CD73 Activity Research Articles

T‐helper cell regulation of CD45 phosphatase activity by galectin‐1 and CD43 governs chronic lymphocytic leukaemia proliferation

SummaryChronic lymphocytic leukaemia (CLL) is characterised by malignant mature‐like B cells. Supportive to CLL cell survival is chronic B‐cell receptor (BCR) signalling; however, emerging evidence demonstrates CLL cells proliferate in response to T‐helper (Th) cells in a CD40L‐dependent manner. We showed provision of Th stimulation via CD40L upregulated CD45 phosphatase activity and BCR signalling in non‐malignant B cells. Consequently, we hypothesised Th cell upregulation of CLL cell CD45 activity may be an important regulator of CLL BCR signalling and proliferation. Using patient‐derived CLL cells in a culture system with activated autologous Th cells, results revealed increases in both Th and CLL cell CD45 activity, which correlated with enhanced downstream antigen receptor signalling and proliferation. Concomitantly increased was the surface expression of Galectin‐1, a CD45 ligand, and CD43, a CLL immunophenotypic marker. Galectin‐1/CD43 double expression defined a proliferative CLL cell population with enhanced CD45 activity. Targeting either Galectin‐1 or CD43 using silencing, pharmacology, or monoclonal antibody strategies dampened CD45 activity and CLL cell proliferation. These results highlight a mechanism where activated Th cells drive CLL cell BCR signalling and proliferation via Galectin‐1 and CD43‐mediated regulation of CD45 activity, identifying modulation of CD45 phosphatase activity as a potential therapeutic target in CLL.

A phase II multi-arm study to test the efficacy of oleclumab and durvalumab in specific sarcoma subtypes.

TPS11594 Background: Anti-PD 1/PD-L1 blockade alone or in combination with anti-CTLA4 have yielded suboptimal results in most sarcoma subtypes. CD73, an ectonucleotidase, catalyzes the rate-limiting step for adenosine production in the extracellular space, which then aids tumors in evading immune recognition and destruction. Oleclumab, a monoclonal antibody (mAb) selectively binds and inhibits the activity of CD73, and preclinical data suggests additive activity with durvalumab, a mAb that blocks PD-L1. We designed a trial combining oleclumab and durvalumab in certain sarcoma subtypes, selected based on modest activity with anti-PDL-1 and intense staining with CD73 in the tumor microenvironment. Methods: This phase 2 study (NCT04668300) is enrolling patients with age ≥18 years with recurrent/metastatic angiosarcoma (cohort 1) or dedifferentiated liposarcoma (DLPS) (cohort 2) and ≥12 years with recurrent/metastatic osteosarcoma (cohort 3), who have received at least one prior systemic therapy but are checkpoint inhibitor naïve and have measurable disease. Each treatment cycle is 28 days with oleclumab administered at 3000 mg i.v. every 2 weeks x 5 doses, and then every 4 weeks and durvalumab administered at 1500 mg i.v every 4 weeks. Tumor assessments are based on RECIST 1.1 and immune-related response criteria (irRC) and performed at baseline, and every 8 weeks after start of therapy, with an additional scan at 12 weeks for confirmation of response. Planned sample size is ≤ 25 pts in each arm. The primary efficacy endpoint for cohorts 1 and 2 is response rate (RR) at 4 months (per RECIST 1.1). The primary efficacy endpoint for cohort 3 is event free survival (EFS) rate at 4 months. If there is a high probability that the RR4 months is unlikely to be at least 20% for cohorts 1 and 2 or the EFS4 months is unlikely to be at least 40% for cohort 3 then the accrual of the corresponding cohort will be halted. The cohorts will be monitored separately for both futility and toxicity in groups of 5 after a minimum of 10 patients have been enrolled in each cohort. Secondary endpoints for the study include safety, best RR by RECIST and irRC, median PFS, and OS. Core needle biopsies and blood samples are collected at baseline and early on-treatment (week 6). Fresh flow cytometry is being performed to assess changes in T-cell activation, proliferation, and function and CD73 expression in the membrane and cytoplasm is being assessed by immunohistochemistry. Localization of tumor-infiltrating lymphocytes and engagement of the PD-1/PDL1 axis is being assessed using multiplex immunofluorescence staining. As of Jan 30th, 2022, twenty-two patients have initiated study treatment, 3 in cohort 1, 12 in cohort 2, and 7 in cohort 3. Clinical trial information: NCT04668300.

A novel humanized antibody targeting CD39 that enhances anti-tumor immunity and the efficacy of cancer immunotherapy.

e14508 Background: Immunotherapy using immune-checkpoint inhibitors (ICIs) has shown remarkable clinical efficacy in patients with cancer. However, it is crucial to utilize appropriate combination of immunotherapy for patient condition, as most patients do not benefit or sustain responses of immunotherapy. We focused on CD39 that is highly expressed in some tumors and tumor infiltrating immune cells (e.g. exhausted T cells and Tregs) as a target for cancer immunotherapy. CD39 is an ectoenzyme that converts extracellular ATP to immunosuppressive adenosine. Since the ATP-adenosine pathway is a key modulator of innate and adaptive immunity within the tumor microenvironment (TME), we presume that anti-CD39 antibodies suppress the production of adenosine and limit the immunosuppressive state in TMEs. Methods: Anti-human CD39 antibodies were screened by sorting B cells of mice immunized with human CD39 protein. The obtained clones that inhibit enzyme activity of CD39 were humanized on IgG1 and IgG4. PBMC were used in proliferation and functional assays. The anti-tumor effect was investigated with mouse xenograft model. Results: Analyzing gene expression profiles from TCGA data, high CD39 expression is associated with a poor prognosis in several cancers. There was a strong correlation between them in multiple myeloma treated with chemotherapies. The expression of CD39 was upregulated in some cancer cell lines treated with chemotherapeutic agents. A novel humanized anti-CD39 antibody, BP1202, which has a high affinity for the recombinant CD39 protein (KD: 481 pM) and inhibits the enzyme activity of CD39 expressed on a cancer cell line (IC50: 1 nM) and human Tregs. The antibody showed a high potency to enhance T cell activity including their proliferation, cytokine production, and cytotoxicity against a cancer cell line. We examined the anti-tumor effect of BP1202 in vivo using tumor-bearing mice and found that the combination treatment of BP1202 with chemotherapeutic agents significantly reduced tumor volume. Conclusions: We developed a new humanized antibody that enhances anti-tumor immunity by inhibiting CD39 expressed on exhausted T cells and intra-tumor T cells. These data suggest the scientific rationale for the clinical development of BP1202 and their use in combination with ICIs, with ATP-adenosine pathway inhibitors, and with chemotherapies. BP1202 is currently being developed as a new antibody agent for cancer immunotherapy.

Tetraspanin CD53 controls Tcell immunity through regulation of CD45RO stability, mobility, and function.

T cells depend on the phosphatase CD45 to initiate Tcell receptor signaling. Although the critical role of CD45 in Tcells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in Tcell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and Tcell activation. CD53-negative Tcells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing Tcells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for Tcell immunity.

The Multifaceted Actions of CD73 During Development and Suppressive Actions of Regulatory T Cells

Adenosine (Ado) has been shown to have immunosuppressive effects in a variety of diseases. It can either be released directly into the extracellular environment by cells, or it can be produced by degradation of ATP within the extracellular spaces. This extracellular pathway is facilitated by the concerted actions of the ectoenzymes CD39 and CD73. In a first step CD39 dephosphorylates ATP to ADP and AMP, respectively, and in a second step CD73 converts AMP to Ado. Thus, activity of CD73 on the cell surface of cells is the rate limiting step in the generation of extracellular Ado. Among T cells, CD73 is most abundantly expressed by regulatory T cells (Tregs) and is even upregulated after their activation. Functionally, the generation of Ado by CD73+ Tregs has been shown to play a role in immune suppression of dendritic cells, monocytes and T cells, and the defined expression of CD73 by Tregs in immunosuppressive environments, such as tumors, made CD73 a novel checkpoint inhibitor. Therefore, therapeutical intervention by anti-CD73 antibodies or by chemical inhibitors of the enzymatic function is currently under investigation in some preclinical animal models. In the following we summarize the expression pattern and the possible functions of CD73 in T cells and Tregs, and exemplify novel ways to manipulate CD73 functions in Tregs to stimulate anti-tumor immunity.

Compound 78c exerts a therapeutic effect on collagen-induced arthritis and rheumatoid arthritis.

Many studies have found that CD38 expression is increased in rheumatoid arthritis (RA), an autoimmune disease in which immune tolerance is dysregulated. Inhibition of CD38 expression or activity can significantly alleviate collagen-induced arthritis (CIA), a well-known animal model used for the study of RA. This study aimed to confirm the therapeutic effect of 78c, a specific inhibitor of CD38, and the role of CD38+ NK cells in immune imbalance in RA. CIA mice were injected with 78c to observe the therapeutic effect. CD38+ NK cells were extracted from human peripheral blood and treated with 78c. The pretreated NK cells were co-cultured with CD4+ T cells. We found that 78c significantly suppressed joint inflammation; reduced the levels of B cells, IL-6 and TNF-α; and increased the levels of IL-10, energy metabolism and spontaneous movement in CIA mice. 78c treatment also increased Treg cell numbers and decreased the Th1/Th2 ratio in the CIA model animals. Moreover, the proportion of CD38+ NK cells was increased in the CIA mice and significantly decreased following 78c treatment. Human CD4+ T cells that were co-cultured with 78c-pretreated CD38+ NK cells differentiated into more Treg cells and had lower Th17/Treg and Th1/Th2 ratios than CD4+ T cells co-cultured with CD38+ NK cells without the pretreatment. Transcriptomic analyses demonstrated that 78c changed expression pro les in CD38+ NK cells. These results suggested that 78c could be used for the treatment of RA and CIA as it alleviates the inhibitory effect of CD38+ NK cells on CD4+ T cell differentiation to Treg cells to restore immune balance.

Structure activity relationship of 3‐methylcytidine‐5’‐α,β‐methylenediphosphates as CD73 inhibitors

Adenosine in the tumor microenvironment acts on A2A and A2B adenosine receptors (ARs) on immune cells (T cells, dendritic cells, NK cells, macrophages and neutrophils) to prevent their activation. Therefore, a means of lowering extracellular adenosine would be beneficial in cancer immunotherapy either as a co‐therapy or monotherapy. Inhibitors of CD73 (ecto‐5’‐nucleotidase, the main extracellular adenosine‐forming enzyme) are already in clinical trials for cancer immunotherapy. Many purine (ADP) analogues have been shown to inhibit this enzyme, but we have explored pyrimidine (UDP/CDP) analogues. We recently reported N4‐substituted 3‐methylcytidine‐5’‐α,β‐methylenediphosphates as potent CD73 inhibitors. We now expand the structure activity relationship (SAR) of pyrimidine nucleotides as human CD73 inhibitors, focusing mainly on cytidine functionalization at the N4 position. 4‐Chloro (MRS4598 16, Ki 0.673 nM) and 4‐iodo (MRS4620 18, Ki 0.436 nM) substitution of the N4‐benzyloxy group increased inhibitory potency Ki by ~20‐fold. Primary alkylamine derivatives coupled through a p‐amido group and varying methylene chain length (24and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. hCD73 X‐ray structures with two of the newly synthesized inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between C‐terminal and N‐terminal domains) as N4‐benzyl groups in potent adenine‐derived inhibitors. Molecular dynamics simulation identified stabilizing interactions and predicted conformational diversity around the N4‐benzyloxy moiety. Thus, by structure‐guided substitution of the N4‐benzyloxy moiety, we have greatly enhanced their inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in human head and neck squamous cell carcinoma (HNSCC) and palatine tonsils in situ.

Lentiviral Mediated ADA2 Gene Transfer Corrects the Defects Associated With Deficiency of Adenosine Deaminase Type 2.

Deficiency of adenosine deaminase type 2 (DADA2) is an autosomal recessive disease caused by bi-allelic loss-of-function mutations in ADA2. Treatment with anti-TNF is effective for the autoinflammatory and vasculitic components of the disease but does not correct marrow failure or immunodeficiency; and anti-drug antibodies cause loss of efficacy over time. Allogeneic haematopoietic stem cell transplantation may be curative, but graft versus host disease remains a significant concern. Autologous gene therapy would therefore be an attractive longer-term therapeutic option. We investigated whether lentiviral vector (LV)–mediated ADA2 gene correction could rescue the immunophenotype of DADA2 in primary immune cells derived from patients and in cell line models. Lentiviral transduction led to: i) restoration of ADA2 protein expression and enzymatic activity; (ii) amelioration of M1 macrophage cytokine production, IFN-γ and phosphorylated STAT1 expression in patient-derived macrophages; and (iii) amelioration of macrophage-mediated endothelial activation that drives the vasculitis of DADA2. We also successfully transduced human CD34+ haematopoietic stem progenitor cells (HSPC) derived from a DADA2 patient with pure red cell aplasia and observed restoration of ADA2 expression and enzymatic activity in CD34+HSPC, alongside recovery of stem-cell proliferative and colony forming unit capacity. These preclinical data now expand the evidence for the efficacy of gene transfer strategies in DADA2, and strongly support clinical translation of a lentivirus-mediated gene therapy approach to treat DADA2.

A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape.

CD73 catalyzes the conversion of ATP to adenosine, which is involved in various physiological and pathological processes, including tumor immune escape. Because CD73 expression and activity are particularly high on cancer cells and contribute to the immunosuppressive properties of the tumor environment, it is considered an attractive target molecule for specific cancer therapies. In line, several studies demonstrated that CD73 inhibition has a significant antitumor effect. However, complete blocking of CD73 activity can evoke autoimmune phenomena and adverse side effects. We developed a CD73-specific antibody, 22E6, that specifically inhibits the enzymatic activity of membrane-tethered CD73 present in high concentrations on cancer cells and cancer cell-derived extracellular vesicles but has no inhibitory effect on soluble CD73. Inhibition of CD73 on tumor cells with 22E6 resulted in multiple effects on tumor cells in vitro, including increased apoptosis and interference with chemoresistance. Intriguingly, in a xenograft mouse model of acute lymphocytic leukemia (ALL), 22E6 treatment resulted in an initial tumor growth delay in some animals, followed by a complete loss of CD73 expression on ALL cells in all 22E6 treated animals, indicating tumor immune escape. Taken together, 22E6 shows great potential for cancer therapy, favorably in combination with other drugs.

CD38 DEFICIENCY PROTECTS MICE FROM AGE-RELATED SPONTANEOUS OA DEVELOPMENT

Purpose: CD38 is recently shown to be the main NADase in mammalian tissue, which contributes substantially to degradation of cellular nicotinamide adenine dinucleotide (NAD+), a key metabolite involved in cellular energy metabolism and adaptive responses of cells to bioenergetics and oxidative stress. CD38 expression and activity are increased during aging, which contributes to age-related decline of NAD+. In our previous studies, we observed an age-related increase in expression of CD38 in human knee cartilage, and that increased CD38 expression in chondrocytes is associated with reduced NAD/NADH levels and increased catabolic responses to pro-inflammatory cytokine IL-1β.

The Ecto-5'nucleotidase/CD73 Mediates Leishmania amazonensis Survival in Macrophages.

Endogenous nucleotides produced by various group of cells under inflammatory conditions act as potential danger signals in vivo. Extracellularly released nucleotides such as ATP are rapidly hydrolyzed to adenosine by the coordinated ectonucleotidase activities of CD39 and CD73. Leishmania is an obligate intracellular parasite of macrophages and capable of modulating host immune response in order to survive and multiply within host cells. In this study, the activity of CD73 induced by Leishmania amazonensis in infected macrophages has been investigated and correlated with parasite survival and infection in vitro. For this, the expression of CD39 and CD73, by flow cytometry, in murine peritoneal macrophages infected with metacyclic promastigotes of L. amazonensis has been analyzed. Our results showed that L. amazonensis-infected macrophages, unlike LPS-treated macrophages, increased CD73 expression. It was also noted that when CD73 enzymatic activity was blocked by α, β-methyleneadenosine 5′-diphosphate sodium salt (APCP), macrophage parasitism was significantly decreased. Interestingly, these effects were not associated with the production of TNF-α, IL-10, or nitric oxide (NO). Together, these data demonstrate that L. amazonensis induces a regulatory phenotype in macrophages, which by activating the CD39/CD73 pathway allows parasite survival through the action of immunomodulatory adenosine receptors.

Abstract WP16: Characterizing Cd38 Expression And Activity In The Brain Of Spontaneously Hypertensive Stroke Prone Rats

Introduction: CD38 is an ectoenzyme that is the main source of age dependent decrease in NAD + levels. Its role in cerebral small vessel disease (cSVD) is unknown. We hypothesize that CD38 is over-expressed and activated in spontaneously hypertensive stroke-prone rat (SHRSP), a model of human cSVD. Methods: 20 SHRSP and 20 age matched male Wistar Kyoto control rats (WKY) were studied. Systolic blood pressure (SBP) was measured weekly using tail-cuff plethysmography. Half of the groups were euthanized at 7 weeks of age (Wks) and the remaining rats were followed until 24 Wks. Routine histology and immunohistochemistry (IHC) were performed for nitric oxide synthase (NOS) subtypes, superoxides (DHE), NO (DAF), and NADPH. IHC was used to detect CD38 expression and co-staining determined expression on endothelial cells (eNOS), astrocytes (GFAP), microglia (Iba1), T cells (CD3), macrophages (CD68), neutrophils (MPO) and neurons (Neuon). CD38 activity was detected in the brain homogenate by using a substrate analog of NAD + with measurement of its conversion to the strongly fluorescent product (ε-ADPR). Results: Compared to WKY, SHRSP developed hypertension between (9 -12) Wks and sustained it afterwards (SBP: 19 Wks: WKY 142.5±11.8 mmHg vs SHRSP 180.3±12.8 mmHg, P<0.0001). At 24 Wks, SHRSP showed histological evidence of cSVD including microbleed, enlarged perivascular spaces, and lipohyalinosis. Compared to WKY, CD38 expression was increased in SHRSP brain (7 Wks: 102.6%, P=0.011, 24 Wks: 83.2%, p=0.0077). CD38 activity was also significantly higher in SHRSP (7 Wks: 11.8%, p=0.012, 24 Wks: 20.2%, p=0.0065). We identified CD38 expression on endothelial cells, astrocytes and microglia. In comparison to WKY, SHRSP showed lower levels of NADPH (7 Wks: 22.3%, p=0.029, 24 Wks: 65.5%, p=0.0065), NO (7 Wks: 39.3% p=0.0008, 24 Wks: 36%, p=0.04), endothelial NOS (7 Wks: 57.5%, p=0.02, 24 Wks: 50.6%, p=0.01) and higher superoxide (7 Wks: 131%, p<0.0001, 24 Wks: 163%, p<0.0001). Conclusions: The brain of SHRSP shows evidence for elevated CD38 activity and expression with associated oxidative stress, depletion of NADPH and eNOS dysfunction compared to normotensive WKY. These results suggest the potential role of CD38 based therapeutics in future management of human cSVD.

Abstract TMP115: Increased Expression Of Cd38 In Activated Astrocytes Contributes To Impaired Synaptic Plasticity Following Middle Cerebral Artery Occlusion

Introduction: Emerging evidence has implicated post-stroke cognitive impairment (PSCI) as a major contributor to long-term disability following acute ischemic stroke. While the hippocampus is shown to contribute to PSCI, the exact mechanisms underlying PSCI have yet to be elucidated. Interestingly, a preclinical model of large artery stroke (middle cerebral artery occlusion; MCAO) causes hippocampal dysfunction, despite direct ischemic insult to brain regions distant from the hippocampus, suggesting the injury causes perturbations in neural circuits. Thus, we utilize electrophysiological recordings of hippocampal LTP as an indicator of network health following ischemia. We hypothesize activated astrocytes in the hippocampus following MCAO increase expression of the ectoenzyme, CD38, which signals to neurons to impair plasticity. Methods: Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared 30 days after recovery from MCAO in adult (8-12 week) mice. Immunohistochemistry was performed to assess hippocampal CD38 expression and astrogliosis. Acute slices were treated with CD38 inhibitors (78c & apigenin) to assess plasticity. Results: Recordings obtained in brain slices 30 days after MCAO exhibited a significant reduction in LTP; 161±9%, n=6 in sham compared to 115±4%, n=7 30 days after MCAO, in both ipsilateral and contralateral hippocampi. Immunohistochemical (IHC) staining indicates CD38 levels are increased and colocalize with activated astrocyte marker, GFAP, 30 days following MCAO. Bath application of CD38 inhibitors, 78c (vehicle n=6, 4.0±8.5% vs. 100nM 78c n=6, 78.3±15.8%; p<0.05) and apigenin (vehicle n=7, 23.9±7% vs. 10μM apigenin n=7, 65.5±2.5%; p<0.05), restored LTP in MCAO brain slices 30 days following injury. Conclusion: These data indicate that MCAO provides a reproducible model of post-stroke memory dysfunction (PSCI) and that remote astrogliosis in the uninjured hippocampus may contribute to altered neuronal function (plasticity). Our data implicates increased levels of CD38 impair plasticity following stroke. Therefore, reducing CD38 activity at chronic timepoints following ischemia may represent a novel strategy to treat the symptoms of PSCI.

Benefits in cardiac function by CD38 suppression: Improvement in NAD+ levels, exercise capacity, heart rate variability and protection against catecholamine-induced ventricular arrhythmias

CD38 enzymatic activity regulates NAD+ and cADPR levels in mammalian tissues, and therefore has a prominent role in cellular metabolism and calcium homeostasis. Consequently, it is reasonable to hypothesize about its involvement in cardiovascular physiology as well as in heart related pathological conditions. AimTo investigate the role of CD38 in cardiovascular performance, and its involvement in cardiac electrophysiology and calcium-handling. Methods and resultsWhen submitted to a treadmill exhaustion test, a way of evaluating cardiovascular performance, adult male CD38KO mice showed better exercise capacity. This benefit was also obtained in genetically modified mice with catalytically inactive (CI) CD38 and in WT mice treated with antibody 68 (Ab68) which blocks CD38 activity. Hearts from these 3 groups (CD38KO, CD38CI and Ab68) showed increased NAD+ levels. When CD38KO mice were treated with FK866 which inhibits NAD+ biosynthesis, exercise capacity as well as NAD+ in heart tissue decreased to WT levels.Electrocardiograms of conscious unrestrained CD38KO and CD38CI mice showed lower basal heart rates and higher heart rate variability than WT mice. Although inactivation of CD38 in mice resulted in increased SERCA2a expression in the heart, the frequency of spontaneous calcium release from the sarcoplasmic reticulum under stressful conditions (high extracellular calcium concentration) was lower in CD38KO ventricular myocytes. When mice were challenged with caffeine-epinephrine, CD38KO mice had a lower incidence of bidirectional ventricular tachycardia when compared to WT ones. ConclusionCD38 inhibition improves exercise performance by regulating NAD+ homeostasis. CD38 is involved in cardiovascular function since its genetic ablation decreases basal heart rate, increases heart rate variability and alters calcium handling in a way that protects mice from developing catecholamine induced ventricular arrhythmias.

Structure-Activity Relationship of 3-Methylcytidine-5'-α,β-methylenediphosphates as CD73 Inhibitors.

We recently reported N4-substituted 3-methylcytidine-5'-α,β-methylenediphosphates as CD73 inhibitors, potentially useful in cancer immunotherapy. We now expand the structure-activity relationship of pyrimidine nucleotides as human CD73 inhibitors. 4-Chloro (MRS4598 16; Ki = 0.673 nM) and 4-iodo (MRS4620 18; Ki = 0.436 nM) substitution of the N4-benzyloxy group decreased Ki by ∼20-fold. Primary alkylamine derivatives coupled through a p-amido group with a varying methylene chain length (24 and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. X-ray structures of hCD73 with two inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between the C-terminal and N-terminal domains) as N4-benzyl groups in adenine inhibitors. Molecular dynamics identified stabilizing interactions and predicted conformational diversity. Thus, by N4-benzyloxy substitution, we have greatly enhanced the inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in tumor tissues in situ.

Adenosinergic Signaling as a Key Modulator of the Glioma Microenvironment and Reactive Astrocytes.

Astrocytes are numerous glial cells of the central nervous system (CNS) and play important roles in brain homeostasis. These cells can directly communicate with neurons by releasing gliotransmitters, such as adenosine triphosphate (ATP) and glutamate, into the multipartite synapse. Moreover, astrocytes respond to tissue injury in the CNS environment. Recently, astrocytic heterogeneity and plasticity have been discussed by several authors, with studies proposing a spectrum of astrocytic activation characterized by A1/neurotoxic and A2/neuroprotective polarization extremes. The fundamental roles of astrocytes in communicating with other cells and sustaining homeostasis are regulated by purinergic signaling. In the CNS environment, the gliotransmitter ATP acts cooperatively with other glial signaling molecules, such as cytokines, which may impact CNS functions by facilitating/inhibiting neurotransmitter release. Adenosine (ADO), the main product of extracellular ATP metabolism, is an important homeostatic modulator and acts as a neuromodulator in synaptic transmission via P1 receptor sensitization. Furthermore, purinergic signaling is a key factor in the tumor microenvironment (TME), as damaged cells release ATP, leading to ADO accumulation in the TME through the ectonucleotidase cascade. Indeed, the enzyme CD73, which converts AMP to ADO, is overexpressed in glioblastoma cells; this upregulation is associated with tumor aggressiveness. Because of the crucial activity of CD73 in these cells, extracellular ADO accumulation in the TME contributes to sustaining glioblastoma immune escape while promoting A2-like activation. The present review describes the importance of ADO in modulating astrocyte polarization and simultaneously promoting tumor growth. We also discuss whether targeting of CD73 to block ADO production can be used as an alternative cancer therapy.

Crowding within synaptic junctions influences the degradation of nucleotides by CD39 and CD73 ectonucleotidases

Synapsed cells can communicate using exocytosed nucleotides like adenosine triphosphate (ATP). Ectonucleotidases localized to synaptic junctions degrade nucleotides into metabolites like adenosine monophosphate (AMP) or adenosine. Oftentimes nucleotide degradation occurs in a sequential manner, of which ATP degradation by CD39 and CD73 is a representative example. Here, CD39 first converts ATP and adenosine diphosphate (ADP) into AMP, after which AMP is dephosphorylated into adenosine by CD73. Hence, the concerted activity of CD39 and CD73 can help shape cellular responses to extracellular ATP. In a previous study, we demonstrated that coupled CD39 and CD73 activity within synapse-like junctions is strongly controlled by the enzymes' co-localization, their surface charge densities, and the electrostatic potential of the surrounding cell membranes. In this study, we demonstrate that crowders within synaptic junctions, which can include globular proteins like cytokines and membrane-bound proteins, impact coupled CD39 and CD73 ectonucleotidase activity and, in turn, the availability of intrasynapse ATP. Specifically, we developed a spatially explicit, reaction-diffusion model for the coupled conversion of ATP → AMP and AMP → adenosine in a model synaptic junction with crowders that is solved via the finite element method. Our modeling results suggest that the association rate for ATP to CD39 is strongly influenced by the density of intrasynaptic protein crowders, as increasing crowder density generally suppressed ATP association kinetics. Much of this suppression can be rationalized based on a loss of configurational entropy. The surface charges of crowders can further influence the association rate, with the surprising result that favorable crowder-nucleotide electrostatic interactions can yield CD39 association rates that are faster than crowder-free configurations. However, attractive crowder-nucleotide interactions decrease the rate and efficiency of adenosine production, which in turn increases the availability of ATP and AMP within the synapse relative to crowder-free configurations. These findings highlight how CD39 and CD73 ectonucleotidase activity, electrostatics, and crowding within synapses influence the availability of nucleotides for intercellular communication.

135P First-in-human phase I trial of SRF617, a potent inhibitor of CD39 activity, as monotherapy or in combination, in patients (pts) with advanced solid tumors

135P First-in-human phase I trial of SRF617, a potent inhibitor of CD39 activity, as monotherapy or in combination, in patients (pts) with advanced solid tumors

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.

Multispecific targeting of glioblastoma with tumor microenvironment-responsive multifunctional engineered NK cells

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking, have rendered glioblastoma (GBM) highly resistant to therapy. To address these obstacles, here we describe a unique, sophisticated combinatorial platform for GBM: a cooperative multifunctional immunotherapy based on genetically engineered human natural killer (NK) cells bearing multiple antitumor functions including local tumor responsiveness that addresses key drivers of GBM resistance to therapy: antigen escape, immunometabolic reprogramming of immune responses, and poor immune cell homing. We engineered dual-specific chimeric antigen receptor (CAR) NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site-specific activity in the tissue, and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising NK cell-based combinatorial strategy that can target multiple clinically recognized mechanisms of GBM progression simultaneously.