Ectoenzyme CD38 Research Articles

Development of endothelial-targeted CD39 as a therapy for ischemic stroke

BackgroundIschemic stroke is characterized by a necrotic lesion in the brain surrounded by an area of dying cells termed the penumbra. Salvaging the penumbra either with thrombolysis or mechanical retrieval is the cornerstone of stroke management. At-risk neuronal cells release extracellular adenosine triphosphate, triggering microglial activation and causing a thromboinflammatory response, culminating in endothelial activation and vascular disruption. This is further aggravated by ischemia-reperfusion injury that follows all reperfusion therapies. The ecto-enzyme CD39 regulates extracellular adenosine triphosphate by hydrolyzing it to adenosine, which has antithrombotic and anti-inflammatory properties and reverses ischemia-reperfusion injury. ObjectivesThe objective off the study was to determine the efficacy of our therapeutic, anti-VCAM-CD39 in ischaemic stroke. MethodsWe developed anti-VCAM-CD39 that targets the antithrombotic and anti-inflammatory properties of recombinant CD39 to the activated endothelium of the penumbra by binding to vascular cell adhesion molecule (VCAM)-1. Mice were subjected to 30 minutes of middle cerebral artery occlusion and analyzed at 24 hours. Anti-VCAM-CD39 or control agents (saline, nontargeted CD39, or anti-VCAM-inactive CD39) were given at 3 hours after middle cerebral artery occlusion. ResultsAnti-VCAM-CD39 treatment reduced neurologic deficit; magnetic resonance imaging confirmed significantly smaller infarcts together with an increase in cerebrovascular perfusion. Anti-VCAM-CD39 also restored blood-brain barrier integrity and reduced microglial activation. Coadministration of anti-VCAM-CD39 with thrombolytics (tissue plasminogen activator [tPA]) further reduced infarct volumes and attenuated blood-brain barrier permeability with no associated increase in intracranial hemorrhage. ConclusionAnti-VCAM-CD39, uniquely targeted to endothelial cells, could be a new stroke therapy even when administered 3 hours postischemia and may further synergize with thrombolytic therapy to improve stroke outcomes.

The ecto-enzyme CD38 modulates CD4T cell immunometabolic responses and participates in HIV pathogenesis.

Despite abundant evidence correlating T cell CD38 expression and HIV infection pathogenesis, its role as a CD4T cell immunometabolic regulator remains unclear. We find that CD38's extracellular glycohydrolase activity restricts metabolic reprogramming after T cell receptor (TCR)-engaging stimulation in Jurkat T CD4 cells, together with functional responses, while reducing intracellular nicotinamide adenine dinucleotide and nicotinamide mononucleotide concentrations. Selective elimination of CD38's ectoenzyme function licenses them to decrease the oxygen consumption rate/extracellular acidification rate ratio upon TCR signaling and to increase cycling, proliferation, survival, and CD40L induction. Pharmacological inhibition of ecto-CD38 catalytic activity in TM cells from chronic HIV-infected patients rescued TCR-triggered responses, including differentiation and effector functions, while reverting abnormally increased basal glycolysis, cycling, and spontaneous proinflammatory cytokine production. Additionally, ecto-CD38 blockage normalized basal and TCR-induced mitochondrial morphofunctionality, while increasing respiratory capacity in cells from HIV+ patients and healthy individuals. Ectoenzyme CD38's immunometabolic restriction of TCR-involving stimulation is relevant to CD4T cell biology and to the deleterious effects of CD38 overexpression in HIV disease.

Dysregulation in CD39/CD73 Axis May Trigger the Upsurge of the Immune Suppressive Agent Adenosine in OSA Patients

IntroductionAlthough higher incidence of cancer represents a major burden for obstructive sleep apnea (OSA) patients, the molecular pathways driving this association are not completely understood. Interestingly, adenosinergic signaling has emerged as a powerful immune checkpoint driving tumor development and progression. MethodsHere, we explored the expression of the adenosinergic ecto-enzymes CD39 and CD73 in T-lymphocytes of OSA patients without any evidence of cancer, as well as their soluble forms in plasma (sCD39 and sCD73), along with adenosine. In addition, we explored the role of intermittent hypoxia (IH) in this context by in vitro models. ResultsOur results showed that CD39 is upregulated while CD73 is downregulated in OSA T-cells’ membrane. Moreover, our findings suggest that IH, through HIF-1, mediates the upregulation of both CD39 and CD73; and that CD73 downregulation could be mediated by a higher release of sCD73 by OSA T-lymphocytes. Importantly, we found that both sCD39 and sCD73 are upregulated in OSA plasma, suggesting T-lymphocytes as a potential source for plasmatic sCD73. Finally, our data propose the alterations in CD39/CD73 axis could underlie the upsurge of adenosine levels in the plasma of OSA patients. ConclusionOur study reveals a hypoxia-mediated alteration of the CD39/CD73 axis in OSA patients, which could trigger ADO upregulation, thus potentially contributing to the immune suppressive environment and ultimately facilitating tumor development and progression. Therefore, our data highlights the need for new longitudinal studies evaluating CD39 and/or CD73 as potential cancer-risk prognostic biomarkers in OSA patients.

CD73-dependent generation of extracellular adenosine by vascular endothelial cells modulates de novo lipogenesis in adipose tissue.

Next to white and brown adipocytes present in white and brown adipose tissue (WAT, BAT), vascular endothelial cells, tissue-resident macrophages and other immune cells have important roles in maintaining adipose tissue homeostasis but also contribute to the etiology of obesity-associated chronic inflammatory metabolic diseases. In addition to hormonal signals such as insulin and norepinephrine, extracellular adenine nucleotides modulate lipid storage, fatty acid release and thermogenic responses in adipose tissues. The complex regulation of extracellular adenine nucleotides involves a network of ectoenzymes that convert ATP via ADP and AMP to adenosine. However, in WAT and BAT the processing of extracellular adenine nucleotides and its relevance for intercellular communications are still largely unknown. Based on our observations that in adipose tissues the adenosine-generating enzyme CD73 is mainly expressed by vascular endothelial cells, we studied glucose and lipid handling, energy expenditure and adaptive thermogenesis in mice lacking endothelial CD73 housed at different ambient temperatures. Under conditions of thermogenic activation, CD73 expressed by endothelial cells is dispensable for the expression of thermogenic genes as well as energy expenditure. Notably, thermoneutral housing leading to a state of low energy expenditure and lipid accumulation in adipose tissues resulted in enhanced glucose uptake into WAT of endothelial CD73-deficient mice. This effect was associated with elevated expression levels of de novo lipogenesis genes. Mechanistic studies provide evidence that extracellular adenosine is imported into adipocytes and converted to AMP by adenosine kinase. Subsequently, activation of the AMP kinase lowers the expression of de novo lipogenesis genes, most likely via inactivation of the transcription factor carbohydrate response element binding protein (ChREBP). In conclusion, this study demonstrates that endothelial-derived extracellular adenosine generated via the ectoenzyme CD73 is a paracrine factor shaping lipid metabolism in WAT.

Altered CD39 and CD73 Expression in Rheumatoid Arthritis: Implications for Disease Activity and Treatment Response.

In rheumatoid arthritis (RA) synovium, ATP, and ADP are released, sparking inflammation. Ectoenzymes CD39 and CD73 metabolize these purine nucleotides, generating anti-inflammatory adenosine. Therefore, dysregulated CD39 and CD73 expression may impact RA development. We assessed CD39 and CD73 expression in peripheral blood from 15 healthy controls (Cs) and 35 RA patients at baseline and after 3 and 6 months of tocilizumab treatment using flow cytometry. Additionally, ectoenzyme expression was examined on cultured T cells to understand activation and IL-6 effects. At baseline, RA patients exhibited a lower CD8+CD39-CD73+ cell percentage, which inversely correlated with DAS28. Additionally, they had lower percentages of Treg CD39+CD73+ and CD39-CD73- cells. Good responders tended to have lower B CD39+CD73+ cell percentages at baseline and 3 months. Additionally, Treg, CD8+ T and B cells inversely correlated with DAS28. T-cell activation increased CD39 and decreased CD73 expression, regardless of IL-6. IL-6 reduced IFNγ-secreting CD4+ T-cell percentage in Cs, but increased the percentage of IFNγ-secreting CD4+ and CD8+ T cells in RA patients. These findings indicate differing CD39 and CD73 expression in RA and Cs, influenced by T-cell activation and IL-6. Correlations between these molecules and RA activity suggest their role in dysregulated inflammation in RA.

Heavy-chain antibody targeting of CD38 NAD+ hydrolase ectoenzyme to prevent fibrosis in multiple organs

The functionally pleiotropic ectoenzyme CD38 is a glycohydrolase widely expressed on immune and non-hematopoietic cells. By converting NAD+ to ADP-ribose and nicotinamide, CD38 governs organismal NAD+ homeostasis and the activity of NAD+-dependent cellular enzymes. CD38 has emerged as a major driver of age-related NAD+ decline underlying adverse metabolic states, frailty and reduced health span. CD38 is upregulated in systemic sclerosis (SSc), a chronic disease characterized by fibrosis in multiple organs. We sought to test the hypothesis that inhibition of the CD38 ecto-enzymatic activity using a heavy-chain monoclonal antibody Ab68 will, via augmenting organismal NAD+, prevent fibrosis in a mouse model of SSc characterized by NAD+ depletion. Here we show that treatment of mice with a non-cytotoxic heavy-chain antibody that selectively inhibits CD38 ectoenzyme resulted in NAD+ boosting that was associated with significant protection from fibrosis in multiple organs. These findings suggest that targeted inhibition of CD38 ecto-enzymatic activity could be a potential pharmacological approach for SSc fibrosis treatment.

Regadenoson for the treatment of COVID-19: A five case clinical series and mouse studies.

Adenosine inhibits the activation of most immune cells and platelets. Selective adenosine A2A receptor (A2AR) agonists such as regadenoson (RA) reduce inflammation in most tissues, including lungs injured by hypoxia, ischemia, transplantation, or sickle cell anemia, principally by suppressing the activation of invariant natural killer T (iNKT) cells. The anti-inflammatory effects of RA are magnified in injured tissues due to induction in immune cells of A2ARs and ecto-enzymes CD39 and CD73 that convert ATP to adenosine in the extracellular space. Here we describe the results of a five patient study designed to evaluate RA safety and to seek evidence of reduced cytokine storm in hospitalized COVID-19 patients. Five COVID-19 patients requiring supplemental oxygen but not intubation (WHO stages 4-5) were infused IV with a loading RA dose of 5 μg/kg/h for 0.5 h followed by a maintenance dose of 1.44 μg/kg/h for 6 hours, Vital signs and arterial oxygen saturation were recorded, and blood samples were collected before, during and after RA infusion for analysis of CRP, D-dimer, circulating iNKT cell activation state and plasma levels of 13 proinflammatory cytokines. RA was devoid of serious side effects, and within 24 hours from the start of infusion was associated with increased oxygen saturation (93.8 ± 0.58 vs 96.6 ± 1.08%, P<0.05), decreased D-dimer (754 ± 17 vs 518 ± 98 ng/ml, P<0.05), and a trend toward decreased CRP (3.80 ± 1.40 vs 1.98 ± 0.74 mg/dL, P = 0.075). Circulating iNKT cells, but not conventional T cells, were highly activated in COVID-19 patients (65% vs 5% CD69+). RA infusion for 30 minutes reduced iNKT cell activation by 50% (P<0.01). RA infusion for 30 minutes did not influence plasma cytokines, but infusion for 4.5 or 24 hours reduced levels of 11 of 13 proinflammatory cytokines. In separate mouse studies, subcutaneous RA infusion from Alzet minipumps at 1.44 μg/kg/h increased 10-day survival of SARS-CoV-2-infected K18-hACE2 mice from 10 to 40% (P<0.001). Infused RA is safe and produces rapid anti-inflammatory effects mediated by A2A adenosine receptors on iNKT cells and possibly in part by A2ARs on other immune cells and platelets. We speculate that iNKT cells are activated by release of injury-induced glycolipid antigens and/or alarmins such as IL-33 derived from virally infected type II epithelial cells which in turn activate iNKT cells and secondarily other immune cells. Adenosine released from hypoxic tissues, or RA infused as an anti-inflammatory agent decrease proinflammatory cytokines and may be useful for treating cytokine storm in patients with Covid-19 or other inflammatory lung diseases or trauma.

Microglia sense and suppress epileptic neuronal hyperexcitability

Microglia are the resident immune cells of the central nervous system, undertaking surveillance role and reacting to brain homeostasis and neurological diseases. Recent studies indicate that microglia modulate epilepsy-induced neuronal activities, however, the mechanisms underlying microglia-neuron communication in epilepsy are still unclear. Here we report that epileptic neuronal hyperexcitability activates microglia and drives microglial ATP/ADP hydrolyzing ectoenzyme CD39 (encoded by Entpd1) expression via recruiting the cAMP responsive element binding protein (CREB)-regulated transcription coactivator-1 (CRTC1) from cytoplasm to the nucleus and binding to CREB. Activated microglia in turn suppress epileptic neuronal hyperexcitability in a CD39 dependent manner. Disrupting microglial CREB/CRTC1 signaling, however, decreases CD39 expression and diminishes the inhibitory effect of microglia on epileptic neuronal hyperexcitability. Overall, our findings reveal CD39-dependent control of epileptic neuronal hyperexcitability by microglia is through an excitation-transcription coupling mechanism.

Abstract B032: Combined A2AR and PARP inhibition in homologous recombination deficient (HRD) castrate-resistant prostate cancer (CRPC)

Abstract Background: In response to cellular death and hypoxia, ATP is passively released into the extracellular space. ATP undergoes rapid stepwise conversion to the potent, immune-suppressive adenosine by ectoenzymes CD39 and CD73. Engagement of adenosine with the A2A and A2B receptors (A2AR, A2BR) promote the suppressive effects of adenosine. Early-phase clinical trials in men with metastatic castration-resistant prostate cancer (CRPC) suggest that A2AR antagonism may mitigate adenosine-driven immunosuppression in the tumor microenvironment (TME). The effect of A2AR antagonism on tumor cells themselves is unknown. Investigating combination treatments to promote both immune and tumor-intrinsic anti-tumor activity, we hypothesize that inhibition of adenosine signaling through the A2AR combined with PARP inhibition is an effective combination therapy for men with homologous repair deficient (HRD) CRPC. Methods: Here, we interrogate publicly available prostate cancer genomic datasets in the cBioPortal to evaluate the incidence of mutational and copy number alterations in the ENTPD1 (CD39), NT5E (CD73), and ADORA2A (A2AR) genes. Using an A2AR antagonist, we examine the effects of blocking A2AR signaling on tumor cell viability and expression of CD39, CD73, A2AR, and PARP expression in two androgen-insensitive human prostate cancer cell lines, DU145 and PC3. In DU145 and PC3 xenograft models, we tested A2AR antagonist, PARP inhibitor, or a combination of A2AR antagonist and PARP inhibitor, compared to vehicle alone on tumor growth. Results: Assessment of mutational and copy number alterations of ENTPD1 (CD39), NT5E (CD73), and ADORA2A (A2AR) in localized and metastatic prostate cancer reveals that the ADORA2A gene is amplified 10 times more frequently (2.4% vs. 0.2%, p&amp;lt;0.01) in the metastatic setting. Stratifying the metastatic cohort by anti-androgen treatments, we found that in patients with prior abiraterone and/or enzalutamide exposure, ADORA2A was more commonly amplified (4.4% vs. 0.7%, p&amp;lt;0.005), whereas ENTPD1 or NT5E were not. In both DU145 and PC3 cell lines, A2AR antagonism drives a dose dependent increase in cell death and expressions of A2AR, CD39, and CD73, especially in the DU145 cell line characterized by multiple missense mutations in key genes in the homologous recombination pathway. In both the DU145 and PC3 xenograft models, treatment with an A2AR antagonist or PARP inhibitor monotherapy did not significantly influence tumor volumes. However, when combined, A2AR antagonism plus PARP inhibition resulted in a 59% reduction in tumor volume (196.3 mm3 vs. 479.6 mm3, p&amp;lt;0.001) in the DU145 model, compared to vehicle-treated control. In the PC3 model, with fewer alterations to the HR pathway, monotherapy also had no influence on tumor volume. When combined, A2AR antagonism plus PARP inhibition caused a 43% reduction in volume (304.1 mm3 vs. 538.2.0 mm3, p&amp;lt;0.05). Conclusion: These data suggest that combined A2AR and PARP inhibition may be an effective therapeutic combination for HRD metastatic castration-resistant prostate cancer. Citation Format: Patrick J. McCann, Wendy Mao, Matthew Chaimowitz, Erika Houthys, Reece Marillier, Shruti Bansal, Julia An, Jason A. Piersaint, Namita Sen, Charles G. Drake, Catherine S. Spina. Combined A2AR and PARP inhibition in homologous recombination deficient (HRD) castrate-resistant prostate cancer (CRPC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B032.

NT5E DNA methylation in papillary thyroid cancer: Novel opportunities for precision oncology

The ectoenzyme CD73, encoded by the NT5E gene, has emerged as a potential prognostic and therapeutic marker for papillary thyroid carcinoma (PTC), which has increased in incidence in recent decades. Here, from The Cancer Genome Atlas Thyroid Cancer (TCGA-THCA) database, we extracted and combined clinical features, levels of NT5E mRNA, and DNA methylation of PTC samples and performed multivariate and random forest analyses to evaluate the prognostic relevance and the potential of discriminating between adjacent non-malignant and thyroid tumor samples. As a result, we revealed that lower levels of methylation at the cg23172664 site were independently associated with BRAF-like phenotype (p = 0.002), age over 55 years (p = 0.012), presence of capsule invasion (p = 0.007) and presence of positive lymph node metastasis (LNM) (p = 0.04). The methylation levels of cg27297263 and cg23172664 sites showed significant and inversely correlations with levels of NT5E mRNA expression (r = −0.528 and r = −0.660, respectively), and their combination was able to discriminate between adjacent non-malignant and tumor samples with a precision of 96%–97% and 84%–85%, respectively. These data suggest that combining cg23172664 and cg27297263 sites may bring new insights to reveal new subsets of patients with papillary thyroid carcinoma.

Abstract 1158: Inosine endows CAR T cells with features of increased stemness and anti-tumor potency

Abstract Chimeric antigen receptors (CARs) are synthetic receptors that link an extracellular tumor specific domain to intracellular signaling domains. Despite of remarkable results against refractory B cell malignancies, intense effort is underway to augment the potency of CAR T cells in order to overcome the suppressive tumor microenvironment, which is associated with T cell exhaustion. Adenosine is a major mediator of immune suppression. Ectoenzyme CD39 plays a central role in the generation of adenosine by catalyzing the metabolism of ATP into ADP/AMP. Then CD73 subsequently metabolizes ADP/AMP into adenosine which mediates immune suppression through adenosine associated receptor signaling. CD39 is also expressed by exhausted CD8+ and tumor reactive T cells within the tumor microenvironment, where it is associated with tumor progression, but it remains unclear whether exhausted and/or tumor reactive CD39+CD8+ T cells mediate immune suppression via the purigenic pathway. We developed a high affinity version of the disialoganglioside (GD2)-targeting chimeric antigen receptor (HA-GD2) that spontaneously clusters on the surface of human T cells in the absence of antigen and mimics chronic antigen exposure leading to T cell exhaustion. Using this model, we demonstrate that exhausted CD39+CD8+ CAR T cells actively produce adenosine and mediate immune suppression. In an attempt to generate adenosine resistance and enhance the function of exhausted CAR T cells, we knocked out CD39, CD73, or A2aR (adenosine A2a receptor) but observed only modest changes in phenotype and transcriptomics. In contrast, overexpression of transmembrane-bound adenosine deaminase, which metabolizes adenosine to inosine, induced higher frequency of stem- and central- like memory T cells, and a simultaneous decrease of exhausted T cell subpopulations. Direct exposure of HA-GD2 CAR T cells to high inosine concentration during cell manufacturing process, lead to a higher frequency of central-like memory cells and significant fitness enhancement associated with broad changes at the metabolic level. RNAseq and cyTOF analysis indicated decreased glycolytic flux, increased mitochondrial activity driven by glutamine and polyamine metabolism. Further, inosine altered the epigenetic state of HA-GD2 CAR T cells. We observed significant enrichment of IRF and NF-κB transcription factor motifs and motifs associated with memory differentiation in T cells grown in the presence of inosine. Finally, we showed that production of exhausted HA-GD2 and clinical GD2 CAR T cells in inosine-containing culture media enhances their in vivo efficacy, leading to improved survival in GD2+ tumor-bearing mice. In conclusion, we propose introducing inosine during GMP cell manufacturing as a novel strategy to improve CAR T function and subsequent clinical outcomes of CAR T cell therapy. Citation Format: Dorota Klysz, Katie Freitas, Meena Malipatlolla, Stefanie Maier, Carley Fowler, Louai Labanieh, Lucille Stuani, Bence Daniel, Katalin Sandor, Peng Xu, Jing Huang, Amaury Leruste, Vimal Keerthi, Nikolaos Gkitsas, Shabnum Patel, Justin Arredondo-Guerrero, Chris Fisher, Kyle Asano, Sunny Patel, Ansuman Satpathy, Kara Davis, Elena Sotillo, Steven Feldman, Crystal Mackall. Inosine endows CAR T cells with features of increased stemness and anti-tumor potency [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 1158.

Adenosine signalling in T-cell activation favours development of IL-17 positive cells with suppressive properties.

Evidence suggests that the anti-inflammatory nucleoside adenosine can shape immune responses by shifting the regulatory (Treg )/helper (Th17) T-cell balance in favour of Treg . Since this observation is based on in vivo and in vitro studies mostly confined to murine models, we comprehensively analysed effects of adenosine on human T-cells. Proliferation, phenotype and cytokine production of stimulated T-cells were assessed by flow cytometry, multiplex assay and ELISA, gene expression profiling was determined by microarray. We found that the pan-adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) skews human CD3+ T-cell responses towards non-inflammatory Th17 cells. Addition of NECA during T-cell activation increased the development of IL-17+ cells with a CD4+ RORγt+ phenotype and enhanced CD161 and CD196 surface expression. Remarkably, these Th17 cells displayed non-inflammatory cytokine and gene expression profiles including reduced Th1/Th17 transdifferentiation, a stem cell-like molecular signature and induced surface expression of the adenosine-producing ectoenzymes CD39 and CD73. Thus, T-cells cultured under Th17-inducing conditions together with NECA were capable of suppressing responder T-cells. Finally, genome-wide gene expression profiling revealed metabolic quiescence previously associated with non-pathogenic Th17 cells in response to adenosine signalling. Our data suggest that adenosine induces non-inflammatory Th17 cells in human T-cell differentiation, potentially through regulation of metabolic pathways.

EXTH-77. TARGETING CD73 IN GBM SENSITIZES TUMORS TO ONCOLYTIC VIRUS THERAPY

Abstract Glioblastoma Multiforme (GBM) is the most aggressive malignant primary brain tumor and has abysmal 5-year overall survival. With the approval of oHSV Imlygic by FDA for metastatic melanoma and more recently, conditional approval of G47Δ, marketed by Daiichi Sankyo for GBM treatment in Japan, oncolytic viral therapy has emerged as a promising biological approach to treat solid tumors. Our laboratory has previously shown that oHSV expressing long isoform of PTEN (PTENα), HSV-P10, has faster kinetics of virus replication associated with enhanced killing of the glioma cells compared to control HSV. Additionally, HSVP-10 is known to increase mitochondrial membrane potential and cellular ATP production while stimulating anti-tumor immune responses in vivo. RNA sequencing of primary GBM cells infected with HSV-P10 shows altered metabolic pathways relative to control HSV infected cells. 13C metabolic flux analysis in uninfected and control or HSV-P10 infected primary GBM cells reveals that while HSV-P10 infection shuttles most of the glutamine towards citrate by reductive carboxylation of α-ketoglutarate resulting in faster replication of HSV-P10, simultaneously it increased glucose utilization and shuttling towards TCA cycle. This corroborates with enhanced mitochondrial activity leading to increased oxidative phosphorylation and increased cellular and extracellular ATP (eATP). eATP binds to purinergic receptors on tumor and immune cells and boosts anti-tumor immunity. However, the use of extracellular ATP in cancer therapy is limited owing to its short half-life and rapid hydrolysis by ectoenzymes CD39 and CD73 into immune-suppressing adenosine. CD73 carries out the conversion of AMP to adenosine, making it a key regulator of this pathway. Using mice with CD73 knocked out globally (CD73KO), we show that combination of CD73 inhibition with HSV-P10 imparts significant survival benefit compared to WT mice treated with HSV-P10. Our findings will further the understanding of oHSV therapy and the role of the ATP/adenosine in the tumor microenvironment.

EXTH-18. TARGETING THE ADENOSINERGIC IMMUNE SUPPRESSION PATHWAY IN HIGH GRADE GLIOMA SYNERGIZES WITH INNATE IMMUNE CHECKPOINT BLOCKADE

Abstract Immune response in the tumor microenvironment is modulated by the conversion of eATP to AMP to adenosine via the ecto-enzymes CD39 and CD73. Overexpression of CD73 in tumors leads to an increase in extracellular adenosine concentration and decrease in eATP, resulting in immune suppression. In this study, CD73 knockouts were created in adult and pediatric glioma cell lines (DIPG17, T3691, BT245) and used to quantify the effects of CD73 knockout on macrophage phagocytosis in the presence of extracellular AMP. In cell line DIPG17, CD73 knockout significantly increased phagocytosis with and without external AMP compared to the WT. In the WT condition, addition of AMP significantly reduced phagocytosis, while this decrease was not significant in the CD73-KO. The addition of anti-CD47 antibody significantly increased phagocytosis with external AMP in the WT condition. Similar results were obtained with cell line T3691. However, CD73 knockout combined with anti-CD47 treatment and external AMP showed significantly greater phagocytosis than the WT condition. This suggests that a combination of anti-CD73 and anti-CD47 treatment may be more effective in the tumor microenvironment. Lastly, in cell line BT245, CD73 knockout significantly increased phagocytosis as seen in the other cell lines. However, no significant difference was observed between WT and CD73-KO with extracellular AMP or anti-CD47. In vivo studies were conducted in orthotopic xenograft mouse models with DIPG17 CD73 knockout cells. Combination treatment with anti-CD47 antibody significantly decreased tumor burden and prolonged survival in the CD73-KO tumors compared to anti-CD47 treated DIPG17-WT tumors. We conclude that the CD73 adenosinergic pathway and the CD47-SIRPα pathway may present a target for immunotherapy in pediatric and adult gliomas.

CHARACTERIZATION OF DIFFERENTIAL EXPRESSION PATTERNS OF THE EXTRACELLULAR PURINERGIC ENZYMES IN COLORECTAL CANCER

The aim of this study is to characterize tumor cell specific expression of purinergic ecto-enzymes CD39 and CD73, and to associate prognostic significance of these expression patterns in colorectal cancer (CRC) patients. Protein and gene expression of the target genes in various CRC cell lines were assessed via Western Blot (WB) analysis and Real Time PCR (RT-PCR). Additionally, tumor vs stromal cell expression of the target genes was analyzed from publicly available patient expression datasets. Finally, the correlation between CD39 and CD73 expression with patient prognosis was analyzed via The Cancer Genome Atlas (TCGA) datasets. In CRC cell lines, CD39 was found to be not expressed at all while CD73 was expressed extensively in most cell lines via WB and RT-PCR analyses. Patient microarray expression data confirmed the results from CRC cell lines that CD39 expression was very low in epithelial/tumor cells relative to other stromal cell types yet CD73 was expressed abundantly in every cell type within patient tumor samples. Interestingly, CD39 expression in patient tumors was correlated with favorable prognosis while CD73 expression was associated with worse prognosis. Although CD39 and CD73 are related enzymes involved in extracellular purinergic signaling, their expression patterns in tumor cells and prognostic effects in patients show opposing outcomes. Therefore, better insights in understanding the functional involvement of purinergic ecto-enzymes in colorectal tumor development is needed via further mechanistic studies.

HSP90α induces immunosuppressive myeloid cells in melanoma via TLR4 signaling

BackgroundTumor cells modulate host immunity by secreting extracellular vesicles (EV) and soluble factors. Their interactions with myeloid cells lead to the generation of myeloid-derived suppressor cells (MDSC), which inhibit the...

LBA17 Primary endpoint results of SYNERGY, a randomized phase II trial, first-line chemo-immunotherapy trial of durvalumab, paclitaxel, and carboplatin with or without the anti-CD73 antibody oleclumab in patients with advanced or metastatic triple-negative breast cancer (TNBC)

Overexpression of the ectoenzyme CD73 is involved in the generation of immunosuppressive adenosine in the tumor microenvironment and is associated with poor prognosis in patients (pts) with TNBC. Blocking CD73 with oleclumab may enhance the antitumor response to the combination of an anti-PD-L1 with chemotherapy in TNBC.

Abstract 1362: Metabolic engineering of CAR-T cells overcomes suppressive adenosine signaling and enhances functionality

Abstract Chimeric Antigen Receptor (CAR) T cell therapy has resulted in remarkable clinical outcomes in the context of acute and chronic lymphoblastic leukemia, but remains unsuccessful in the treatment of solid tumors. One reason for this failure is thought to be T cell dysfunction or exhaustion promoted by suppressive soluble factors within the tumor microenvironment (TME). High extracellular levels of the immunosuppressive factor adenosine (Ado) are generated in the TME via breakdown of ATP by ecto-enzymes CD39 and CD73 expressed on tumor-infiltrating immune cells. Binding of extracellular Ado to its receptor A2a on T cells results in inhibition of proliferation and effector function. Interestingly, CD39 has recently been described as a surrogate marker of exhaustion on human CAR-T cells and non-engineered T cells. Therefore, we hypothesized that CD39 expression on exhausted CAR-T cells promotes dysfunction through generation of extracellular adenosine. Using an in vitro model of T cell exhaustion, whereby human T cells express a CAR that tonically signals in an antigen-independent manner (HA CAR), we demonstrate that exhausted HA CAR T cells actively hydrolyze extracellular ATP via their elevated expression of CD39 and CD73. Moreover, exhausted CD39+ CAR T cells upregulate several genes associated with a Treg phenotype at the mRNA and protein levels, suggesting that this cell population might be suppressive. To assess whether CD39+/CD73+ CAR T cells exhibit suppressive functions, we co-cultured them with non-exhausted CD19-CAR T cells. Indeed, proliferation and secretion of IL-2 by CD19 CAR T cells were diminished when they were co-cultured with exhausted CD39+ CAR T cells, and that this suppression is dependent on the A2a receptor. Using this knowledge, we used gene-editing and overexpression approaches to engineer CAR-T cells with resistance to suppressive adenosine signaling. In contrast to genetic deletion of CD39 or CD73, which did not alleviate CAR T cell dysfunction, genetic deletion of adenosine receptor A2aR in exhausted CAR T cells resulted in phenotypic changes and a modest improvement in tumor-specific killing. Further, ectopic overexpression of adenosine deaminase (ADA) in CAR T cells led to decreased exhaustion marker expression and significantly enhanced effector function. These data indicate that ADA overexpression is an innovative approach to increase the functionality of CAR T cells through avoidance of suppressive adenosine signaling, and provides proof-of-concept that metabolic engineering of CAR-T cells can pave the way for responses in patients with solid tumors. Citation Format: Dorota Klysz, Meena Malipatlolla, Katherine Freitas, Malek Bashti, Louai Labanieh, Peng Xu, Cecilia Ramello, Amaury Lerust, Hyatt Balke Want, Kaithlen Zen Pacheco, Evan W. Weber, Shabnum Patel, Steven Feldman, Elena Sotillo, Crystal L. Mackall. Metabolic engineering of CAR-T cells overcomes suppressive adenosine signaling and enhances functionality [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 1362.

Abstract 5504: Adoptive cell therapy with tumor-specific Th9 cells induces viral mimicry to restrain acquired resistance from antigen-loss-variant tumor cells

Abstract Adoptive cell therapy (ACT) with tumor-specific T cells expressing T-cell-receptors (TCR) or chimeric antigen receptors (CAR) has not mediated sustained responses in solid tumors. Several factors limit the efficacy of ACT in solid tumors and lead to primary resistance and acquired resistance (refers to recurrence after the initial response). These include but not limited to the insufficient proliferation of T cells, the low capacity of T cells to penetrate stroma-rich solid tumors, and heterogeneity of antigen expression in solid tumors, which allows the escape of T cell attack by tumor cells when becoming antigen-loss-variant cancer cell (ALV). Hence, it is urgent to develop novel ACT strategies to eradicate solid tumors and prevent acquired resistance. We have previously reported (Yong Lu, Cancer Cell, 2018) that tumor-specific IL-9-producing CD4+ Th9 cells represent a novel T cell paradigm for ACT - they are less exhausted, fully cytolytic, and hyperproliferative. This hyperproliferation of Th9 cells is driven by a unique Th9 master transcriptional factor Pu.1 mediated Pu.1-Traf6-NFκB signaling to ensure Th9 cells persist as long as “stem cell-like” T cells, which is not presented in other known CD4+ T cells subsets. In our most recent study, we demonstrate that murine and human Th9 cells, but not Th1/Tc1 or Th17 cells, expressing tumor-specific T-cell-receptors (TCR) or chimeric antigen receptors (CAR), eradicate advanced tumors that contain ALVs. This unprecedented antitumor capacity of Th9 cells is attributed to both enhanced direct tumor cell killing and bystander antitumor effects promoted by intratumor release of IFNα/β. Mechanistically, tumor-specific Th9 cells increase the intratumor accumulation of extracellular ATP (eATP, released from dying tumor cells), because of a unique feature of Th9 cells that lack the expression of ATP degrading ectoenzyme CD39. Intratumor enrichment of eATP promotes the monocyte infiltration and stimulates their production of IFNα/β by inducing eATP-endogenous retrovirus-TLR3/MAVS pathway activation. These results identify tumor-specific Th9 cells as a unique T cell subset endowed with the unprecedented capacity to eliminate ALVs for curative responses. Citation Format: Yong Lu. Adoptive cell therapy with tumor-specific Th9 cells induces viral mimicry to restrain acquired resistance from antigen-loss-variant tumor cells [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 5504.

A TNFR2-Specific TNF Fusion Protein With Improved In Vivo Activity.

Tumor necrosis factor (TNF) receptor-2 (TNFR2) has attracted considerable interest as a target for immunotherapy. Indeed, using oligomeric fusion proteins of single chain-encoded TNFR2-specific TNF mutants (scTNF80), expansion of regulatory T cells and therapeutic activity could be demonstrated in various autoinflammatory diseases, including graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). With the aim to improve the in vivo availability of TNFR2-specific TNF fusion proteins, we used here the neonatal Fc receptor (FcRn)-interacting IgG1 molecule as an oligomerizing building block and generated a new TNFR2 agonist with improved serum retention and superior in vivo activity.MethodsSingle-chain encoded murine TNF80 trimers (sc(mu)TNF80) were fused to the C-terminus of an in mice irrelevant IgG1 molecule carrying the N297A mutation which avoids/minimizes interaction with Fcγ-receptors (FcγRs). The fusion protein obtained (irrIgG1(N297A)-sc(mu)TNF80), termed NewSTAR2 (New selective TNF-based agonist of TNF receptor 2), was analyzed with respect to activity, productivity, serum retention and in vitro and in vivo activity. STAR2 (TNC-sc(mu)TNF80 or selective TNF-based agonist of TNF receptor 2), a well-established highly active nonameric TNFR2-specific variant, served as benchmark. NewSTAR2 was assessed in various in vitro and in vivo systems.ResultsSTAR2 (TNC-sc(mu)TNF80) and NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) revealed comparable in vitro activity. The novel domain architecture of NewSTAR2 significantly improved serum retention compared to STAR2, which correlated with efficient binding to FcRn. A single injection of NewSTAR2 enhanced regulatory T cell (Treg) suppressive activity and increased Treg numbers by > 300% in vivo 5 days after treatment. Treg numbers remained as high as 200% for about 10 days. Furthermore, a single in vivo treatment with NewSTAR2 upregulated the adenosine-regulating ectoenzyme CD39 and other activation markers on Tregs. TNFR2-stimulated Tregs proved to be more suppressive than unstimulated Tregs, reducing conventional T cell (Tcon) proliferation and expression of activation markers in vitro. Finally, singular preemptive NewSTAR2 administration five days before allogeneic hematopoietic cell transplantation (allo-HCT) protected mice from acute GvHD.ConclusionsNewSTAR2 represents a next generation ligand-based TNFR2 agonist, which is efficiently produced, exhibits improved pharmacokinetic properties and high serum retention with superior in vivo activity exerting powerful protective effects against acute GvHD.