CD73 Inhibitor Research Articles

Adenosine restrains ILC2-driven allergic airway inflammation via A2A receptor.

Although group 2 Innate Lymphoid Cells (ILC2s) play important roles in driving the pathogenesis of allergic airway inflammation, the molecular mechanisms regulating ILC2 responses remain to be fully elucidated. Adenosine signaling is emerging as an important factor to limit excessive inflammation and tissue damage, its role in ILC2-driven airway inflammation remains to be understood. Here we identify adenosine as a negative regulator of ILC2s and allergic airway inflammation. Elevation of adenosine was observed in lungs after protease papain challenge. Adenosine receptor A2A was abundantly expressed in lung ILC2s. The adenosine analog NECA significantly suppress ILC2s responses and relieved airway inflammation induced by IL-33 or papain. Conversely, blockage of adenosine synthesis by CD73 inhibitor APCP or deficiency of A2A aggravated murine airway inflammation. Adoptive transfer of ILC2s into immunodeficiency NCG mice demonstrated that the regulation of ILC2 by adenosine was cell intrinsic. Mechanistic studies showed that the effects of adenosine on ILC2s were associated with changes in transcriptional profiling, and the elevation of intracellular cAMP and resulted NF-κB downregulation. These observations indicate that adenosine-A2A signaling is a negative regulator of ILC2s, which confers protection against airway inflammation and represents a novel therapeutic target for controlling asthma.

Synthesis and Studies of Potential Inhibitors of CD73 Based on a Triazole Scaffold

AbstractThe ecto‐5′‐nucleotidase CD73 is involved in the production of immunosuppressive adenosine in the tumoral microenvironment and recently became a validated target in immuno‐oncology. To avoid formation of CD73‐produced adenosine, several series of potential inhibitors of the target enzyme based on a triazole scaffold were synthetized and evaluated on recombinant purified hCD73 and in cell‐based assays.

Combined Blockade of TIGIT and CD39 or A2AR Enhances NK-92 Cell-Mediated Cytotoxicity in AML.

This study aimed to characterize different natural killer (NK) cell phenotypes on bone marrow and peripheral blood cells from acute myeloid leukemia (AML) patients and healthy donors (HDs). Our data show that CD56dimCD16− and CD56brightCD16− NK cells represent the predominant NK cell subpopulations in AML, while the CD56dimCD16+ NK cells are significantly reduced compared to HDs. Moreover, TIGIT+ and PVRIG+ cells cluster on the CD56dimCD16+ subset whereas CD39+ and CD38+ cells do so on CD56brightCD16− NK cells in AML. Furthermore, functional effects of (co-)blockade of TIGIT and CD39 or A2AR on NK cell functionality were analyzed. These experiments revealed that the single blockade of the TIGIT receptor results in an increased NK-92 cell-mediated killing of AML cells in vitro. Combined targeting of CD39 or A2AR significantly augments the anti-TIGIT-mediated lysis of AML cells. Our data indicate that distinct NK cell subsets in AML exhibit different immunosuppressive patterns (via the TIGIT/PVRIG receptors and the purinergic pathway). In summary, we conclude that TIGIT, CD39, and A2AR constitute relevant inhibitory checkpoints of NK cells in AML patients. A combinatorial blockade synergistically strengthens NK-92 cell-mediated cytotoxicity. As inhibitors of TIGIT, CD39, and A2AR are clinically available, studies on their combined use could be conducted in the near future.

Generation of Non-Nucleotide CD73 Inhibitors Using a Molecular Docking and 3D-QSAR Approach.

Radiotherapy and chemotherapy are conventional cancer treatments. Around 60% of all patients who are diagnosed with cancer receive radio- or chemotherapy in combination with surgery during their disease. Only a few patients respond to the blockage of immune checkpoints alone, or in combination therapy, because their tumours might not be immunogenic. Under these circumstances, an increasing level of extracellular adenosine via the activation of ecto-5’-nucleotidase (CD73) and consequent adenosine receptor signalling is a typical mechanism that tumours use to evade immune surveillance. CD73 is responsible for the conversion of adenosine monophosphate to adenosine. CD73 is overexpressed in various tumour types. Hence, targetting CD73’s signalling is important for the reversal of adenosine-facilitated immune suppression. In this study, we selected a potent series of the non-nucleotide small molecule inhibitors of CD73. Molecular docking studies were performed in order to examine the binding mode of the inhibitors inside the active site of CD73 and 3D-QSAR was used to study the structure–activity relationship. The obtained CoMFA (q2 = 0.844, ONC = 5, r2 = 0.947) and CoMSIA (q2 = 0.804, ONC = 4, r2 = 0.954) models showed reasonable statistical values. The 3D-QSAR contour map analysis revealed useful structural characteristics that were needed to modify non-nucleotide small molecule inhibitors. We used the structural information from the overall docking and 3D-QSAR results to design new, potent CD73 non-nucleotide inhibitors. The newly designed CD73 inhibitors exhibited higher activity (predicted pIC50) than the most active compound of all of the derivatives that were selected for this study. Further experimental studies are needed in order to validate the new CD73 inhibitors.

CD73 Inhibition Overcomes Immunosuppression and Triggers Autologous T-Cell Mediated Multiple Myeloma Cell Lysis in the Bone Marrow Milieu

CD73 Inhibition Overcomes Immunosuppression and Triggers Autologous T-Cell Mediated Multiple Myeloma Cell Lysis in the Bone Marrow Milieu

Modulation of Purinergic Signaling Prior to Transplantation Both in Hematopoietic Stem Progenitor Cells (HSPCs) or Recipient Bone Marrow (BM) Microenvironment - As Novel Strategies to Accelerate and Facilitate HSPCs Homing and Engraftment

Background. The success rate of hematopoietic stem cell transplantation strongly depends on the number of transplanted hematopoietic stem/progenitor cells (HSPCs) and their speed of engraftment after infusion to the myeloablated transplant recipient. Therefore, clinical outcomes will benefit from accelerating the speed of homing and engraftment rate of transplanted HSPCs. This is important when the number of available HSPCs is low, as seen after poor harvest from BM, poor mobilization efficiency of the donor, and a low number of HSPCs present in the available umbilical cord blood (UCB) unit for an adult recipient. Our recent research demonstrated that purinergic signaling involving extracellular adenosine triphosphate (eATP) and its extracellular metabolite adenosine (eAdo) play a significant opposite role in homing/engraftment of HSPCs - reviewed in Curr Opin Hematol 2021, 28:251-261. To explain this eATP released from the cells of conditioned for transplantation by myeloablation recipient's BM facilitates homing of HSPCs, and subsequently becomes metabolized by cell surface ectonucleotidases CD39 and CD73 to eAdo, that inhibits this process. Therefore, eATP and eAdo upregulated in PB and BM modulate homing/engraftment in i) infused to the recipient donor-derived HSPCs and ii) in recipient BM microenvironment - in an opposite way. We also reported that the beneficial effect of eATP on homing/engraftment of HSPCs depends on the promotion of membrane lipid raft formation on the surface of HSPCs that incorporate homing receptors for their optimal interaction with BM released homing chemoattractants. This process is promoted by eATP activated Nlrp3 inflammasome. On the other hand, Nlrp3 inflammasome and membrane lipid raft formation are inhibited by eAdo in heme oxygenase-1 (HO-1)-dependent manner (Leukemia 2020; 34:1512-1523). Hypothesis. We hypothesized that proper modulation of eATP - eAdo signaling both at the level of transplanted HSPCs and recipient BM microenvironment will speed up the seeding efficiency of transplanted cells to BM niches. Material and Methods. We exposed HSPCs before transplantation ex vivo to i) exogenous eATP or ii) small molecular CD39 and CD73 inhibitors. We also inhibited CD39 and CD73 in transplant recipients BM at the time of myeloablative conditioning. In addition, we also activated ex vivo Nlrp3 inflammasome in HSPCs to be transplanted by specific activator nigericin. In control experiments, eATP stimulated Nlrp3 inflammasome activity was inhibited by the HO-1 activator that is CoPP. Homing of HSPCs was evaluated by measuring a number of donor-derived fluorochrome-labeled cells and clonogenic progenitors in BM of myeloablated hosts at 24 hours after transplantation. Early engraftment was assessed by counting the number of CFU-S and clonogeneic progenitors 12 days after transplantation and by evaluating kinetics of recovery of PB hematopoietic cell counts. Finally, while activation of Nlrp3 inflammasome was assessed by immunofluorescence assay, membrane lipid raft formation was evaluated by confocal microscopy. Results. We noticed that homing and engraftment of HSPCs was significantly accelerated after i) short exposure before transplantation to eATP, ii) inhibition of eAdo formation by CD39, and CD73 inhibitors, and iii) activation of Nlrp3 inflammasome by nigericin. Similarly, inhibition of eAdo formation in recipient BM microenvironment of transplanted mice by CD39 and CD73 inhibitors also improved homing and engraftment efficiency. This correlated with activation in the eATP-dependent manner of Nlrp3 inflammasome in HSPCs followed by membrane lipid raft formation. In the BM microenvironment, upregulation of eATP and inhibition of eAdo also enhanced expression of homing chemoattractants. Conclusions. Since all purinergic signaling modifiers employed in our studies are non-toxic against HSPCs, our data obtained in the animal model indicates that modulation of purinergic signaling before transplantation in HSPCs as well as in BM of the myeloablated recipient would significantly accelerate hematopoietic recovery after hematopoietic transplantation. DisclosuresNo relevant conflicts of interest to declare.

Plasma NTPDase1 Activity Regulates Platelet Purinergic Signaling in Sickle Cell Disease

Acute systemic painful vaso-occlusive episode (VOE) often serves as an antecedent to acute chest syndrome (ACS), which is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. Based on clinical epidemiology, ACS is often preceded by thrombocytopenia and involves massive thrombosis across pulmonary artery branches in 10-20% of ACS patients. Although, released during hemolysis, adenosine diphosphate (ADP) is known to activate platelets by stimulating their P2Y1 and P2Y12 purinergic receptors, antagonists of P2Y12 have not shown any benefit in ACS therapy, justifying the need for better understanding of purinergic signaling in SCD. Ecto-nucleoside-tri-phosphate-diphosphohydrolase-1 (E-NTPDase1; CD39) maintains ADP homeostasis by degrading excessive ADP. Though CD39 inhibits ADP-dependent platelet activation and vascular thrombosis, its role in ASC is still unidentified.Here, we use intravital lung microscopy in transgenic humanized SCD mice to show that intravascular (IV) administration of ADP triggered pulmonary thrombosis in control mice but failed to trigger pulmonary thrombosis in SCD mice. Identical to intravital findings, IV ADP administration also evoked transient thrombocytopenia in control but not SCD mice, while, IV collagen led to comparable drop in platelet count in both SCD and control mice. In vitro turbidimetric aggregation study yet again demonstrated impaired SCD mouse platelet response to ADP, which was significantly augmented by CD39 inhibitor (sodium metatungstate, POM-1). Indeed, we found significantly higher plasma levels and activity of CD39 in SCD mice compared to control mice using ELISA and malachite green assays, respectively. Hemin, a major host-derived damage associated molecular pattern (DAMP) in SCD, was incubated with Human Lung MicroVascular Endothelial Cell (HMVEC-L) to assess CD39 expression using western blotting. Hemin (10 -100 µM) in a dose dependent manner decreased CD39 levels in HMVEC-L.Our current findings suggest that elevated CD39 plasma levels and activity possibly prevents ADP-mediated platelet aggregation and pulmonary thrombosis in SCD. We demonstrated that SCD milieu promotes loss of endothelial CD39, which may be directly associated with increased CD39 plasma levels and activity. Current study explains why P2Y12 blockers are not effective in SCD therapy and warrant the need for further studies to understand the role of purinergic signaling in pathogenesis of ACS. DisclosuresSundd: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring Inc: Research Funding; Bayer: Research Funding.

Immunotherapy Strategy Targeting Programmed Cell Death Ligand 1 and CD73 with Macrophage-Derived Mimetic Nanovesicles to Treat Bladder Cancer.

Combination immunotherapy is a promising strategy to remove the inhibitory effect of the tumor microenvironment on immune effector cells, improving the efficacy of immune checkpoint inhibitor treatment in bladder cancer. However, it is challenging to deliver multiple drugs to the tumor tissue effectively and simultaneously to ensure optimal therapeutic effects. Macrophage-derived exosome-mimetic nanovesicles (EMVs) were designed and validated as a nanoplatform for coloading and delivery of the CD73 inhibitor (AB680) and the monoclonal antibody to programmed cell death ligand 1 (aPDL1). The tumor-targeting, biosafety, and therapeutic effects of these nanocomplexes (AB680@EMVs-aPDL1), as a combined immunotherapy strategy for bladder cancer, were assessed in vitro and in vivo. Our results indicate that the nanodrug system was highly stable, provided adequate biosafety, and enhanced tumor targeting in a mouse model of bladder cancer. Moreover, the CD73 inhibitor reduced extracellular adenosine production, and the combination therapy significantly promoted the activation and infiltration of cytotoxic T-lymphocytes, which helped to optimally suppress tumor growth and extend median survival in vivo. Therefore, using EMVs to deliver a combination of aPDL1 and the CD73 inhibitor may be a useful combined immunotherapy strategy for treating bladder cancer.

Identification of Novel CD39 Inhibitors Based on Virtual Screening and Enzymatic Assays.

The accumulation of adenosine in the tumor microenvironment mediates immunosuppression and promotes tumor growth and proliferation. Intervention of the adenosine pathway is an important direction of antitumor immunity research. CD39 is an important ecto-nucleotidases for adenosine generation, therefore targeting the CD39-adenosine pathway is an emerging immune checkpoint for anticancer treatment. However, currently no CD39 inhibitor has been approved by the U.S. Food and Drug Administration. The development of CD39 drugs is urgent for clinical application. In this study, we combined homology modeling, virtual screening, and in vitro enzymatic activity to characterize the structural features of the CD39 protein and identify a triazinoindole-based compound as a CD39 inhibitor. The identified inhibitor and one of its analogues could effectively prevent the enzymatic activity of CD39 with IC50 values of 27.42 ± 5.52 and 79.24 ± 12.21 μM, respectively. At the same time, the inhibitor significantly inhibited the adenosine monophosphate production in colorectal cancer cell lines (HT29 and MC38) and thereafter prevented cell proliferation. Molecular docking studies, mutagenesis, and microscale thermophoresis indicated that residues such as R85 could be the main contributor in binding triazinoindole compounds. The binding mode can potentially be utilized for hit-to-lead optimization, and the identified inhibitor can be further tested for its anticancer activity in vivo or may serve as a chemical agent to study CD39-related functions.

Modeling codelivery of CD73 inhibitor and dendritic cell-based vaccines in cancer immunotherapy

Dendritic cells (DCs) are the dominant class of antigen-presenting cells in humans; therefore, a range of DC-based approaches have been established to promote an immune response against cancer cells. The efficacy of DC-based immunotherapeutic approaches is markedly affected by the immunosuppressive factors related to the tumor microenvironment, such as adenosine. In this paper, based on immunological theories and experimental data, a hybrid model is designed that offers some insights into the effects of DC-based immunotherapy combined with adenosine inhibition. The model combines an individual-based model for describing tumor-immune system interactions with a set of ordinary differential equations for adenosine modeling. Computational simulations of the proposed model clarify the conditions for the onset of a successful immune response against cancer cells. Global and local sensitivity analysis of the model highlights the importance of adenosine blockage for strengthening effector cells. The model is used to determine the most effective suppressive mechanism caused by adenosine, proper vaccination time, and the appropriate time interval between injections.

CD39+ Fibroblasts Enhance Myofibroblast Activation by Promoting IL-11 Secretion in Hypertrophic Scars

Fibroblasts (Fbs) are critical to hypertrophic scar (HTS) formation and were recently shown to be highly heterogeneous. However, Fb heterogeneity in HTSs has not been fully elucidated. In this study, we observed an increased fraction of CD39+ Fbs in HTS after screening four Fb subtypes (CD26+, CD36+, FAP+, and CD39+). CD39+ Fbs, enriched in the upper dermis, were positively correlated with scar severity. The transcriptional analysis of CD39+ and CD39- Fbs sorted from HTS revealed that IL-11 was more highly expressed in CD39+ Fbs. We then showed that IL-11 was upregulated in HTSs and that its expression was induced by TGFβ1 invitro. TGFβ1 also stimulated the expression of CD39 at the transcriptional and protein levels, mediating the maintenance of the CD39+ phenotype. Furthermore, IL-11 facilitated myofibroblast activation and extracellular matrix production in both CD39+ and CD39- Fbs. Interestingly, CD39+ Fbs secreted more IL-11 on TGFβ1 treatment and were less responsive to IL-11 than CD39- Fbs. Notably, a CD39 inhibitor effectively reduced stretch-induced scar formation and attenuated bleomycin-induced skin fibrosis, suggesting an antiscarring approach by targeting CD39+ Fbs.

A Body of Circumstantial Evidence for the Irreversible Ectonucleotidase Inhibitory Action of FSCPX, an Agent Known as a Selective Irreversible A1 Adenosine Receptor Antagonist So Far.

In previous studies using isolated, paced guinea pig left atria, we observed that FSCPX, known as a selective A1 adenosine receptor antagonist, paradoxically increased the direct negative inotropic response to A1 adenosine receptor agonists (determined using concentration/effect (E/c) curves) if NBTI, a nucleoside transport inhibitor, was present. Based on mathematical modeling, we hypothesized that FSCPX blunted the cardiac interstitial adenosine accumulation in response to nucleoside transport blockade, probably by inhibiting CD39 and/or CD73, which are the two main enzymes of the interstitial adenosine production in the heart. The goal of the present study was to test this hypothesis. In vitro CD39 and CD73 inhibitor assays were carried out; furthermore, E/c curves were constructed in isolated, paced rat and guinea pig left atria using adenosine, CHA and CPA (two A1 adenosine receptor agonists), FSCPX, NBTI and NBMPR (two nucleoside transport inhibitors), and PSB-12379 (a CD73 inhibitor), measuring the contractile force. We found that FSCPX did not show any inhibitory effect during the in vitro enzyme assays. However, we successfully reproduced the paradox effect of FSCPX in the rat model, mimicked the “paradox” effect of FSCPX with PSB-12379, and demonstrated the lipophilia of FSCPX, which could explain the negative outcome of inhibitor assays with CD39 and CD73 dissolved in a water-based solution. Taken together, these three pieces of indirect evidence are strong enough to indicate that FSCPX possesses an additional action besides the A1 adenosine receptor antagonism, which action may be the inhibition of an ectonucleotidase. Incidentally, we found that POM-1 inhibited CD73, in addition to CD39.

A Novel Antagonistic CD73 Antibody for Inhibition of the Immunosuppressive Adenosine Pathway.

Despite some impressive clinical results with immune checkpoint inhibitors, the majority of patients with cancer do not respond to these agents, in part due to immunosuppressive mechanisms in the tumor microenvironment. High levels of adenosine in tumors can suppress immune cell function, and strategies to target the pathway involved in its production have emerged. CD73 is a key enzyme involved in adenosine production. This led us to identify a novel humanized antagonistic CD73 antibody, mAb19, with distinct binding properties. mAb19 potently inhibits the enzymatic activity of CD73 in vitro, resulting in an inhibition of adenosine formation and enhanced T-cell activation. We then investigated the therapeutic potential of combining CD73 antagonism with other immune modulatory and chemotherapeutic agents. Combination of mAb19 with a PD-1 inhibitor increased T-cell activation in vitro Interestingly, this effect could be further enhanced with an agonist of the adenosine receptor ADORA3. Adenosine levels were found to be elevated upon doxorubicin treatment in vivo, which could be blocked by CD73 inhibition. Combining CD73 antagonism with doxorubicin resulted in superior responses in vivo Furthermore, a retrospective analysis of rectal cancer patient samples demonstrated an upregulation of the adenosine pathway upon chemoradiation, providing further rationale for combining CD73 inhibition with chemotherapeutic agents.This study demonstrates the ability of a novel CD73 antibody to enhance T-cell function through the potent suppression of adenosine levels. In addition, the data highlight combination opportunities with standard of care therapies as well as with an ADORA3 receptor agonist to treat patients with solid tumors.

987P A new therapeutic antibody against CD73 that ameliorates the tumor microenvironment and improves the efficacy of cancer immunotherapy

CD73 is expressed on certain types of leukocytes and tumor cells and is known as a poor prognostic factor in patients. CD73 catalyzes conversion of extracellular adenosine monophosphate (AMP) to adenosine (Ado) which accumulates in the tumor microenvironment (TME). Ado suppresses immune activation through Ado receptors, allowing tumors to escape from the host immune system. Suppressing CD73 activity provokes T-cell activation and reverses immune suppression exerted by Ado. Thus, the inhibition of CD73 by anti-CD73 antibodies may provide potent cancer immunotherapy options.

Anti-Inflammatory Effects of Endogenously Released Adenosine in Synovial Cells of Osteoarthritis and Rheumatoid Arthritis Patients.

Exogenous adenosine and its metabolite inosine exert anti-inflammatory effects in synoviocytes of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. We analyzed whether these cells are able to synthesize adenosine/inosine and which adenosine receptors (ARs) contribute to anti-inflammatory effects. The functionality of synthesizing enzymes and ARs was tested using agonists/antagonists. Both OA and RA cells expressed CD39 (converts ATP to AMP), CD73 (converts AMP to adenosine), ADA (converts adenosine to inosine), ENT1/2 (adenosine transporters), all AR subtypes (A1, A2A, A2B and A3) and synthesized predominantly adenosine. The CD73 inhibitor AMPCP significantly increased IL-6 and decreased IL-10 in both cell types, while TNF only increased in RA cells. The ADA inhibitor DAA significantly reduced IL-6 and induced IL-10 in both OA and RA cells. The A2AAR agonist CGS 21680 significantly inhibited IL-6 and induced TNF and IL-10 only in RA, while the A2BAR agonist BAY 60-6583 had the same effect in both OA and RA. Taken together, OA and RA synoviocytes express the complete enzymatic machinery to synthesize adenosine/inosine; however, mainly adenosine is responsible for the anti- (IL-6 and IL-10) or pro-inflammatory (TNF) effects mediated by A2A- and A2BAR. Stimulating CD39/CD73 with simultaneous ADA blockage in addition to TNF inhibition might represent a promising therapeutic strategy.

Substrate binding modes of purine and pyrimidine nucleotides to human ecto-5\u2032-nucleotidase (CD73) and inhibition by their bisphosphonic acid derivatives

Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5′-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the Ki value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.

A Novel CD73 Inhibitor SHR170008 Suppresses Adenosine in Tumor and Enhances Anti-Tumor Activity with PD-1 Blockade in a Mouse Model of Breast Cancer.

IntroductionCD73 and adenosine support growth-promoting neovascularization, metastasis, and survival in cells, and promote anti-PD-1 mAb therapy-induced immune escape. Consequently, developing a CD73 inhibitor as monotherapy and a potential beneficial combination partner with immune-checkpoint inhibitors needs investigation.MethodsCD73 inhibitors were evaluated in vitro with soluble and membrane-bound CD73 enzymes, as well as its PD biomarker responses in human peripheral blood mononuclear cells (PBMC) by flow cytometry and ELISA. The binding modes of the molecules were analyzed via molecular modeling. The anti-tumor activity and synergistic effect of SHR170008 in combination with anti-PD-1 mAb were evaluated in a syngeneic mouse breast cancer model.ResultsSHR170008 was discovered during the initial structural modifications on the link between the ribose and the α-phosphate of AMPCP, which significantly improved the stability of the compound confirmed by the metabolite identification study. Further modifications on the adenine base of AMPCP improved the potency due to forming stronger interactions with CD73 protein. It exhibited potent inhibitory activities on soluble and endogenous membrane-bound CD73 enzymes, and induced IFNγ production, reversed AMP-suppressed CD25+ and CD8+/CD25+ expression, and enhanced granzyme B production on CD8+ T cells in human PBMC. SHR170008 showed dose-dependent anti-tumor efficacy with suppression of adenosine in the tumors in EMT6 mouse breast tumor model. The increase of adenosine in tumor tissue by anti-PD-1 mAb alone was suppressed by SHR170008 in the combination groups. Simultaneous inhibition of CD73 and PD-1 neutralization synergistically enhanced antitumor immunity and biomarkers in response, and exposures of SHR170008 were correlated with the efficacy readouts.ConclusionOur findings suggest that CD73 may serve as an immune checkpoint by generating adenosine, which suppresses the antitumor activity of anti-PD-1 mAb, and inhibition of CD73 may be a potential beneficial combination partner with immune-checkpoint inhibitors to improve their therapeutic outcomes in general.

Istradefylline, an adenosine A2a receptor antagonist, ameliorates neutrophilic airway inflammation and psoriasis in mice

AbstractObjectiveExtracellular adenosine is produced from secreted adenosine triphosphate by cluster of differentiation (CD)39 and CD73. Both are critical nucleotide metabolizing enzymes of the adenosine generating pathway and are secreted by neuronal or immune cells. Adenosine plays a role in energy processes, neurotransmission and endogenous regulation of inflammatory responses. Istradefylline is a selective adenosine A2a receptor antagonist used for the treatment of Parkinson's disease. We have reported that adenosine primes hypersecretion of interleukin (IL)‐17A through the adenosine A2a receptor. Istradefylline, as well as an inhibitor of CD39 (ARL67156) and an inhibitor of CD73 (AMP‐CP), suppressed IL‐17A production, and the administration of istradefylline to mice with experimental autoimmune encephalomyelitis led to the marked amelioration of the disease. These previous results suggest that adenosine is an endogenous modulator of neutrophilic inflammation. We investigated the effect of istradefylline, ARL67156 and AMP‐CP on other mouse models of neutrophilic inflammation.MethodsWe tested the effect of istradefylline, ARL67156 and AMP‐CP on ovalbumin‐induced neutrophilic airway inflammation or imiquimod‐induced psoriasis in mice. The two mice models received these drugs orally or percutaneously, respectively. The production of IL‐17A in the lung and ear thickness were used as an index of the effects.ResultsWe show that istradefylline, ARL67156 and AMP‐CP suppressed the ovalbumin‐induced IL‐17A production in the lung and imiquimod‐induced psoriasis.ConclusionThese results show that adenosine‐mediated IL‐17A production plays a role in neutrophilic inflammation models, and furthermore, istradefylline, ARL67156 and AMP‐CP are effective in animal models of neutrophilic inflammation. Some clinical relevancies in coronavirus disease 2019 are discussed.

The Preventive Effect of the Phenotype of Tumour-Associated Macrophages, Regulated by CD39, on Colon Cancer in Mice

Background: This study was designed to investigate the effect of cluster differentiation (CD)39 and CD73 inhibitors on the expresion of tumour-associated macrophages (TAMs), M1- versus M2-tumour phenotypes in mice with colon cancer. Methods: An in vitro study of co-culture with colon cancer cells and immune cells from the bone marrow (BM) of mice was performed. After the confirmation of the effect of polyoxotungstate (POM-1) as an inhibitor of CD39 on TAMs, the mice were randomly divided into a control group without POM-1 and a study group with POM-1, respectively, after subcutaneous injection of CT26 cells. On day 14 after the injection, the mice were sacrificed, and TAMs were evaluated using fluorescence-activated cell sorting. Results: In the in vitro study, the co-culture with POM-1 significantly increased the apoptosis of CT26 cells. The cell population from the co-culture with POM-1 showed significant increases in the expression of CD11b+ for myeloid cells, lymphocyte antigen 6 complex, locus C (Ly6C+) for monocytes, M1-tumour phenotypes from TAMs, and F4/80+ for macrophages. In the in vivo study, tumour growth in the study group with POM-1 was significantly limited, compared with the control group without POM-1. The expressions of Ly6C+ and major histocompatibility complex class II+ for M1-tumour phenotypes from TAMs on F4/80+ from the tumour tissue in the study group had significantly higher values compared with the control group. Conclusion: The inhibition of CD39 with POM-1 prevented the growth of colon cancer in mice, and it was associated with the increased expression of M1-tumour phenotypes from TAMs in the cancer tissue.

Abstract LB163: Blocking adenosine production with ORIC-533, a CD73 inhibitor with best-in-class properties, reverses immunosuppression in high-AMP environments

Abstract Adenosine is recognized for its anti-inflammatory and immunosuppressive properties. Signaling through adenosine receptors expressed on immune cells initiates a cascade that diminishes activation and proliferation of cytotoxic T-cells, impairs activity of natural killer cells and CD4+ effector T-cells, and promotes the expansion of immunosuppressive cell types. CD73, a cell surface ecto-5'-nucleotidase, converts AMP to adenosine and is a major catalyst of adenosine generation in the tumor microenvironment. Overexpression of CD73 is observed in many solid tumors and may correlate with unfavorable clinical outcomes; therefore, reducing the level of adenosine via inhibition of CD73 is a strategy for enhancing antitumor immune responses and overcoming therapeutic resistance. We have developed ORIC-533, a potent, orally bioavailable, AMP-competitive, small molecule inhibitor of CD73. ORIC-533 is highly selective for CD73 and exhibits picomolar potency in biochemical and cellular assays, blocking adenosine production from AMP. Expression of CD73 on immune cells is sufficient to drive immunosuppression in vitro when AMP is present. We have previously shown that ORIC-533 can rescue activation of CD8+ T- cells exposed to AMP and restore proliferation, cytokine secretion and CD25 expression at low nanomolar concentrations. To understand the role of CD73 in dendritic cell (DC) priming of helper CD4+ T-cells, we utilized co-culture of allogeneic immature DC and CD4+ T-cells, where AMP to adenosine conversion severely suppresses activation of helper CD4+ T-cells. Treatment with ORIC-533 completely restored activation of CD4+ T-cells in this setting. To better define the biologically relevant AMP concentrations likely to be encountered in tumors, we determined the concentration of AMP in a panel of primary patient tumors. We discovered that intratumoral AMP levels can reach up to 500 micromoles, emphasizing the requirement for CD73 inhibitors to be efficacious in a high AMP environment. We previously demonstrated that nanomolar concentrations of ORIC-533, unlike other CD73 inhibitors tested, can efficiently rescue cytotoxic T-cell function in the presence of millimolar AMP concentrations. Surprisingly, in these studies we found that inhibitors of adenosine receptors, A2A or A2A/B, can only rescue CD8+ T-cell function in the context of low micromolar AMP or adenosine and thus are likely to be ineffective in patient tumors with high AMP. Taken together, these preclinical results indicate the CD73 inhibitor ORIC-533 has best-in-class properties in reversing immunosuppression in tumors. ORIC-533 is currently undergoing GLP studies to support an IND filing in the first half of 2021. Citation Format: Dena Sutimantanapi, Chelsea Chen, Bob Warne, Alex Pankov, Jae Chang, Lori Friedman, Melissa Junttila, Tatiana Zavorotinskaya. Blocking adenosine production with ORIC-533, a CD73 inhibitor with best-in-class properties, reverses immunosuppression in high-AMP environments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB163.