppressive adenosine. Specifically, ZIF-90, an ATP-responsive consumer, is synthesized as the core carrier to encapsulate AB680 (CD73 inhibitor) and then coated with an iron-polyphenol layer to prepare the ICD inducer (AZTF), which is further grafted onto prebiotic bacteria via the esterification reaction to obtain the engineered biohybrid (Bc@AZTF). Particularly, the designed Bc@AZTF can actively enrich in tumor sites and respond to the acidic tumor microenvironment to offload AZTF nanoparticles, which can consume intracellular ATP (iATP) content and simultaneously inhibit the ATP-adenosine axis to reduce the accumulation of adenosine, thereby alleviating adenosine-mediated immunosuppression and strikingly amplifying ICD effect. Importantly, the synergy of anti-PD-1 (αPD-1) with Bc@AZTF not only establishes a collaborative antitumor immune network to potentiate effective tumoricidal immunity but also activates long-lasting immune memory effects to manage tumor recurrence and rechallenge, presenting a new paradigm for ICD treatment combined with adenosine metabolism.

Adenosine analogs have immunomodulatory antiviral properties through the Adenosine A2A Receptor pathway

Abstract The COVID-19 pandemic has unveiled an urgent need for novel antivirals to control emerging infectious diseases. Classic antivirals are often insufficient to clear infections in the absence of an effective immune response. Immunotherapy could complement the use of antivirals; however, its application to infectious diseases remains largely unexplored. Adenosine analogs (AAs) are antiviral drugs which resemble the structure of adenosine, a metabolite which suppresses the immune response through activation of the Adenosine A2A Receptor (A2AR) in immune cells. We hypothesized that selected AAs have previously unrecognized immunomodulatory properties by acting as A2AR antagonists. In this study, we used molecular docking modeling to screen AAs for their capacity to bind to A2AR and test AAs for immunomodulatory properties in cell-based and in vivo assays. We found that selected AAs restored T-cell functions from adenosine-mediated immunosuppression in T-cells, suggesting that they are endowed with immunomodulatory properties through A2AR antagonism, in addition to their intrinsic antiviral properties. These immunomodulatory functions contribute to the antiviral activity of the AAs as we found that the analogs could restore antiviral T-cell responses in COVID-19 models. In conclusion, selected AAs could be used as novel dual – immunomodulatory and intrinsic – antiviral drugs and could represent effective therapies against emerging viral diseases and future pandemics.

Abstract PR15: Adenosine inhibits STING driven anti-tumor immunity in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressed tumor immune microenvironment (TIME) where combinations of chemotherapy and immune checkpoint blockade (ICB) have shown limited efficacy. Standard of care FOLFIRINOX chemotherapy promotes replication stress associated cell death leading to an increase in extracellular ATP in the TIME and subsequent inflammation that favors anti-tumor immunity. Concomitantly, replication stress from chemotherapy also generates aberrant cytosolic DNA, which stimulates the production of 2’-3’ cGAMP, an immunotransmitter that activates STING in cells in the TIME. STING activation leads to the induction of Type 1 Interferon and a host of anti-tumor immune responses. Moreover, ATP augments leukocyte uptake of cGAMP, further increasing STING activation and promoting tumor immunity. However, acute ATP-dependent inflammation is self-limiting when ATP is inevitably converted to immunosuppressive adenosine (Ado) by extracellular ectonucleotidases (notably, CD39 and CD73) which are highly expressed in PDAC. As evidence of the tumor-promoting role of Ado in PDAC, we show in a Phase I neoadjuvant clinical trial for borderline resectable tumors (n=20) that resistance to neoadjuvant FOLFIRINOX and PD-1 inhibition is associated with an increase in adenosine-related gene expression. We deciphered a novel mechanism of adenosine-mediated immunosuppression whereby signaling through Ado receptors interrupts STING activation by cGAMP. Specifically, we show that NECA, a potent adenosine receptor agonist, inhibits phosphorylation of IRF3 downstream of STING activation and consequently blocks transcription of STING-driven IFNB1 in RAW264.7 macrophages, murine bone marrow-derived macrophages, and CD14+ human PBMCs. Under these same conditions, NECA increases IL-6 and IL-1B, highlighting a complex cytokine milieu controlled by ATP metabolism and STING signaling. These effects are reversed with the addition of AB928, an adenosine receptor antagonist. We considered that boosting cGAMP in the PDAC TIME might be sufficient to offset the effects of Ado signaling. In support of this approach, we show that blocking hydrolysis of cGAMP by inhibiting ENPP1 augments the efficacy of FOLFIRINOX against syngeneic murine PDAC tumors, despite the likely presence of adenosine. These findings support the conclusion that adenosine signaling interrupts anti-tumor STING signaling and demonstrate that ENPP-1 inhibition may provide therapeutic benefits by boosting STING and reducing adenosine during chemotherapy treatment in PDAC patients. Citation Format: Alykhan Premji, Jason Link, Takanobu Yamao, Khalid Rashid, Evan Abt, Amanda Labora, Charlotte Chan, Thuc Le, Zev Wainberg, Caius Radu, Timothy Donahue. Adenosine inhibits STING driven anti-tumor immunity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR15.

A2AR eGFP reporter mouse enables elucidation of A2AR expression dynamics during anti-tumor immune responses

There is significant clinical interest in targeting adenosine-mediated immunosuppression, with several small molecule inhibitors having been developed for targeting the A2AR receptor. Understanding of the mechanism by which A2AR is regulated has been hindered by difficulty in identifying the cell types that express A2AR due to a lack of robust antibodies for these receptors. To overcome this limitation, here an A2AR eGFP reporter mouse is developed, enabling the expression of A2AR during ongoing anti-tumor immune responses to be assessed. This reveals that A2AR is highly expressed on all tumor-infiltrating lymphocyte subsets including Natural Killer (NK) cells, NKT cells, γδ T cells, conventional CD4+ and CD8+ T lymphocytes and on a MHCIIhiCD86hi subset of type 2 conventional dendritic cells. In response to PD-L1 blockade, the emergence of PD-1+A2AR- cells correlates with successful therapeutic responses, whilst IL-18 is identified as a cytokine that potently upregulates A2AR and synergizes with A2AR deficiency to improve anti-tumor immunity. These studies provide insight into the biology of A2AR in the context of anti-tumor immunity and reveals potential combination immunotherapy approaches.

CD39 Expression in Peripheral T Cells is Associated with Clinicopathological Characteristics in Patients with Cervical Cancer.

CD39 is an inhibitory checkpoint exerting rate-limiting effects on the ATP-adenosine pathway. It can be targeted to block adenosine-mediated immunosuppression. To analyze the relationship between the CD39 expression and clinicopathological characteristics including FIGO stage, lymph node and distant metastasis, and to further explore its potential role in cervical cancer. Peripheral blood was collected from 59 healthy people and 43 patients with cervical cancer. The percentage and absolute counts of CD3-positive, CD4-positive and CD8-positive T lymphocytes, CD4/CD8 ratio and the percentage of the CD39+ T cells in T lymphocytes were assessed by flow cytometry, and their correlations with clinical parameters were analyzed. Absolute numbers of CD8+ T lymphocytes, CD4/CD8 ratios, and the percentage of the CD39+ T cells were linked with FIGO stage, lymph node metastasis, and distant metastasis. The total numbers of CD8+ T lymphocytes were significantly higher in the peripheral blood of patients with cervical cancer in the early and middle stages than in the advanced stage. In addition, patients with early and middle-stage cervical cancer had considerably lower percentage of CD4+ CD39 + and CD8 + CD39 + T lymphocytes than those with advanced cervical cancer. These results suggest that the absolute counts of CD8+ T lymphocytes may be associated with the patient's prognosis and that the CD39 molecule, expressed on the surface of CD8+ T cells, is also related to FIGO stage, lymph node metastasis, and distant metastasis. CD39 expression on CD8-positive T cells exhibits a negative correlation with the number of CD8-positive T lymphocytes.

Abstract 4092: Development of nucleotide metabolic reprogrammed CAR T cell therapy suitable for solid tumors

Abstract CAR T cell therapy has represented an exciting breakthrough in the treatment of patients with hematologic malignancies. In contrast, the antitumor activity of CAR T cell therapy in solid tumors has been modest so far in clinical studies. The suboptimal clinical efficacy is a result of multiple factors including limited T cell trafficking, persistence, and the immunosuppressive tumor microenvironment (TME). While current strategies utilize costimulatory molecules and cytokines to activate CAR T cells, a key but largely overlooked problem of CAR T cell therapy is nutrient competition between tumor cells and T cells in the nutrient-poor TME. In our previous studies, a metabolic screen identified inosine as an alternative fuel for T cells which can support T cell growth and function in the absence of glucose. T cells could metabolize inosine into hypoxanthine and phosphorylated ribose by purine nucleoside phosphorylase. Moreover, we demonstrated that T cells have advantage over cancer cells in utilizing inosine, and supplementation with inosine enhances the antitumor efficacy of PD-1 mAb and adoptive T cell transfer in mouse models. In this study, we therefore engineered CAR T cells to express CD26 on the cell surface and a secreted adenosine deaminase (ADA) fused with an anchor. These metabolic reprogrammed CAR (designated as MRCAR) T cells exerts their activities through different mechanisms. ADA irreversibly converts adenosine to inosine, overcoming adenosine-mediated immunosuppression and providing inosine for CAR T cell growth, while CD26 induces a rich chemokine receptor profile enabling CAR T cells to traffic to solid tumors. We demonstrated that CD26-overexpressed CAR T cells displayed superior migration capacity and resisted TGF-beta suppression, while the CD26 expression on unmodified CAR T cells was significantly down-regulated by TGF-beta. We further proved that ADA is conditionally secreted in stress condition (i.e., low pH, lack of nutrient and oxygen) based on ADA’s biological characteristics, suggesting that ADA activates CAR T cells as a trans-signaling to the CAR construct in a tumor specific manner. Remarkably, we observed that the MRCAR T cells inhibit tumor growth more effectively than unmodified CAR T cells in both Huh7 human hepatocellular carcinoma (GPC3-MRCAR) and A549 human non-small cell lung cancer (HER2-MRCAR) mouse models. Thus, MR-CAR T cell therapy represents a promising approach to improve CAR T cell therapy against solid tumors. Citation Format: Yue Hu, Abhijit Sarkar, Kevin Song, Magnus Hook, Andras Heczey, Xiaotong Song. Development of nucleotide metabolic reprogrammed CAR T cell therapy suitable for solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4092.

176P Phase I study of INCB106385 alone or in combination with immunotherapy in advanced solid tumors

A2A/A2B receptors drive adenosine-mediated immunosuppression in the tumor microenvironment; blockade has shown clinical activity in advanced solid tumors. This ongoing, 2-part (dose-escalation/expansion) phase 1 study is investigating the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of INCB106385, an oral, potent, selective dual A2A/A2B receptor antagonist, alone or combined with an anti-PD-1 antibody retifanlimab for advanced solid tumors (NCT04580485). Part 1 (dose-escalation) monotherapy results are reported herein.

Overcoming high level adenosine-mediated immunosuppression by DZD2269, a potent and selective A2aR antagonist

BackgroundAdenosine is a potent immunosuppressant whose levels in the tumor microenvironment (TME) are often much higher than those in normal tissues. Binding of adenosine to its receptor A2aR activates a cascade of genes and leads to immunosuppression. In addition, immune checkpoint blockage markedly increases A2aR expression in T cells, which could dampen their anti-tumor response. Several A2aR antagonists are under clinical development, but with limited clinical benefit reported so far. These A2aR antagonists showed much diminished activity at high adenosine levels found in TME, which may explain their clinical underperformance. We report the discovery and early clinical development of DZD2269, a novel A2aR antagonist which can fully block A2aR mediated immunosuppression commonly found in TME. Adenosine stimulates phosphorylation of cyclic AMP response element binding protein (CREB) in T cells and inhibits anti-tumor cytokine secretion in PBMCs in a dose-dependent manner. DZD2269 was able to reverse the immunosuppression induced by high concentrations of adenosine, as demonstrated by inhibiting CREB phosphorylation in T cells, restoring Th1 cytokine secretion in PBMCs, and stimulating dendritic cells (DCs) maturation. As a single agent, DZD2269 showed anti-tumor growth in multiple syngeneic mouse tumor models, and more profound anti-tumor effects were observed when DZD2269 was in combination with immune checkpoint inhibitors, radiotherapy, or chemotherapy. A good PK/PD relationship was observed in these animal models. In the phase 1 clinical study, downregulation of pCREB was detected in human T cells, consistent with preclinical prediction. Our data support further clinical development of DZD2269 in patients with cancer.MethodsThe selectivity of DZD2269 for adenosine receptors was tested in engineered cell lines, and its efficacy in blocking A2aR signaling and reversing adenosine-mediated immunosuppression was assessed in human T cells and peripheral blood mononuclear cells (PBMCs). The anti-tumor effects of DZD2269 were evaluated in multiple syngeneic mouse models as a single agent as well as in combination with chemotherapy, radiotherapy, or immune checkpoint inhibitors. A phase 1 study in healthy volunteers (NCT04932005) has been initiated to assess safety, pharmacokinetics (PK) and pharmacodynamics (PD) of DZD2269.ResultsAdenosine stimulates phosphorylation of cyclic AMP response element binding protein (CREB) in T cells and inhibits anti-tumor cytokine secretion in PBMCs in a dose-dependent manner. DZD2269 was able to reverse the immunosuppression induced by high concentrations of adenosine, as demonstrated by inhibiting CREB phosphorylation in T cells, restoring Th1 cytokine secretion in PBMCs, and stimulating dendritic cells (DCs) maturation. As a single agent, DZD2269 showed anti-tumor growth in multiple syngeneic mouse tumor models, and more profound anti-tumor effects were observed when DZD2269 was in combination with immune checkpoint inhibitors, radiotherapy, or chemotherapy. A good PK/PD relationship was observed in these animal models. In the phase 1 clinical study, downregulation of pCREB was detected in human T cells, consistent with preclinical prediction.ConclusionDZD2269 is a novel A2aR antagonist which can fully block A2aR mediated immunosuppression commonly found in TME. Clinical development of DZD2269 in patients with cancer is warranted (NCT04634344).

Abstract 256: Dual A2aR/A2bR antagonism with etrumadenant (AB928) eliminates the suppressive effects of adenosine on immune and cancer cells in the tumor microenvironment

Abstract INTRODUCTION: Tumors employ many strategies to attenuate immune responses. High levels of extracellular adenosine generated in the tumor microenvironment engage A2a and A2b adenosine receptors on immune cells, resulting in immunosuppression. Expression of A2aR and A2bR can vary by cell type, with T cells predominantly expressing A2aR while myeloid cells express both A2aR and A2bR. We have previously shown that etrumadenant, a dual A2aR/A2bR antagonist, blocks the immunosuppressive effects of adenosine in immune cells and enhances anti-tumor immune responses in mouse syngeneic tumors. Using dual and selective adenosine receptor antagonists, we assessed the contribution of these receptors to adenosine-mediated phenotypes in immune and cancer cells. METHODS: Human CD8+ T cells were isolated from healthy human blood and activated using CD3/CD28/CD2 stimulation and cytokines were analyzed by cytokine bead array at 72 hours. Primary human dendritic cells (DC) were isolated from healthy blood and matured with LPS/IFN-γ for 24 hours. Cancer cell lines were purchased from ATCC. The adenosine analogue NECA was used to stimulate A2aR/A2bR-mediated signaling. RESULTS: Activated human CD8+ T cells stimulated in the presence of NECA showed suppression of activation markers (CD69) and cytokine production (IFN-γ, IL-2 and granzyme B). As expected, we observed similar rescue of this phenotype with both etrumadenant and an A2aR-specific antagonist owing to the sole expression of A2aR on T cells. In contrast, primary DC have comparable expression of A2aR and A2bR, suggesting that dual blockade may provide greater resistance to adenosine-mediated suppression than A2aR antagonism. Indeed, etrumadenant was able to attenuate the adenosine-mediated upregulation of IL-10 and enhance IL-12p70 production, whereas a comparable A2aR-specific antagonist showed no significant rescue versus NECA-stimulated controls. These observations may be extended to suppressive myeloid populations as well as tumor-resident macrophages and myeloid-derived suppressor cells isolated from mouse syngeneic tumors, which have very high expression of both A2aR and A2bR. RNA-sequencing identified a cassette of genes regulated by adenosine-signaling in these cells, which were largely reversed by etrumadenant. Finally, cultured human cancer cell lines have high expression of A2bR, which has been implicated in driving their tumorigenesis. Etrumadenant reversed adenosine-stimulated gene expression changes in non-small cell lung cancer cell lines, restoring enriched pathways driven by adenosine signaling. CONCLUSIONS: Taken together, these results show an important role for A2aR/A2bR in adenosine-mediated immunosuppression and provide a mechanistic rationale for stimulation of anti-tumor immune responses with the dual adenosine receptor antagonist etrumadenant. Citation Format: Sachie Marubayashi, Bindi Patel, Livia Yamashiro, Dana Piovesan, Sean Cho, Jenna Jeffrey, Manmohan Leleti, Jay Powers, Matt Walters, Daniel DiRenzo. Dual A2aR/A2bR antagonism with etrumadenant (AB928) eliminates the suppressive effects of adenosine on immune and cancer cells in the tumor microenvironment [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 256.

ARC-8: Phase I/Ib study to evaluate safety and tolerability of AB680 + chemotherapy + zimberelimab (AB122) in patients with treatment-naive metastatic pancreatic adenocarcinoma (mPDAC).

404 Background: AB680, a potent, selective small-molecule inhibitor of soluble and membrane-bound CD73, targets a major pathway of extracellular adenosine production with the aim of eliminating adenosine-mediated immunosuppression within the tumor microenvironment. In mPDAC, programmed cell death protein-1 (PD-1) axis inhibitors have limited clinical activity as monotherapies or combined with standard-of-care (SOC) chemotherapy. KRAS mutations, present in >90% of invasive PDACs, are associated with significantly elevated CD73 expression and poor clinical outcomes. Thus, mPDAC may be particularly sensitive to CD73 inhibition combined with SOC chemotherapy + anti–PD-1 antibody (zimberelimab [Zim]) treatment. Methods: ARC-8 (NCT04104672) is an ongoing phase 1b, dose escalation and expansion study in patients (pts) with treatment-naive mPDAC. In the dose escalation, AB680 (25, 50, 75, or 100 mg) is administered intravenously once every 2 weeks (Q2W) with standard doses of nab-paclitaxel/gemcitabine (NP/Gem) + Zim (240 mg) in a 3+3 design to determine the recommended dose for expansion (RDE). In the dose expansion, AB680 will be administered at the RDE with NP/Gem + Zim. Adverse events (AEs) and dose-limiting toxicities (DLTs) are recorded and graded per NCI CTCAE 5.0. AB680 pharmacokinetics, pharmacodynamics, and biomarker evaluations are also being performed throughout the study. Clinical activity is assessed every 8 weeks per RECIST v1.1. Results: As of 04SEPT2020, enrolled patients include 4 in Cohort 1 (25 mg AB680), 6 in Cohort 2 (50 mg AB680), and 3 in Cohort 3 (75 mg AB680). Initial AE profiles were similar to those observed for NP/Gem. The most common treatment-related AEs were fatigue (n=6, 43%), anemia (n=4, 29%), and neutrophil count decrease (n=4, 29%); anemia (n=2, 14%) was the most common treatment-related grade 3/4 AE. Initial pharmacodynamic data (50 mg dose) indicated excellent peripheral target coverage at AB680 trough concentrations. Best overall responses for 9 evaluable pts were 3 with partial response (1 in Cohort 1; 2 in Cohort 2), including a complete response of a target lesion, and 5 with stable disease (1 in Cohort 1; 4 in Cohort 2). One pt in Cohort 1 discontinued the study due to progressive disease. As of the cutoff date, 1 DLT (Grade 2 autoimmune hepatitis) occurred in Cohort 2; the event completely resolved with steroids and the pt was able to resume study treatment. No DLTs were reported in Cohort 3. Conclusions: Preliminary results from ARC-8 indicate that AB680, the first clinical-stage small-molecule CD73 inhibitor, in combination with SOC chemotherapy + Zim has a manageable safety profile consistent with that expected for each agent alone and demonstrates early signals of clinical activity. Dose escalation to 100 mg AB680 (Cohort 4) is ongoing to inform RDE selection. Clinical trial information: NCT04104672.

Abstract 5555: In vivo and in vitro characterization of RVU330 best-in-class dual A2A/A2B adenosine receptor antagonist

Abstract The immunosuppressive role of adenosine attracts attention of many researchers as an interesting target for anticancer therapy. High concentrations of adenosine detected in many tumor models affect several subpopulations of infiltrating immune cells e.g. T lymphocytes, dendritic cells and macrophages. In these cells, adenosine signals via either A2A or A2B receptor, therefore the highest efficiency in reversal of adenosine mediated immunosuppression should be achieved by dual antagonism to both receptors. Here, we present data for best-in-class dual A2A/A2B antagonist RVU330 that exerts nanomolar potency in tumor-like adenosine-rich environment. By head-to-head comparison of our in-house developed dual A2A/A2B antagonist to compounds currently pursued in clinical development, we have demonstrated the best-in-class characteristics of our molecule. RVU330 maintains sub- or low nanomolar antagonistic potency in a high adenosine conditions on both A2A and A2B receptor. Multilevel pharmacological characterization spans from receptor binding and cAMP accumulation (assays in cell lines overexpressing target receptors) to functional assays in primary human immune cells which demonstrate the reversibility of immune suppression induced by adenosine. Our compound shows superior activity in antagonizing adenosine-mediated immunosuppression in CD4+ and CD8+ T-cells (IL2 and TNFα production), monocyte derived dendritic cells - moDCs (IL12 and TNFα production, driven mainly by A2B receptors) and macrophages (suppression of VEGF release by M2). High activity in human whole blood assay demonstrates that low nanomolar concentration is sufficient to fully antagonize adenosine. Therapeutic efficacy of RVU330 has been confirmed in syngeneic in vivo mouse model. New best-in-class dual A2A/A2B adenosine receptor antagonists RVU330 combines two crucial activities which addresses distinct, cell type mediated mode of action of adenosine related immunosupression. The dual A2A/A2B antagonistic profile as well as the ability to inhibit both receptors at very high adenosine conditions are key features differentiating RVU330 from other adenosine receptor antagonist. The RVU330 is currently being evaluated in preclinical studies. Citation Format: Michał Gałęzowski, Katarzyna Dziedzic, Paulina Węgrzyn, Aniela Gołas, Magdalena Bońkowska, Karolina Grycuk, Mateusz Ogórek, Aneta Bobowska, Joanna Szeremeta-Spisak, Marcin Nowogródzki, Grzegorz Satała, Iwona Łozińska-Raj, Przemysław Wyrębek, Marcelina Dudek, Anita Janiga, Marek Wronowski, Magdalena Zastawna, Mateusz Świrski, Luigi Stasi, Peter Littlewood, Krzysztof Brzózka, Mateusz O. Nowak. In vivo and in vitro characterization of RVU330 best-in-class dual A2A/A2B adenosine receptor antagonist [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 5555.

Abstract 1071: Targeting A2aR in mouse Pten-deficient prostate cancer

Abstract Inactivation of PTEN occurs frequently in advanced human prostate cancer and can promote an immunosuppressive tumor microenvironment (TME). Extracellular adenosine can be produced by tumors which can in turn modulate anti-inflammatory and immunosuppressive effects via interactions with the A2a receptor (A2aR). Here, we used a transgenic mouse model of Pten-deficient prostate cancer to characterize adenosine-mediated immunosuppression in the TME and evaluate the effects of A2aR blockade. Transcriptomic analysis of normal wildtype prostate and Pten-deficient prostate tumors revealed enriched signatures for genes downregulated after a A2aR knockdown or upregulated after A2aR stimulation. Moreover, androgen withdrawal led to increased expression of the A2aR-stimulated responsive genes Cxcl2/3/5, Il1b, S100a8, Ptgs2 and Thbs1. Immunohistochemical staining of CD73, the ecto-enzyme responsible for producing extracellular adenosine, showed heterogeneous staining ranging from negative to focally strong in epithelial cancer cells and was strongly expressed in immune cells. A2aR was also strongly expressed on immune infiltrates. We used the oral A2aR antagonist AZD4635 to determine the immunosuppressive activity of extracellular adenosine and evaluate its antitumor activity in the context of castration-naïve (CNPC), castration-sensitive (CSPC) and castration-resistant (CRPC) prostate cancer. Four weeks of treatment with AZD4635 led to a 12.1% (P=0.140) reduction of tumor burden compared to control mice. Notably a subset of these mice (4/8) had tumor reductions greater than 20%. For the CSPC/CRPC models, mice were orchidectomized and treated with apalutamide with or without AZD4635 for four or eight weeks to represent CSPC or CRPC phenotypes, respectively. In these settings, treatment with AZD4635 improved the reduction of tumor burden by 16.4% (P=0.165) and 16.8% (P=0.123) in CSPC and CRPC, respectively. Despite similar reduction rates between the cohorts, a greater proportion of AZD4635 treated mice were below the median distribution in the CRPC setting compared to the CSPC setting, 50% (4/8) vs 70% (7/10), respectively. Immunophenotyping showed an increase in the abundance of dendritic cells in tumors and tumor draining lymph nodes of AZD4635-treated mice in the CPNC setting. Whereas a shift in M2 to M1 macrophage, decrease of PD1hi CD8 T cells and an increase in genes related to cytotoxic lymphocyte activity (Gzma, Gzmb, Prf1, Klrg1, and B3gat1) were observed in AZDD4635 treated mice in the CRPC setting. Decreased expressions of immunosuppressive genes related to T regulatory cells (Ctla4, Il2ra, Itga2, Tigit, Foxp3) were noted in both CSPC and CRPC settings. Our findings show that targeting extracellular adenosine with AZD4635 is effective in a subset of mouse Pten-deficient tumors and provides evidence that suggests context-specific immune modulating activity by extracellular adenosine in prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Alwin Schuller, Kazuto Nishio, Hirotsugu Uemura. Targeting A2aR in mouse Pten-deficient prostate cancer [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 1071.

Abstract A10: The dual A2aR/A2bR antagonist AB928 reverses adenosine-mediated immune suppression and inhibits tumor growth in vivo

Abstract Introduction: High levels of immunosuppressive adenosine are found in the tumor microenvironment, reaching 50-100 μM in experimental models. Adenosine exerts its effects on immune cells primarily through the adenosine receptors A2aR/A2bR, which increase intracellular levels of cyclic AMP, leading to CREB phosphorylation (pCREB). We have previously shown that the dual A2aR/A2bR antagonist AB928 is capable of inhibiting adenosine-induced pCREB in healthy human volunteer (HV) blood lymphocytes. AB928 has also been shown to relieve adenosine-mediated T-cell suppression in vitro and exhibit combinatorial effects with standard-of-care chemotherapeutics in mouse syngeneic tumor models. Herein, we show that AB928 is capable of inhibiting NECA-induced gene expression changes and CREB phosphorylation in non-small cell lung carcinoma (NSCLC) patient whole blood (WB). Additionally, observations from our in vitro human studies showing the combinatorial effect of AB928 and α-PD-1 were reproduced in B16F10 syngeneic tumors. Methods: Human WB was stimulated with 5 μM of NECA and flow cytometry was used to quantify AB928-mediated inhibition of pCREB and CD3ζ phosphorylation. B16F10 tumors were treated with α-PD-1 +/- AB928 and gene expression was determined from excised mouse tumors using the nCounter PanCancer panel. Results: To ensure AB928 can successfully inhibit the high levels of intratumoral adenosine, we found that 5 μM NECA provides maximal stimulation and is significantly more potent (>20 fold) than adenosine in the pCREB assay. Additional experiments demonstrated that AB928 has comparable potency in NECA-stimulated WB from both HV and NSCLC patients. In addition to blocking downstream signaling, NanoString analysis showed that AB928 could prevent NECA-stimulated gene expression changes in NSCLC WB. We also found that NECA stimulation, alone or in combination with PD-1 inhibition, significantly reduced proximal TCR signaling, leading to reduced levels of CD3ζ phosphorylation at TYR142 (pTYR142). These reduced pTYR142 levels, with and without α-PD-1, could be significantly rescued by AB928, suggesting that blocking adenosine immunosuppression may provide additional benefit to PD-1 inhibition in tumors. Consistent with these results, AB928 was capable of suppressing growth (volume in mm3) of B16-F10 tumors both as a single agent (vehicle: 462 +/- 58; AB928: 292 +/- 55; p<0.05) or in combination with α-PD-1 therapy (α-PD-1: 341 +/- 60; AB928+α-PD-1: 123 +/- 30; p<0.05). Further, NanoString analysis of the tumors showed an increased “T-cell Functions” score with both α-PD-1 and AB928 monotherapy, which was further enhanced in combination treatment. Conclusions: Collectively, these results support a role for AB928 in relieving adenosine-mediated immunosuppression by blocking A2aR/A2bR-induced signaling events, gene expression changes, and suppressing tumor growth in vivo. Citation Format: Daniel M. DiRenzo, Nell Narasappa, Dana Piovesan, Devika Ashok, Park Adam, Jenna L. Jeffrey, Manmohan R. Leleti, Joanne B.L. Tan, Lisa Seitz, Steve W. Young, Jay P. Powers, Matthew J. Walters. The dual A2aR/A2bR antagonist AB928 reverses adenosine-mediated immune suppression and inhibits tumor growth in vivo [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A10.

Abstract A87: The A2AR antagonist AZD4635 prevents adenosine-mediated immunosuppression in tumor microenvironment and enhances antitumor immunity partly by enhancing CD103+ dendritic cells

Abstract Adenosine signaling via the adenosine 2A receptor (A2AR) is emerging as an important regulatory mechanism of immune responses. Adenosine levels are increased in the tumor microenvironment by the CD39/CD73 axis and adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects that promote tumor growth and limit the efficacy of immune checkpoint inhibitors. We provide evidence that AZD4635, an oral A2AR antagonist, inhibits downstream signaling causing enhanced antigen presentation by CD103+ DCs and increased T-cell activation. In our in vitro assays, NECA (a stable analog of adenosine) caused potent suppression of CD3/CD28 stimulated IFNγ secretion in T cells with 1 μM NECA leading to 80% suppression of IFNγ secretion. Incubation of CD8+ T cells with AZD4635 restored IFNγ secretion back to baseline levels with an observed EC50 of 0.053μM and 0.598 μM at 0.1 and 1 μM NECA, respectively. Treatment of mice bearing CT26 colorectal carcinoma and MC38 colorectal carcinoma with AZD4635 monotherapy led to tumor growth inhibition with (TGI) of 44% and 73%, respectively, and in combination with anti-PD-1 with a TGI of 73% and 91%, respectively. Ex vivo analysis of MC38 OVA tumor-bearing mice showed increase in intratumoral CD8 T cells and CD103+DCs in treated groups compared to control. In particular, combination treatment increased the frequency of OVA antigen specific CD8+ T cells as measured by % of cells expressing the activation marker CD25 (2-fold increase) or the proliferation marker Ki67 (2-fold increase). Aligned with increased tumor antigen-specific T-cell response was a 2-fold increase of intratumoral CD103+ DCs. CD103+DCs play a critical role in tumor antigen trafficking and cross-priming to T cells, which are crucial for successful tumor rejection. In vitro, treatment of CD103+ DCs with NECA suppressed about 50% of pinocytosis (uptake of fluorescent beads) and antigen presentation (OVA peptides), which was almost completely rescued by AZD4635 treatment. We extend these studies into human DCs, and show adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen presentation and T-cell costimulation by DCs, leading to 50% increase in priming and expansion of antigen-specific T cells compared to adenosine-treated group. Our results support the role of adenosine signaling as an intrinsic negative regulator of CD103+ DC maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. Our preclinical data support the evaluation of AZD4635 as a single agent and in combination with durvalumab (αPD-L1 antibody) in patients with solid malignancies (www.clinicaltrials.gov- NCT02740985, NCT03980821). Citation Format: Dinesh Chandra, Christine M. Barbon, Alexandra Borodovsky, Yanjun Wang, Minwei Ye, Laura Prickett, Kris Sachsenmeier, Wenlin Shao, Carl Barrett, Stephen Fawell, Deanna A. Mele, Alwin Schuller. The A2AR antagonist AZD4635 prevents adenosine-mediated immunosuppression in tumor microenvironment and enhances antitumor immunity partly by enhancing CD103+ dendritic cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A87.

Abstract LB-192: The A2AR antagonist AZD4635 prevents adenosine-mediated immunosuppression of CD103+ dendritic cells

Abstract Adenosine signaling is a normal physiologic process preventing autoimmunity, that is co-opted by tumors as an immune escape mechanism. Dendritic cells (DC), generated from human monocytes or mouse bone marrow, are susceptible to the immunosuppressive effects of extracellular adenosine. In vitro studies using a stable analog of adenosine, 5’-N-ethylcarboxamidoadenosine (NECA), demonstrate DC dysfunction in differentiation and of priming naïve T cells. This negatively effects the critical role of DCs to drive antigen-specific tumor cytotoxic T cell responses under conditions of high extracellular adenosine. AZD4635 (HTL-1071) is a selective oral A2AR antagonist, currently in clinical trials as a single agent and in combination with durvalumab (anti-PD-L1 Ab) in patients with solid malignancies. We have reported previously that AZD4635 treatment led to reduction in tumor growth as monotherapy and in combination with anti-PD-L1 Ab in several syngeneic mouse tumor models. Antitumor activity by AZD4635 was supported by the observation that AZD4635 treated tumors had increased expression of genes associated with immune activation and increased expression of co-stimulatory molecules on antigen presenting cells (APCs). We now extend our previous findings by demonstrating improved antigen presentation in antigen-specific mouse (OVA) and human (Melan-A) systems and show reversed dysfunction in mouse CD103+ crosspresenting/crosspriming DC. In this report, we tested the ability of AZD4635 to prevent adenosine-mediated immunosuppression of antigen specific responses in both mouse and human DCs. We demonstrate that AZD4635 could significantly prevent NECA-mediated immuno-suppression of critical aspects of CD103+ DC phenotype and function in vitro. These included CD103+ DC differentiation and costimulatory molecule expression in FLT-3L/GM-CSF differentiated mouse bone marrow cultures, reduced pinocytosis of ovalbumin and subsequently reduced antigen processing and presentation, leading to weaker OT-I T cell functional responses in vitro. NECA also affected the ability of CD103+ DC to crosspresent antigens from dying MC38-Ova tumor cells and crossprime OT-I T cells in vitro. In vivo, immunophenotyping showed that mice bearing MC38-OVA tumors treated with AZD4635 and anti-PD-L1 had increased antigen-specific T cells and CD103+ DC in tumors. CD103+ DC from TdLN of AZD4635 treated mice elicited proliferation of OT-I T cells without exogenously added antigen. In co-cultures of naïve human CD8+ T cells and DCs containing an HLA-A2-restricted Melan-A peptide, NECA abrogated the ability of monocyte-derived DC to prime antigen specific CD8+ T cells. Adenosine antagonism by AZD4635 improved antigen presentation and costimulation by DCs, leading to better priming and expansion of antigen specific T cells. In summary, we report that AZD4635’s MOA includes restoration of DC function in the elicitation of antigen-specific T cell responses. Citation Format: Christine M. Barbon, Alexandra Borodovsky, Yanjun Wang, Laura Prickett, Kris Sachsenmeier, Alwin Schuller, Wenlin Shao, Carl Barrett, Stephen Fawell, Deanna A. Mele. The A2AR antagonist AZD4635 prevents adenosine-mediated immunosuppression of CD103+ dendritic cells [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 LB-192.

Abstract P4-06-19: The effect of relieving adenosine-mediated immunosuppression on trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (T-ADCC) against HER2+ breast cancer cell lines

Abstract Introduction: Trastuzumab (T) is a monoclonal antibody therapy used in the treatment of HER2+ breast cancer. T inhibits HER2 intracellular signalling and is capable of engaging the immune system through ADCC. Adenosine is an important negative regulator of the immune response through its interaction with the A2A receptor (A2AR, ADORA2A). Relieving adenosine-mediated immunosuppression by inhibiting A2AR may improve NK cell-mediated T-ADCC against HER2+ breast cancer cells. In addition, we have previously shown that SKBR3 cells resistant to the EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib are less sensitive to T-ADCC and showed increased A2AR protein levels. This study examines the effects of inhibiting A2AR signalling on NK cell-mediated T-ADCC against treatment naïve HER2+ breast cancer cell lines HCC1954 and SKBR3 and lapatinib and afatinib (irreversible pan-HER-family TKI)-resistant sublines of HCC1954 and SKBR3. Methods: HER2+ breast cancer cell lines SKBR3 and HCC1954 were exposed to afatinib (150nM) or lapatinib (1μM) for 6 months to generate TKI-resistant SKBR3-A and HCC1954-L cell lines. Acid-phosphatase-based proliferation assays were used to confirm resistance to TKI treatment. Western blotting was used to examine A2AR and HER2 protein levels in cell lines. NK cells were isolated from healthy volunteer whole blood by MACSxpress isolation kits. Immune cell-mediated cytotoxicity was determined at a 1:1 (NK cell: TC) ratio over 12 hours using a flow cytometry-based method. Direct cytotoxicity and T-ADCC were determined +/- A2AR agonist CGS21680 (1 μM) and/or A2AR antagonist preladenant (100 nM) for all cell lines. Experiments were carried out three times with three separate volunteer samples with representative results presented. Results: HCC1954-L cells were 5.3-fold resistance to lapatinib (IC50 1.65 μM +/- 0.22 μM) vs. HCC1954 (IC50 0.31 μM +/- 0.15 μM). SKBR3-A cells were 33-fold resistant to afatinib (IC50 0.28 μM +/- 0.006 nM) vs. the parental SKBR3 cell line (IC50 0.009 μM +/- 0.006 μM). SKBR3 and HCC1954 expressed detectable protein levels of A2AR. A2AR and HER2 levels were not significantly changed between parental and resistant cell lines. Levels of direct cytotoxicity and T-ADCC elicited by NK cells were higher against SKBR3-A (p=0.002) and HCC1954-L cells (p=0.0004) than parental cell lines. The A2AR agonist CGS21680 alone had inconsistent effects on direct cytotoxicity and T-ADCC in all cell lines tested. The addition of A2AR antagonist preladenant to CGS21680, but not preladenant alone, increased T-ADCC against the parental HCC1954 cells by 12.7 +/- 3.4% and parental SKBR3 cells by 9.5 +/- 3.6%. T-ADCC levels in the targeted therapy-resistant HCC1954-L and SKBR3-A cell lines were not impacted by the CGS21680/preladenant combination. Conclusions: A HER2-targeted therapy resistance phenotype is associated with increased T-ADCC in the models tested. Inhibition of activated A2AR can increase T-ADCC elicited by NK cells against treatment naïve HER2+ breast cancer cell lines but not TKI-resistant sublines. Further work is warranted to examine the impact of targeting A2AR in HER2+ breast cancer. Citation Format: Gaynor N, Noone J, Monedero J, Murphy EE, O'Gorman DJ, Crown J, Collins DM. The effect of relieving adenosine-mediated immunosuppression on trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (T-ADCC) against HER2+ breast cancer cell lines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-19.

Abstract A162: AB928, a dual antagonist of the A2aR and A2bR adenosine receptors, relieves adenosine-mediated immune suppression

Abstract Introduction: Adenosine, generated through the hydrolysis of extracellular adenosine monophosphate (AMP) by the ecto-nucleotidase CD73, is an important mechanism for immunosuppression in cancer development. Adenosine’s suppressive effects on immune cells are driven primarily through 2 of the 4 adenosine receptors, A2aR and A2bR. We have previously shown that adenosine-mediated suppression of T-cells can be blocked by the dual A2aR/A2bR antagonist, AB928. Herein, we show that AB928 is capable of relieving adenosine-mediated immune suppression using human in vitro cell cultures, advanced gene expression studies, and mouse syngeneic tumor models. Methods: The ability of AB928 to inhibit adenosine-mediated suppression of dendritic cell function in vitro was assessed using human monocyte-derived dendritic cells (moDC). Briefly, moDC were generated from freshly isolated CD14+ monocytes and differentiated with IL-4/GM-CSF for 7 days +/- adenosine/EHNA +/- AB928. Cells were then taken for NanoString analysis or placed in a mixed lymphocyte reaction (MLR) with CD4+ T-cells. Mouse syngeneic tumor studies were conducted using C57BL/6 mice inoculated with mouse mammary tumor AT3-OVA or melanoma B16-F10 cells. Tumors were subsequently treated with doxorubicin, oxaliplatin, or α-PD-1 +/- AB928. Results: Quantitative immunohistochemistry and analysis of public gene expression databases identified individual human tumor types that express high levels of adenosine processing enzymes. Most notably, non-small cell lung, renal, triple-negative breast, ovarian, colorectal, and gastroesophageal cancers were found to have the most favorable expression profiles for interventions targeting the adenosine pathway. Additionally, a high degree of correlation was found between transcript and protein measurements for CD73 (r2 = 0.87), illustrating the robust and reproducible nature of these techniques. In human in vitro cell cultures, moDC differentiated in the presence of adenosine showed a decreased ability to stimulate IFN-γ secretion from allogenic CD4+ T-cells in a MLR. This suppression was significantly reversed by addition of AB928. Next, multiplexed gene expression profiling using NanoString identified a cassette of 39 genes (>2.0 fold change, p <0.05) that were regulated by adenosine during moDC differentiation. Similarly to the MLR results, AB928 showed rescue of these gene expression changes, further supporting the role for A2aR/A2bR in adenosine-mediated suppression of dendritic cell maturation. The antitumor effects of AB928 were demonstrated in vivo using AT3-OVA tumors treated with anthracycline chemotherapy. Concurrent treatment with AB928 and chemotherapy resulted in significantly reduced tumor volume when compared to chemotherapy alone (doxorubicin: 86 +/- 12 mg; AB928 + doxorubicin: 28 +/- 2 mg; p<0.001); similar results were observed for the combination of AB928 and oxaliplatin. Further, gene expression analysis and histologic analyses showed increased immune cell infiltrate and stromal response in tumors treated with chemotherapy and AB928 in combination. AB928 was also capable of suppressing growth of B16-F10 tumors both as a single agent or in combination with α-PD-1 therapy (α-PD-1: 324 +/- 54 mm3; AB928 + α-PD-1: 180 +/- 35 mm3; p<0.05). Conclusions: Collectively, these results support the importance of targeting both A2aR and A2bR to relieve adenosine-mediated immunosuppression in the context of cancer biology. Furthermore, AB928 increases the antitumor immune response leading to suppressed tumor growth and increased immune cell infiltration in mouse syngeneic tumors. AB928 has successfully completed a phase 1 study in healthy volunteers and has begun clinical trials in subjects with cancer. Citation Format: Daniel DiRenzo, Dana Piovesan, Joanne Tan, Dillon H. Miles, Manmohan R. Leleti, Timothy Park, Ferdie Soriano, Bryan Handlos, Jenna L. Jeffrey, Ehesan U. Sharif, Brandon R. Rosen, Ulrike Schindler, Jay P. Powers, Matthew J. Walters. AB928, a dual antagonist of the A2aR and A2bR adenosine receptors, relieves adenosine-mediated immune suppression [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A162.