CD73 Inhibitor Research Articles

Microalgae-Derived Carotenoid Extract and Biomass Reduce Viability, Induce Oxidative Stress, and Modulate the Purinergic System in Two Melanoma Cell Lines

Cutaneous melanoma (CM) is an aggressive and metastatic tumor, resulting in high mortality rates. Despite significant advances in therapeutics, the available treatments still require improvements. Thus, purinergic signaling emerged as a potential pathway to cancer therapy due to its involvement in cell communication, proliferation, differentiation, and apoptosis. In addition, due to safety and acceptable clinical tolerability, carotenoids from microalgae have been investigated as adjuvants in anti-melanoma therapy. Then, this work aimed to investigate the in vitro anti-melanogenic effect of carotenoid extract (CA) and total biomass (BM) of the Scenedesmus obliquus microalgae on two cutaneous melanoma cell lines (A375 and B16F10). Cells were cultivated under ideal conditions and treated with 10, 25, 50, and 100 μM of CA or BM for 24 h. The effects of the compounds on viability, oxidant status, and purinergic signaling were verified. The IC50 cell viability results showed that CA and BM decreased B16F10 viability at 24.29 μM and 74.85 μM, respectively and decreased A375 viability at 73.93 μM and 127.80 μM, respectively. Carotenoid treatment for 24 h in B16F10 and A375 cells increased the release of reactive oxygen species compared to the control. In addition, CA and BM isolated or combined with cisplatin chemotherapy (CIS) modulated the purinergic system in B16F10 and A375 cell lines through P2X7, A2AR, CD39, and 5′-nucleotidase. They led to cell apoptosis and immunoregulation by activating A2A receptors and CD73 inhibition. The results disclose that CA and BM from Scenedesmus obliquus exhibit an anti-melanogenic effect, inhibiting melanoma cell growth.

CD73/adenosine dynamics in treatment-induced pneumonitis: balancing efficacy with risks of adverse events in combined radio-immunotherapies.

Consolidation with PD-1/PD-L1-based immune checkpoint blockade after concurrent platinum-based chemo-radiotherapy has become the new standard of care for advanced stage III unresectable non-small cell lung cancer (NSCLC) patients. In order to further improve therapy outcomes, innovative combinatorial treatment strategies aim to target additional immunosuppressive barriers in the tumor microenvironment such as the CD73/adenosine pathway. CD73 and adenosine are known as crucial endogenous regulators of lung homeostasis and inflammation, but also contribute to an immunosuppressive tumor microenvironment. Furthermore, the CD73/adenosine pathway can also limit the immune-activating effects of cytotoxic therapies by degrading the pro-inflammatory danger molecule ATP, which is released into the tumor microenvironment and normal lung tissue upon therapy-induced cell damage. Thus, while targeting CD73 may enhance the efficacy of radio-immunotherapies in cancer treatment by mitigating tumor immune escape and improving immune-mediated tumor killing, it also raises concerns about increased immune-related adverse events (irAEs) in the normal tissue. In fact, combined radio-immunotherapies bear an increased risk of irAEs in the lungs, and additional pharmacologic inhibition of CD73 may further enhance the risk of overwhelming or overlapping pulmonary toxicity and thereby limit therapy outcome. This review explores how therapeutic interventions targeting CD73/adenosine dynamics could enhance radiation-induced immune activation in combined radio-immunotherapies, whilst potentially driving irAEs in the lung. We specifically investigate the interactions between radiotherapy and the CD73/adenosine pathway in radiation pneumonitis. Additionally, we compare the incidence of (radiation) pneumonitis reported in relevant trials to determine if there is an increased risk of irAEs in the clinical setting. By understanding these dynamics, we aim to inform future strategies for optimizing radio-immunotherapy regimens, ensuring effective cancer control while preserving pulmonary integrity and patient quality of life.

T Cell-Derived Apoptotic Extracellular Vesicles Ameliorate Bone Loss via CD39 and CD73-Mediated ATP Hydrolysis.

Osteoporosis is a major public health concern characterized by decreased bone density. Among various therapeutic strategies, apoptotic extracellular vesicles (ApoEVs) have emerged as promising agents in tissue regeneration. Specifically, T cell-derived ApoEVs have shown substantial potential in facilitating bone regeneration. However, it remains unclear whether ApoEVs can promote bone mass recovery through enzymatic activity mediated by membrane surface molecules. Therefore, this study aimed to investigate whether T cell-derived ApoEVs could promote bone mass recovery in osteoporosis mice and reveal the underlying mechanisms. ApoEVs were isolated through sequential centrifugation, and their proteomic profiles were identified via mass spectrometry. Western blot and immunogold staining confirmed the enrichment of CD39 and CD73 on ApoEVs. The role of CD39 and CD73 in hydrolyzing adenosine triphosphate (ATP) to adenosine was evaluated by quantifying the levels of ATP and adenosine. Inhibitors of CD39 and CD73, and an A2BR antagonist were used to explore the molecular mechanism of ApoEVs in promoting bone regeneration. ApoEVs significantly reduced bone loss and promote the osteogenic differentiation of BMMSCs in ovariectomy (OVX) mice. We observed increased levels of extracellular ATP and a decrease in CD39 and CD73, key enzymes in ATP-to-adenosine conversion in bone marrow of OVX mice. We found that ApoEVs are enriched with CD39 and CD73 on their membranes, which enable the hydrolysis of extracellular ATP to adenosine both in vitro and in vivo. The adenosine generated by ApoEVs inhibits the inflammatory response and promotes osteogenesis through A2BR and downstream PKA signaling. T cell-derived ApoEVs are enriched with CD39 and CD73, enabling them to hydrolyze extracellular ATP to adenosine, thereby promoting bone regeneration via A2BR and PKA signaling pathway. Our data underscore the substantive role of T cell-derived ApoEVs to treat osteoporosis, thus providing new ideas for the development of ApoEVs-based therapies in tissue regeneration.

Co‐Delivery of aPD‐L1 and CD73 Inhibitor Using Calcium Phosphate Nanoparticles for Enhanced Melanoma Immunotherapy with Reduced Toxicity

AbstractMelanoma, a malignant skin tumor, presents significant treatment challenges, particularly in unresectable and metastatic cases. While immune checkpoint inhibitors (ICIs) targeting PD‐1/PD‐L1 have brought new hope, their efficacy is limited by low response rates and significant immune‐mediated adverse events (irAEs). Through multi‐omics data analysis, it is discovered that the spatial co‐localization of CD73 and PD‐L1 in melanoma correlates with improved progression‐free survival (PFS), suggesting a synergistic potential of their inhibitors. Building on these insights, a novel therapeutic strategy using calcium phosphate (CaP) nanoparticles is developed for the co‐delivery of aPD‐L1 and APCP, a CD73 inhibitor. These nanoparticles, constructed via a biomineralization method, exhibit high drug‐loading capacity and pH‐responsive drug release. Compared to free aPD‐L1, the CaP‐delivered aPD‐L1 effectively avoids systemic side effects while significantly enhancing anti‐tumor efficacy, surpassing even a 20‐fold dose of free aPD‐L1. Furthermore, the co‐delivery of aPD‐L1 and APCP via CaP nanoparticles demonstrates a synergistic anti‐tumor effect, with substantial immune activation and prevention of tumor recurrence through immune memory effects. These findings suggest that the co‐delivery of aPD‐L1 and APCP using CaP nanoparticles is a promising approach for improving melanoma immunotherapy, achieving enhanced efficacy and reduced toxicity.

Development of a hydrogel-based three-dimensional (3D) glioblastoma cell lines culture as a model system for CD73 inhibitor response study

BackgroundDespite the development of various therapeutic approaches over the past decades, the treatment of glioblastoma multiforme (GBM) remains a major challenge. The extracellular adenosine-generating enzyme, CD73, is involved in the pathogenesis and progression of GBM, and targeting CD73 may represent a novel approach to treat this cancer. In this study, three-dimensional culture systems based on three hydrogel compositions were characterized and an optimal type was selected to simulate the GBM microenvironment. In addition, the effect of a CD73 inhibitor on GBM cell aggregates and spheroids was investigated as a potential therapeutic approach for this disease.MethodsRheology measurements, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and cell proliferation assays were performed to analyze the synthesized hydrogel and select an optimal formulation. The viability of tumor cells in the optimal hydrogel was examined histologically and by confocal microscopy. In addition, the sensitivity of the tumor cells to the CD73 inhibitor was investigated using a cell proliferation assay and real-time PCR.ResultsThe data showed that the hydrogel containing 5 wt% gelatin and 5 wt% sodium alginate had better rheological properties and higher cell viability. Therefore, it could provide a more suitable environment for GBM cells and better mimic the natural microenvironment. GBM cells treated with CD73 inhibitors significantly decreased the proliferation rate and expression of VEGF and HIF1-α in the optimal hydrogel.ConclusionOur current research demonstrates the great potential of CD73 inhibitor for clinical translation of cancer studies by analyzing the behavior and function of 3D tumor cells, and thus for more effective treatment protocols for GBM.

Abstract PR003: Structure-guided design and optimization of small molecule CD73 inhibitors with excellent drug-like properties: discovery of quemliclustat

Abstract In the tumor microenvironment, high concentrations of extracellular adenosine promote tumor proliferation through various immunosuppressive mechanisms. High expression of CD73—an ecto-nucleotidase that produces adenosine from adenosine monophosphate—has been associated with poorer prognosis in several tumor types. Herein, we describe the drug discovery efforts that resulted in the discovery of quemliclustat, a highly potent and selective inhibitor of CD73. As a small molecule with excellent drug-like properties, quemliclustat offers several advantages over CD73 antibodies in development, such as greater inhibition of CD73 enzymatic activity (both soluble and membrane-bound), deeper tumor penetration, and the potential for both IV and oral formulations. Quemliclustat was the first small molecule CD73 inhibitor to enter clinical development and is currently being evaluated in various Phase 1/2 studies in advanced solid tumors, including lung and pancreatic cancers. Recent data in metastatic pancreatic cancer are supportive of further study in this disease setting. Citation Format: Jenna Jeffrey Structure-guided design and optimization of small molecule CD73 inhibitors with excellent drug-like properties: discovery of quemliclustat. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr PR003

Bacteria-based biohybrids for remodeling adenosine-mediated immunosuppression to boost radiotherapy-triggered antitumor immune response.

Radiotherapy (RT) can trigger immunogenic cell death (ICD) in tumor cells and release adenosine triphosphate (ATP) to activate antitumor immunity. However, the formation of immunosuppressive adenosine (ADO) mediated by ectonucleotidases including CD39 and CD73, can exacerbate the immunosuppressive effects. Herein, a radiosensitizer-based metal-organic framework (MOF) composed of bismuth (Bi) and ellagic acid (EA) was synthesized in situ on the surface of Escherichia coli Nissle 1917 (EcN) to serve as a carrier for the CD39 inhibitor sodium polyoxotungstate (POM-1). This therapeutic platform, acting as a radiosensitizer, significantly enhances cytotoxicity against tumor cells while effectively inducing ICD and releasing high concentrations of ATP. Subsequently, the released POM-1 increases the levels of pro-inflammatory extracellular ATP while preventing tumor immunosuppression caused by the accumulation of ADO. Additionally, as a natural immune adjuvant, EcN further promotes the maturation of dendritic cells (DCs) and the infiltration of cytotoxic T lymphocytes (CTLs). As a result, such treatment initiates the destruction of established tumor growth and induces strong abscopal effects, leading to a significant inhibition of tumor metastases. This strategy presents a bacterial-based biohybrid system that facilitates RT-induced ICD while simultaneously limiting the degradation of ATP into ADO, thereby achieving sustained anti-tumor immunity.

Adenosine signaling in tumor-associated macrophages and targeting adenosine signaling for cancer therapy.

This review examined the critical role of adenosine signaling in modulating the behavior of tumor-associated macrophages (TAMs), a key determinant of the tumor microenvironment (TME). Adenosine is an immunosuppressive metabolite that is highly enriched in the TME due to elevated expression of adenosine triphosphatase (ATPase). Adenosine influences polarization of TAMs through A2A and A2B receptors, which drives a phenotype that supports tumor progression and immune evasion. The adenosine-mediated regulation of TAMs significantly suppresses the TME, dampening the efficacy of current immunotherapies. Targeting the adenosine pathway has shown potential in preclinical studies through reversal of the immunosuppressive microenvironment and antitumor immune response enhancement. Clinical trials are currently underway to determine the impact of A2A receptor antagonists, and CD39 and CD73 inhibition, enzymes that are pivotal in adenosine production, in various cancers. The current understanding of the CD39-CD73-adenosine axis in TAM regulation and the emerging strategies targeting adenosine signaling pathway for therapeutic intervention are the subjects of this review. The current clinical trials focusing on adenosine pathway inhibitors in combination with existing therapies to improve clinical outcomes are summarized and the need for continued research to refine these approaches for cancer treatment is emphasized.

A self-gelling hemostatic powder boosting radiotherapy-elicited NK cell immunity to combat postoperative hepatocellular carcinoma relapse.

Liver resection represents a main curative treatment for patients with early-stage hepatocellular carcinoma (HCC), but there is a rather high incidence of postoperative HCC relapse, which severely shortens long-term survival time. Currently, no standard adjuvant strategies are available for preventing HCC relapse in clinical practice. Impaired natural killer (NK) cell anti-tumor immunity has been disclosed as a crucial root of HCC relapse, indicating that reinstating NK cell anti-tumor immunity may show promise to curb HCC relapse. Coincidently, mounting evidence shows that radiotherapy (RT) can trigger NK cell anti-tumor immunity, though its mechanisms have never been completely elucidated. Herein, we uncover that RT can induce immunogenic cell death and activate cGAS-STING pathway in HCC cells to elicit NK cell anti-tumor immunity. However, RT is also revealed to enhance autophagy and CD73 expression in HCC cells, as well as neutrophil extracellular traps (NETs) formation, which largely limits RT-induced activation of NK cell anti-tumor immunity. Therefore, a cocktail of autophagy inhibitor 3-methyladenine, CD73 inhibitor ARL 67156 trisodium and NETs lyase DNase I may sensitize RT to reinvigorate NK cell anti-tumor immunity and thus prevent HCC relapse postresection. To minimize therapy-related side effects, a nanocomposite powder encapsulating such a triple-drug cocktail is developed. This powder can rapidly form adhesive hydrogel in situ after applied to surgical margin, consequently fulfilling liver-localized sustained drug delivery. Importantly, it can sensitize RT to reinstate NK cell anti-tumor immunity to combat postoperative HCC relapse in Heap1-6-HCC murine model. Besides, this powder can also generate rapid hemostasis in rat and porcine models. Altogether, this work provides an innovative strategy to thwart postoperative HCC relapse and bleeding.

The evolution of BRAF-targeted therapies in melanoma: overcoming hurdles and unleashing novel strategies.

Melanoma, a highly aggressive form of skin cancer, poses a significant global health burden, with 331,647 new cases and 58,645 deaths reported in 2022. The development of melanoma is influenced by various factors, including sunlight exposure and BRAFV600 mutations that activate the MAPK/ERK pathway. The introduction of BRAF and MEK inhibitors has revolutionized the treatment landscape for melanoma patients. However, innate and acquired therapeutic resistance remains a significant challenge. This review provides a comprehensive overview of the current state of BRAF-targeted therapies in melanoma, highlighting the efficacy and limitations of FDA-approved combinations of BRAF and MEK inhibitors such as vemurafenib, dabrafenib, trametinib, and cobimetinib. The review also explores the off-target effects of BRAF inhibitors on endothelial cells, emphasizing the need for more selective therapies to minimize vascular complications and metastatic potential. The article also discusses potential druggable targets, including ERK5, CD73, ALDH1A1, PLA1A, and DMKN, which are promising in addressing diagnostic hurdles and guiding personalized therapeutic decisions. Recent studies on regorafenib, ERK5 signaling, and CD73 inhibition are highlighted as novel strategies to overcome resistance and improve treatment outcomes. The review also delves into the role of advanced therapeutic tools, such as mRNA vaccines and CRISPR-Cas9, in revolutionizing personalized oncology by targeting specific genetic mutations and enhancing immune responses against melanoma. The ongoing synergy between advancing research, targeted interventions, strategic treatment combinations, and cost-effectiveness evaluations offers a promising pathway to elevate patient outcomes in the persistent battle against melanoma significantly.

Discovery of Novel 5-(Pyridazin-3-yl)pyrimidine-2,4(1H,3H)-dione Derivatives as Potent and Orally Bioavailable Inhibitors Targeting Ecto-5'-nucleotidase.

Ecto-5-nucleotidase (CD73) is overexpressed in a variety of cancers and associated with the immunosuppressive tumor microenvironment, making it an attractive target for cancer immunotherapy. Herein, we designed and synthesized a series of novel (pyridazine-3-yl)pyrimidine-2,4(1H,3H)-dione derivatives as CD73 inhibitors. These compounds exhibited remarkable inhibitory activity against CD73 in both enzymatic biochemical and cellular assays. Among them, compound 35j proved to be one of the most potent inhibitors and an uncompetitive inhibitor with no obvious cytotoxicity. This compound showed high metabolic stability in rat liver microsomes and favorable pharmacokinetic profiles in rats (T1/2 = 3.37 h, F = 50.24%). Importantly, orally administered 35j significantly inhibited tumor growth in the triple-negative breast cancer 4T1 mouse model (TGI = 73.6%, 50 mg/kg). Immunoassays suggested that 35j remarkably increased the infiltration of positive immune cells, thereby reinvigorating antitumor immunity. These results demonstrate that 35j is a potent CD73 inhibitor worthy of further development.

CD39 inhibitor (POM-1) enhances radiosensitivity of esophageal squamous cell carcinoma (ESCC) cells by promoting apoptosis through the Bax/Bcl-2/Caspase 9/Caspase 3 pathway

CD39 inhibitor (sodium polyoxotungstate, POM-1) has been reported to have antitumor effects. However, the synergistic effect of POM-1 with radiotherapy requires further elucidation. This study aimed to investigate the role and the molecular mechanism of POM-1 in esophageal squamous cell carcinoma (ESCC) radiosensitization. Firstly, the expression of CD39 in ESCC cells and normal esophageal epithelial cells were detected. Then radioresistant ESCC cells (Eca109R and KYSE150R) were constructed and CD39 expression was analyzed. Furthermore, the effect of POM-1 on radiosensitivity for parent cells and radioresistant cells were observed. Then, we analyzed the effect of POM-1 and CD39 siRNA on radiotherapy-induced apoptosis and determined whether POM-1 modulated the radioresistance of ESCC cells depending on the apoptotic signaling pathway. Finally, we validated the synergistic effect of POM-1 combined with radiotherapy in vivo. Our results showed that CD39 was highly expressed in ESCC cells and radioresistant ESCC cells (p < 0.05). POM-1 reduced radioresistance and proliferation of parent cells and radioresistant cells (p < 0.05). Further mechanistic exploration showed that inhibition of CD39 promoted radiation-induced apoptosis (p < 0.05). Bax knockdown reversed the effect of POM-1 on ESCC cells (p < 0.01). Animal experiments also validated that radiotherapy combined with POM-1 enhanced tumor inhibition in vivo (p < 0.05). These results suggested that POM-1 had synergistic effect with radiotherapy by enhancing cell apoptosis through Bax/Bcl-2 signal pathway in ESCC. The combination of POM-1 and radiotherapy is expected to enhance the anti-tumor effect in ESCC.

Adenosine A2B receptor activation regulates the balance between T helper 17 cells and regulatory T cells, and inhibits regulatory T cells exhaustion in experimental autoimmune myositis.

Idiopathic inflammatory myopathy (IIM) is a systemic autoimmune disease characterized by skeletal muscle involvement. This study aimed to investigate the role of adenosine receptor signalling pathways in the development of experimental autoimmune myositis (EAM). An ecto-5'-nucleotidase (CD73) inhibitor, adenosine receptor agonists, a hypoxia-inducible factor-1α (HIF-1α) inhibitor or a vehicle were administered to control and EAM mice. Murine splenic CD4+ or regulatory T cells (Tregs) were isolated using magnetic beads and subsequently stimulated with an adenosine A2B receptor agonist, a HIF-1α inhibitor, or vehicle in vitro. In cross-sectional studies, we collected 64 serum samples (69% female, 49±9years), 63 peripheral blood samples (70% female, 50±11years), and 34 skeletal muscle samples (71% female, 63±6years) from patients with IIM. Additionally, 35 serum samples and 30 peripheral blood samples were obtained from age- and sex-matched healthy controls, and six quadriceps muscle samples were collected from patients with osteoarthritis to serve as the normal group. Patients with IIM exhibited increased CD73 [dermatomyositis (DM), polymyositis (PM): P<0.01; immune-mediated necrotizing myopathy (IMNM): P<0.0001] and adenosine deaminase (ADA) expression (DM: P<0.001; PM, IMNM: P<0.0001) in the skeletal muscles, and serum ADA levels [56.7 (95% CI: 53.7, 58.7) vs. 198.8 (95% CI: 186.2, 237.3) ng/μL, P<0.0001]. Intervention with a CD73 inhibitor exacerbated (P=0.0461), whereas adenosine receptor agonists (A1: P=0.0009; A2B: P<0.0001; A3: P=0.0001) and the HIF-1α inhibitor (P=0.0044) alleviated skeletal muscle injury in EAM mice. Elevated expression of programmed cell death protein-1 (PD1: P=0.0023) and T-cell immunoglobulin and mucin-domain containing-3 (TIM3: P<0.0001) in skeletal muscles of patients with IIM were correlated with creatine kinase levels (PD1, r=0.7072, P<0.0001; TIM3, r=0.4808, P=0.0046). PD1+CD4+ (r=0.3243, P=0.0115) and PD1+CD8+ (r=0.3959, P=0.0017) T cells were correlated with Myositis Disease Activity Assessment Visual Analogue Scale scores (muscle) in IIM. The exhausted Tregs were identified in the skeletal muscles of patients with IIM. Activation of the A2B adenosine receptor downregulated HIF-1α (protein or mRNA level, P<0.01), resulting in decreased T helper cell 17 (Th17) (13.58% vs. 5.43%, P=0.0201) and phosphorylated-signal transducer and activator of transcription 3 (p-STAT3)+ Th17 (16.32% vs. 6.73%, P=0.0029), decreased exhausted Tregs (PD1+ Tregs: 53.55% vs. 40.28%, P=0.0005; TIM3+ Tregs: 3.93% vs. 3.11%, P=0.0029), and increased Tregs (0.45% vs. 2.89%, P=0.0006) in EAM mice. The exhausted T cells may be pathogenic in IIM,andthe activation of adenosine A2B receptor signalling pathway can regulate Th17/Treg balance and inhibit Tregsexhaustion, thereby slowing EAM disease progression.

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

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

Design, synthesis and structure-activity relationship of malonic acid non-nucleoside derivatives as potent CD73 inhibitors

High levels of extracellular adenosine in tumor microenvironment (TME) has extensive immunosuppressive effect. CD73 catalyzes the conversion of AMP into adenosine and regulates its production. Inhibiting CD73 can reduce the level of adenosine and reverse adenosine-mediated immune suppression. Therefore, CD73 has emerged as a valuable target for cancer immunotherapy. Here, a new series of malonic acid non-nucleoside derivatives were designed, synthesized and evaluated as CD73 inhibitors. Among them, compounds 18 and 19 exhibited significant inhibition activities against hCD73 with IC50 values of 0.28 μM and 0.10 μM, respectively, suggesting the feasibility of replacing the benzotriazole moiety in the lead compound. This study explored the novelty and structural diversity of CD73 inhibitors.

Boost Infiltration and Activity of T Cells via Inhibiting Ecto-5'-nucleotidase (CD73) Immune Checkpoint to Enhance Glioblastoma Immunotherapy.

The currently available immune checkpoint therapy shows a disappointing therapeutic efficacy for glioblastoma multiforme (GBM), and it is of great importance to discover better immune checkpoints and develop innovative targeting strategies. The discovered metabolic immune checkpoint ecto-5-nucleotidase (CD73) in a tumor contributes to its immune evasion due to the dysregulation of extracellular adenosine (ADO), which significantly inhibits the function of antitumor T cells and increases the activity of immunosuppressive cells. Herein, we drastically inhibit the expression of CD73 to reduce the production of ADO by using versatile Au@Cu2-xSe nanoparticles (ACS NPs). ACS NPs can decrease the expression of CD73 by alleviating the tumor hypoxia through their Fenton-like reaction to weaken the ADO-driven immunosuppression for enhancing antitumor T cell infiltration and activity of GBM. The copper ions (Cu2+) released from ACS NPs can chelate with disulfide, leading to the formation of cytotoxic bis(N,N-diethyldithiocarbamate)-copper complex (CuET), which can be combined with radiotherapy to recruit more antitumor T cells to infiltrate into the tumor site. Based on the inhibition of CD73 to promote the infiltration and activity of antitumor T cells, a cascade of enhancing GBM immunotherapy effects can be achieved. The significant increase in CD8+ T and CD4+ T cells within the tumor and the memory T cells in the spleen effectively reduces tumor size by 92%, which demonstrates the excellent efficacy of immunotherapy achieved by a combination of metabolic immune checkpoint CD73 inhibition with chemoradiotherapy. This work demonstrates that modulation of CD73-mediated tumor immunosuppression is an important strategy of improving the outcome of GBM immunotherapy.

Enhancing protein productivities in CHO cells through adenosine uptake modulation – Novel insights into cellular growth and productivity regulation

Maximizing production potential of recombinant proteins such as monoclonal antibodies (mAbs) in Chinese Hamster Ovary (CHO) cells is a key enabler of reducing cost of goods of biologics. In this study, we explored various strategies to utilize adenosine mediated effects in biologics manufacturing processes. Results show that supplementation of adenosine increases specific productivity by up to two-fold while also arresting cell growth. Introducing adenosine in intensified perfusion processes in a biphasic manner significantly enhanced overall productivity. Interestingly, adenosine effect was observed to be dependent on the cell growth state. Using specific receptor antagonists and inhibitors, we identified that ENTs (primarily Slc29a1) mediate the uptake of adenosine in CHO cell cultures. Transcriptomics data showed an inverse correlation between Slc29a1 expression levels and peak viable cell densities. Data suggests that in fed-batch cultures, adenosine can be produced extracellularly. Blocking Slc29a1 using ENT inhibitors such as DZD and DP alone or in combination with CD73 inhibitor, PSB12379, resulted in a twofold increase in peak viable cell densities as well as productivities in fed batch – a novel strategy that can be applied to biologics manufacturing processes. This is the first study that suggests that adenosine production/accumulation in CHO cell cultures can potentially regulate the transition of CHO cells from exponential to stationary phase. We also demonstrate strategies to leverage this regulatory mechanism to maximize the productivity potential of biologics manufacturing processes.

A Phase Ib/II Randomized Clinical Trial of Oleclumab with or without Durvalumab plus Chemotherapy in Patients with Metastatic Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma upregulates CD73, potentially contributing to immune surveillance evasion. Combining oleclumab (CD73 inhibitor) and durvalumab with chemotherapy may identify an effective treatment option. We describe a multicenter phase Ib/II randomized clinical trial in patients with metastatic pancreatic ductal adenocarcinoma, untreated (cohort A) or previously received gemcitabine-based chemotherapy (cohort B; NCT03611556). During escalation, patients received oleclumab 1,500 or 3,000 mg, durvalumab 1,500 mg, and gemcitabine plus nab-paclitaxel (GnP; cohort A; n = 14) or modified FOLFOX (cohort B; n = 11). During expansion, cohort A patients (n = 170) were randomized to GnP (arm A1), oleclumab [recommended phase II dose (RP2D)] with GnP (arm A2), or oleclumab (RP2D) with durvalumab plus GnP (arm A3). Primary objectives were safety (escalation) and objective response rate (expansion). Secondary objectives included progression-free survival (PFS) and overall survival (OS). During escalation, 1/11 patients from cohort B (oleclumab 3,000 mg) experienced two dose-limiting toxicities. Oleclumab's RP2D was 3,000 mg. During expansion, grade ≥3 treatment-related adverse events occurred in 67.7% (42/62) of patients in A1, 73.7% (28/38) in A2, and 77.1% (54/70) in A3. The objective response rate was 29.0%, 21.1%, and 32.9% in A1, A2, and A3, respectively (A1 vs. A3; P = 0.650). PFS [HR = 0.72; 95% confidence interval (CI), 0.47, 1.11] and OS (HR = 0.75; 95% CI, 0.50-1.13) were similar for A3 versus A1. Patients with high CD73 expression had improved PFS and OS in A3 versus A1, although this should be interpreted with caution. Although the safety profile was acceptable, this study did not meet its primary efficacy endpoint.

Preventive Treatment with a CD73 Small Molecule Inhibitor Enhances Immune Surveillance in K-Ras Mutant Pancreatic Intraepithelial Neoplasia.

Immunoprevention is an emerging consideration for solid tumors, including pancreatic ductal adenocarcinoma (PDAC). We and others have shown that Kras mutations in genetic models of spontaneous pancreatic intraepithelial neoplasia (PanIN), which is a precursor to PDAC, results in CD73 expression in the neoplastic epithelium and some populations of infiltrating immune cells, including macrophages and CD8 T cells. CD73 is an ecto-enzyme that converts extracellular adenosine monophosphate to adenosine, a critical immune inhibitory molecule in PDAC. We hypothesized inhibition of CD73 would reduce the incidence of PanIN formation and alter the immune microenvironment. To test our hypothesis, we used the KrasG12D; PdxCre1 (KC) genetically engineered mouse model and tested the utility of AB-680, a small molecule inhibitor targeting CD73, to inhibit PanIN progression. AB-680, or vehicle control, was administered using oral gavage delivery 3 days/week at 10 mg/kg, beginning when the mice were 2 months old and lasting 3 months. We euthanized the mice at 5 months old. In the KC model, we quantified significantly less pancreatitis, early and advanced PanIN, and quantified a significant increase in M1 macrophages in AB-680-treated mice. Single-cell RNA sequencing (scRNA-seq) of pancreata of AB-680-treated mice revealed increased infiltration of CD4+ T cells, CD8+ T cells, and mature B cells. The scRNA-seq analysis showed that CD73 inhibition reduced M2 macrophages, acinar, and PanIN cell populations. CD73 inhibition enhanced immune surveillance and expanded unique clonotypes of TCR and BCR, indicating that inhibition of CD73 augments adaptive immunity early in the neoplastic microenvironment. Prevention Relevance: Previous studies found PanIN lesions in healthy pancreata. Not all progress to PDAC, suggesting a window for enhanced antitumor immunity through immunoprevention therapy. CD73 inhibition in our study prevents PanIN progression, reduces immune-suppressive macrophages and expands TCR and BCR unique clonotypes, highlighting an encouraging therapeutic avenue for high-risk individuals.

Assessment of ATP metabolism to adenosine by ecto-nucleotidases carried by tumor-derived small extracellular vesicles.

Immunosuppression is a hallmark of cancer progression. Tumor-derived small extracellular vesicles (sEV), also known as TEX, produce adenosine (ADO) and can mediate tumor-induced immunosuppression.Here, the ATP pathway of ADO production (ATP ADP AMP ADO) by ecto-nucleotidases carried on the sEV surface was evaluated by a method using N6-etheno-ATP (eATP) and N6-etheno-AMP (eAMP) as substrates for enzymatic activity. The "downstream" N6-etheno-purines (ePurines) were measured by high performance liquid chromatography with fluorescence detection (HPLC-FL).Human melanoma cell-derived TEX (MTEX) metabolized eATP to N6-etheno-ADP (eADP), eAMP and N6-etheno-Adenosine (eADO) more robustly than control keratinocyte cell-derived sEV (CEX); due to accelerated conversion of eATP to eADP and eADP to eAMP. MTEX and CEX similarly metabolized eAMP to eADO. Blocking of the ATP pathway with the selective CD39 inhibitor ARL67156 or pan ecto-nucleotidase inhibitor POM-1 normalized the ATP pathway but neither inhibitor completely abolished it. In contrast, inhibition of CD73 by PSB12379 or AMPCP abolished eADO formation by both MTEX and CEX, suggesting that targeting CD73 is the preferred approach to eliminating ADO produced by ecto-nucleotidases located on the sEV surface.The noninvasive, sensitive, and specific assay assessing ePurine metabolism ± ecto-nucleotidase inhibitors in TEX enables the personalized identification of ecto-nucleotidase activity primarily involved in ADO production in patients with cancer. The assay could guide precision medicine by determining which purine is the preferred target for inhibitory therapeutic interventions.