Adenosine Production Research Articles

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.

Extracellular adenosine oppositely regulates the purinome machinery in glioblastoma and mesenchymal stem cells.

Glioblastoma (GB) is a lethal brain tumor that rapidly adapts to the dynamic changes of the tumor microenvironment (TME). Mesenchymal stem/stromal cells (MSCs) are one of the stromal components of the TME playing multiple roles in tumor progression. GB progression is prompted by the immunosuppressive microenvironment characterized by high concentrations of the nucleoside adenosine (ADO). ADO acts as a signaling molecule through adenosine receptors (ARs) but also as a genetic and metabolic regulator. Herein, the effects of high extracellular ADO concentrations were investigated in a human glioblastoma cellular model (U343MG) and MSCs. The modulation of the purinome machinery, i.e., the ADO production (CD39, CD73, and adenosine kinase [ADK]), transport (equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2)), and degradation (adenosine deaminase [ADA]) were investigated in both cell lines to evaluate if ADO could affect its cell management in a positive or negative feed-back loop. Results evidenced a different behavior of GB and MSC cells upon exposure to high extracellular ADO levels: U343MG were less sensitive to the ADO concentration and only a slight increase in ADK and ENT1 was evidenced. Conversely, in MSCs, the high extracellular ADO levels reduced the ADK, ENT1, and ENT2 expression, which further sustained the increase of extracellular ADO. Of note, MSCs primed with the GB-conditioned medium or co-cultured with U343MG cells were not affected by the increase of extracellular ADO. These results evidenced how long exposure to ADO could produce different effects on cancer cells with respect to MSCs, revealing a negative feedback loop that can support the GB immunosuppressive microenvironment. These results improve the knowledge of the ADO role in the maintenance of TME, which should be considered in the development of therapeutic strategies targeting adenosine pathways as well as cell-based strategies using MSCs.

Abstract We063: CD73 EXPRESSION ON NEUTROPHILS PROTECTS AGAINST CARDIOMYOCYTE DAMAGE DURING INVASIVE STREPTOCOCCUS PNEUMONIAE INFECTION

In recent years, Streptococcus pneumoniae (pneumococcus)-induced cardiac events have emerged as one of the most serious and life-threatening infection outcomes of invasive pneumococcal disease (IPD), leading to major adverse cardiac events (MACE) with high mortality rates. S. pneumoniae has the ability to invade myocardium and damage cardiomyocytes through the release of bacterial factors and formation of bacterial-filled cardiac microlesions. We previously found that polymorphonuclear cells (PMNs) or neutrophils are crucial for host defense against S. pneumoniae lung infection and that extracellular adenosine (EAD) production, by the exonucleosidase CD73, controlled the anti-bacterial functions of these cells. The objective of this study was to explore the role of PMNs and CD73 in cardiac damage during invasive pneumococcal infection. Upon intra-peritoneal ( i.p .) injection with TIGR4, an invasive pneumococcal strain, C57BL/6 mice showed an increased influx of PMNs into the cardiac tissue as determined by flow cytometry. However, the increased PMN numbers failed to contain bacterial burden in the heart and showed a positive correlation with serum levels of the cardiac damage marker Troponin-1 as determined through ELISA. Depletion of PMNs prior infection reduced pneumococcal burden in the heart and lowered the Troponin-1 levels suggesting that PMNs induce cardiac damage during infection. While exploring the mechanisms underlying the detrimental PMN response, we found that the late stage of IPD was associated with reduced CD73 expression on PMNs. The role of CD73 in regulating cardiac damage was tested in vivo using CD73 -/- mice which had significantly higher bacterial burden and cardiac damage compared to wild-type mice despite similar PMN numbers. The role of CD73 expression on PMNs was also tested ex vivo using the HL-1 cardiomyocyte cell line which showed increased LDH release upon S. pneumoniae infection in presence of CD73 -/- PMNs, a phenotype reversed by supplementation of exogenous adenosine. Similar iincreased damage in presence of CD73 -/- PMNs was seen with C57BL/6 Mouse Primary Cardiac Microvascular Endothelial Cells which lead to increased migration of beads across endothelium. Our findings suggest that dysregulation of CD73 on PMNs during invasive pneumococcal infection leads to an inability to control bacterial numbers and increased cardiac damage, and that the EAD-pathway can be a potential pharmacological target to limit disease severity.

Ectonucleotidases in Ischemia Reperfusion Injury: Unravelling the Interplay With Mitochondrial Dysfunction in Liver Transplantation

Ischemia-reperfusion injury (IRI) profoundly impacts organ transplantation, especially in orthotopic liver transplantation (OLT). Disruption of the mitochondrial respiratory chain during ischemia leads to ATP loss and ROS production. Reperfusion exacerbates mitochondrial damage, triggering the release of damage-associated molecular patterns (DAMPs) and inflammatory responses. Mitochondrial dysfunction, a pivotal aspect of IRI, is explored in the context of the regulatory role of ectonucleotidases in purinergic signaling and immune responses. CD39, by hydrolyzing ATP and ADP; and CD73, by converting AMP to adenosine, emerge as key players in mitigating liver IRI, particularly through ischemic preconditioning and adenosine receptor signaling. Despite established roles in vascular health and immunity, the impact of ectonucleotidases on mitochondrial function during hepatic IRI is unclear. This review aims to elucidate the interplay between CD39/73 and mitochondria, emphasizing their potential as therapeutic targets for liver transplantation.This article explores the role of CD39/73 in tissue hypoxia, emphasizing adenosine production during inflammation. CD39 and CD73 upregulation under hypoxic conditions regulate immune responses, demonstrating protective effects in various organ-specific ischemic models. However, prolonged adenosine activation may have dual effects, beneficial in acute settings but detrimental in chronic hypoxia.Herein, we raise questions about ectonucleotidases influencing mitochondrial function during hepatic IRI, drawing parallels with cancer cell responses to chemotherapy.The review underscores the need for comprehensive research into the intricate interplay between ectonucleotidases, mitochondrial dynamics, and their therapeutic implications in hepatic IRI, providing valuable insights for advancing transplantation outcomes.

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.

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

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

IFN-γ-Preconditioned Human Gingival-Derived Mesenchymal Stromal Cells Inhibit Plasmacytoid Dendritic Cells via Adenosine.

Plasmacytoid dendritic cells (pDCs) are vital players in antiviral immune responses because of their high levels of IFN-α secretion. However, this attribute has also implicated them as critical factors behind the immunopathogenesis of inflammatory diseases, and no currently available therapy can efficiently inhibit pDCs' aberrant activation. Mesenchymal stromal cells (MSCs) possess stromal immunomodulatory functionality, regulating immune cell activation through several mechanisms, including the adenosinergic (CD39/CD73/adenosine) pathway. The IFN-γ preconditioning of bone marrow MSCs improves their inhibitory properties for therapy applications; however, isolating human gingival tissue-derived MSCs (hGMSCs) is more accessible. These cells have shown better immunomodulatory effects, yet the outcome of IFN-γ preconditioning and its impact on the adenosinergic pathway has not been evaluated. This study first validated the immunoregulatory properties of primary-cultured hGMSCs, and the results showed that IFN-γ preconditioning strengthens CD39/CD73 coexpression, adenosine production, and the regulatory properties of hGMSC, which were confirmed by describing for the first time their ability to reduce pDC activation and their IFN-α secretion and to increase the frequency of CD73+ pDC. In addition, when CD73's enzymatic activity was neutralized in hGMSCs, adenosine production and the IFN-γ preconditioning effect were restrained. This evidence might be applied to design hGMSCs- and adenosine-based immunotherapeutic strategies for treating inflammatory disorders that are associated with pDC overactivation.

Effect of cancer immunotherapy with CD39/PD-L1 bispecific antibody OriA631-b.

e14508 Background: Despite significant advancements in cancer immunotherapy through the use of immune checkpoint inhibitors, there remains a need for more robust strategies to enhance anti-tumor immune responses. This study introduces and characterizes OriA631-B, an innovative CD39/PD-L1 bispecific antibody. By simultaneously targeting the metabolic checkpoint CD39 and the immune checkpoint PD-L1, this novel antibody has the potential to significantly augment anti-tumor efficacy, particularly in cold tumors characterized by a scarcity of infiltrating immune cells. The development of OriA631-B marks a breakthrough in concurrently addressing two crucial immunosuppressive pathways. Methods: The creation of OriA631-B involved cutting-edge antibody engineering methodologies to guarantee optimal binding affinity and specificity for its target antigens, CD39 and PD-L1. With meticulous design and refinement, OriA631-B was developed to exhibit robust binding capabilities to both CD39 and PD-L1, enabling simultaneous and precise recognition. Leveraging advanced antibody engineering techniques was instrumental in crafting OriA631-B as a potent tool with heightened binding properties, broadening its potential applications across diverse therapeutic contexts. Results: In vitro investigations revealed the efficacy of OriA631-B, derived from the foundation of a clinically validated in-house generated PD-L1 antibody (YN035). This innovative antibody effectively thwarted CD39-mediated adenosine production, resulting in heightened effector T cell function and proliferation. In vivo efficacy assessments conducted on syngeneic hot tumor models MC38 and Hepa1-6 underscored the remarkable effectiveness of OriA631, the CD39 monoclonal antibody (mAb), in controlling tumor growth. Notably, OriA631-B, the CD39/PD-L1 bispecific antibody, exhibited even more profound impacts, significantly restraining tumor growth beyond the effects observed with single-target treatments in the hot tumor model or combination therapies in both the hot tumor model MC38 and the cold tumor model CT26. These findings underscore the potent therapeutic potential of OriA631-B and its capacity to exert robust anti-tumor activity by concurrently targeting CD39 and PD-L1. Additionally, safety profiling revealed an acceptable toxicity profile for OriA631-B, with no significant adverse events observed during the study. Conclusions: In summary, OriA631-B demonstrates potential as an innovative immunotherapeutic strategy for cancer treatment by concurrently targeting CD39-mediated immune suppression and PD-L1-mediated T cell exhaustion. Further exploration, including clinical trials, is imperative to fully uncover the therapeutic capabilities of this groundbreaking bispecific antibody in cancer immunotherapy.

Discovery of Novel Non-Nucleoside Inhibitors Interacting with Dizinc Ions of CD73.

High extracellular concentrations of adenosine triphosphate (ATP) in the tumor microenvironment generate adenosine by sequential dephosphorylation of CD39 and CD73, resulting in potent immunosuppression to inhibit T cell and natural killer (NK) cell function. CD73, as the determining enzyme for adenosine production, has been shown to correlate with poor clinical tumor prognosis. Conventional inhibitors as analogues of adenosine 5'-monophosphate (AMP) may have a risk of further metabolism to adenosine analogues. Here, we report a new series of malonic acid non-nucleoside inhibitors coordinating with zinc ions of CD73. Compound 12f was found to be a superior CD73 inhibitor (IC50 = 60 nM) by structural optimization, and its pharmacokinetic properties were investigated. In mouse tumor models, compound 12f showed excellent efficacy and reversal of immunosuppression in combination with chemotherapeutic agents or checkpoint inhibitors, suggesting that it deserves further development as a novel CD73 inhibitor.

Adenosine in Interventional Cardiology: Physiopathologic and Pharmacologic Effects in Coronary Artery Disease.

This review article focuses on the role of adenosine in coronary artery disease (CAD) diagnosis and treatment. Adenosine, an endogenous purine nucleoside, plays crucial roles in cardiovascular physiology and pathology. Its release and effects, mediated by specific receptors, influence vasomotor function, blood pressure regulation, heart rate, and platelet activity. Adenosine therapeutic effects include treatment of the no-reflow phenomenon and paroxysmal supraventricular tachycardia. The production of adenosine involves complex cellular pathways, with extracellular and intracellular synthesis mechanisms. Adenosine's rapid metabolism underscores its short half-life and physiological turnover. Furthermore, adenosine's involvement in side effects of antiplatelet therapy, particularly ticagrelor and cangrelor, highlights its clinical significance. Moreover, adenosine serves as a valuable tool in CAD diagnosis, aiding stress testing modalities and guiding intracoronary physiological assessments. Its use in assessing epicardial stenosis and microvascular dysfunction is pivotal for treatment decisions. Overall, understanding adenosine's mechanisms and clinical implications is essential for optimizing CAD management strategies, encompassing both therapeutic interventions and diagnostic approaches.

Exploiting the roles of nitrogen sources for HEA increment in Cordyceps cicadae.

Cordyceps cicadae, as a new food ingredient, is a valuable edible and medicinal fungi. However, its resources are severely depleted due to environmental limitations and excessive harvesting practices. N6-(2-hydroxyethyl) adenosine (HEA), as an important product of Cordyceps cicadae, has the potential to be used in medical industry due to its diverse disease curing potential. However, the disclosure of HEA synthesis still severely limited its application until now. In this study, the kinetic curves for adenosine and HEA under shaker fermentation were explored. The kinetics of HEA and adenosine production exhibited a competitive pattern, implicating a possibility of sharing a same step during their synthesis. Due to HEA as a derivative of nitrogen metabolism, the effect of different nitrogen sources (peptone, yeast extract, ammonium sulfate, diammonium oxalate monohydrate, ammonium citrate dibasic, and ammonium citrate tribasic) on HEA production in Cordyceps cicadae strain AH 10-4 had been explored under different incubation conditions (shaker fermentation, stationary fermentation, and submerged fermentation). Our results indicated that the complex organic nitrogen sources were found to improve the accumulation of HEA content under shaker fermentation. In contrast, the optimal nitrogen source for the accumulation of HEA under stationary fermentation and submerged fermentation was ammonium citrate tribasic. But submerged fermentation obviously shortened the incubation time and had a comparable capacity of HEA accumulation by 2.578 mg/g compared with stationary fermentation of 2.535 mg/g, implicating a possibility of scaled-up production of HEA in industry by submerged fermentation. Based on the dramatic HEA production by ammonium sulfate as nitrogen resources between stationary and shaker fermentations, alanine, aspartate and glutamate as well as arginine metabolic pathway were related to the production of HEA by comparative transcriptome. Further investigation indicated that glutamic acid, which is an analog of Asp, showed an optimum production of HEA in comparison with other amino acids.

Biomimetic nanodrug blocks CD73 to inhibit adenosine and boosts antitumor immune response synergically with photothermal stimulation

Combination of tumor immunotherapy with photothermal therapy (PTT) is a feasible tactic to overcome the drawback of immunotherapy such as poor immune response. Via triggering the immunogenic cells death (ICD), PTT can stimulate the activity of immune cells, but meanwhile, the level of adenosine is elevated via the CD73-induced decomposition of ATP which is overexpressed accompanying with the PTT process, resulting in negative feedback to impair the immune stimulation. Herein, we developed a novel biomimetic photothermal nanodrug to specifically block CD73 for inhibition of adenosine production and more efficient priming of the suppressive immune microenvironments. The nanodrug, named as AptEM@CBA, is constructed by encapsulation of photothermal agent black phosphorus quantum dots (BPQDs) and selective CD73 inhibitor α, β-Methyleneadenosine 5′-diphosphate (AMPCP) in chitosan nanogels, which are further covered with aptamer AS1411 modified erythrocyte membrane (EM) for biomimetic camouflage. With AS1411 induced active targeting and EM induced long blood circulation time, the enrichment of the nanodrug tumor sites is promoted. The photothermal treatment promotes the maturation of dendritic cells. Meanwhile, the release of AMPCP suppress the adenosine generation via CD73 blockade, alleviating the impairment of adenosine to dendritic cells and suppressing regulatory T cells, synergically stimulate the activity of T cells. The combination of CD73 blockade with PTT, not only suppresses the growth of primary implanted tumors, but also boosts strong antitumor immunity to inhibit the growth of distal tumors, providing good potential for tumor photoimmunotherapy.

GRHL2 suppression of NT5E/CD73 in breast cancer cells modulates CD73-mediated adenosine production and T cell recruitment

Tumor tissues often contain high extracellular adenosine, promoting an immunosuppressed environment linked to mesenchymal transition and immune evasion. Here, we show that loss of the epithelial transcription factor, GRHL2, triggers NT5E/CD73 ecto-enzyme expression, augmenting the conversion of AMP to adenosine. GRHL2 binds an intronic NT5E sequence and is negatively correlated with NT5E/CD73 in breast cancer cell lines and patients. Remarkably, the increased adenosine levels triggered by GRHL2 depletion in MCF-7 breast cancer cells do not suppress but mildly increase CD8 Tcell recruitment, a response mimicked by a stable adenosine analog but prevented by CD73 inhibition. Indeed, NT5E expression shows a positive rather than negative association with CD8 Tcell infiltration in breast cancer patients. These findings reveal a GRHL2-regulated immune modulation mechanism in breast cancers and show that extracellular adenosine, besides its established role as a suppressor of Tcell-mediated cytotoxicity, is associated with enhanced Tcell recruitment.

Multifaceted role of inosine in complex diseases and human health.

Purines are chemical compounds integral to health and are crucial for the synthesis of nucleic acids. They are part of DNA and RNA and participate in various metabolic and signaling processes. They also function as neurotransmitters and serve as co-substrates for activating many metabolites. Inosine, a purine nucleoside, is a breakdown product of adenosine with similar properties and a much longer half-life (15 h vs ∼5 s) than adenosine. The purpose of this narrative review is to discuss the metabolic effects of inosine and highlight its beneficial properties and implication in complex diseases such as obesity, type 2 diabetes, cancers, cardiovascular diseases, and neurodegenerative diseases. A search was performed for purine- and inosine-related articles on the University of North Carolina (UNC) Health Sciences Library, PubMed, and Google Scholar sites. Inosine is involved in the regulation of RNA editing, metabolic enzyme activity, and signaling pathways. Animal and cell culture studies have shown inosine to be anti-inflammatory, immunomodulatory, and neuroprotective, and serving as a critical regulator of immune checkpoint inhibition therapeutic response in various tumor types. Recent studies have also implicated inosine in increasing energy expenditure, browning of adipose tissue, and improving leptin sensitivity. Human studies, however, have been limited to urate-elevating properties of inosine. These findings make inosine relevant to many complex diseases, and need to be translated to humans. Future studies should be conducted to investigate the mechanisms underlying the role of inosine in adiposity, inflammation, oxidative stress, and neuronal function.

Abstract LB131: CBO421, a novel drug Fc-conjugate, inhibits the enzymatic activity of CD73 and triggers CD73 internalization

Abstract Background: CD73 (5’-nucleotidase or NT5E) is a cell surface enzyme responsible for the rate limiting step in adenosine production; the conversion of adenosine monophosphate (AMP) into adenosine. Immunosupressive adenosine contributes to immune evasion in solid tumors by accumulating within the tumor microenvironment and thereby inhibiting immune effector cells (e.g., CD8+ T cells). CD73 expression levels correlate with a worse prognosis in specific cancer types, including breast cancer. Adenosine production via CD73 can be therapeutically inhibited by two independent mechanisms: (1) enzyme inhibition, and (2) receptor internalization and subsequent degradation. CBO421, a drug Fc-conjugate (DFC), comprised of a novel small molecule CD73 inhibitor stably conjugated to an immune-silent human IgG1 Fc, achieves both mechanisms, combining the strengths of small molecule inhibitors and CD73-targeting monoclonal antibodies (mAbs) with potential best-in-class efficacy. Methods: The activities of CBO421, a commercially sourced CD73 small molecule inhibitor, quemliclustat (AB680), and biosimilar anti-CD73 mAb, oleclumab (MEDI9447), were investigated. Enzymatic inhibition of soluble CD73 and membrane-bound CD73 on MDA-MB-231 cells was determined. Receptor-mediated internalization of CD73 was assessed using the human triple-negative breast cancer cell line (TNBC), MDA-MB-231, by flow cytometry. Cells were treated with CBO421 or oleclumab. CD73 internalization was measured as the decrease in CD73 surface receptor signal over a 6 h time-course using an anti-human CD73 detection antibody with non-overlapping epitopes to CBO421 or oleclumab. Results: CD73 inhibition by CBO421 of soluble CD73 or CD73 expressed on the surface of the MDA-MB-231 cell line was comparable or superior to AB680 or oleclumab. Receptor internalization activities of CBO421 and oleclumab were compared. CBO421 and oleclumab internalization percentages were determined at 1 h (52.3% vs 26.7%), 4 h (80.8% vs 38.0%), and 6 h (84.8% vs 42.9%) respectively, with CBO421 demonstrating superior maximal receptor internalization. The unconjugated hIgG1 Fc negative control did not exhibit any receptor-mediated internalization of CD73, as expected. IC50 values were also calculated for CBO421 and oleclumab at 1 h (0.20 nM vs 0.88 nM), 4 h (0.07 nM vs 0.27 nM), and 6 h (0.08 nM vs 0.14 nM), respectively. Conclusions: CBO421 demonstrated potent and complete CD73 enzyme inhibition and robust CD73 receptor internalization in CD73+ cancer cells that was superior to oleclumab, thereby further differentiating CBO421 from existing CD73-targeting therapeutics currently in clinical development. Based on these compelling results and additional emerging data, CBO421 is progressing as a potential best-in-class clinical development candidate for the treatment of solid cancers. Citation Format: Elizabeth Abelovski, Nicholas Dedeic, Simon Döhrmann, James Levin, Amanda Almaguer, Doug Zuill, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Jeffrey B. Locke, Jason N. Cole, Leslie W. Tari. CBO421, a novel drug Fc-conjugate, inhibits the enzymatic activity of CD73 and triggers CD73 internalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB131.

Abstract LB129: Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies

Abstract CD38 and CD47 are co-expressed in various hematologic malignancies including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), T-cell ALL and B-cell chronic lymphocytic leukemia (CLL). We present, herein, several CD38 antibody - CD47 ligand trap bispecific antibodies (BsAbs) in an ADCC-enhanced IgG1 form that harbor high CD38 binding affinity, low CD47 binding affinity, along with negligible affinity to red blood cells (RBCs) and no induction of RBCs agglutination. Using flow cytometry and Biolayer interferometry (BLI), we confirmed that our BsAbs can simultaneously bind to both CD38 and CD47, and that binding to either antigen first does not prevent binding to the other antigen subsequently. On CD47+/CD38low Jurkat (T leukemic) cells, our BsAbs reveal less blocking effect on the CD47/SIRPα interaction than a monospecific CD47 ligand trap molecule based on which our BsAbs were designed; conversely, on CD38/CD47 double-positive Reh (pro-B ALL) cells, our BsAbs reveal 200-500-fold greater blocking effect on the CD47/SIRPα interaction than the same monospecific CD47 ligand trap molecule, indicating that the inhibition of CD47/SIRPα by our BsAbs is depended on the engagement of CD38 on tumor cells. On CD47+/CD38+ Daudi (B NHL) lymphoma cells and L-1236 (B Hodgkin) lymphoma cells, four of our BsAbs inhibited CD38 enzymatic activity, which is responsible for immune-suppressive adenosine production in tumor microenvironment. Our BsAbs presented potent dose-dependent antibody-dependent cellular cytotoxicity (ADCC) against CD47+/CD38+ Raji (B NHL) and NCI-H929 (B plasmacytoma myeloma) cells, while revealing negligible ADCC activity against CD47+/CD38− cells, indicating low off-tumor toxicity. Likewise, our BsAbs also harbor potent antibody-dependent cellular phagocytosis (ADCP) activity against Raji NHL cells, varied degree of complement-dependent cytotoxicity (CDC) activity and direct apoptosis induction against Daudi NHL cells. In a mouse xenograft model harboring NCI-H929 multiple myeloma, our BsAbs produced more potent tumor growth inhibition, than monospecific anti-CD47 ligand trap, anti-CD38 (isatuximab) or combination of isatuximab and anti-CD47 ligand trap, at the same molecular dose per body weight. Notably, clone IMM5605-12C10 eradicated all tumors and achieved 100% complete response rate in a group of six mice. In summary, simultaneous targeting CD38 and CD47 using our CD38 antibody - CD47 ligand trap bispecific design in an ADCC-enhanced IgG1 form presents a novel multimodal approach for treating hematologic malignancies that are resistant to anti-CD38 antibodies. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Yanan Yang, Huiqin Guo, Dandan Liu, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB129.

Abstract LB348: Discovery and in vitro efficacy validation of anti-CD39 monoclonal antibody for improving the tumor microenvironment

Abstract In the tumor microenvironment (TME), the accumulation of adenosine inhibits anti-tumor immune responses, thereby reducing the effectiveness of immune-based cancer therapies. CD39 is involved in inducing excessive adenosine production in tumor cells and immune cells, causing an immune-suppressive environment. As a result, cancer cells are able to evade the immune system, and immune cells are impaired in their ability to kill cancer cells. Therefore, the discovery of outstanding antibodies targeting CD39 has great potential for cancer immunotherapy. To this end, we have discovered antibodies that effectively inhibit the hydrolysis of ATP to adenosine.We confirmed the binding affinity of the anti-CD39 mAb to the CD39 antigen. BLI measurements revealed that anti-CD39 mAb had binding kinetics of KD = 9.40 x 10-11 M, which is better than those of comparative antibodies, competitor 1 (2.39x10-10 M) and competitor 2 (2.27x10-9 M). The cell binding affinity of anti-CD39 mAb was evaluated using SK-MEL-28 cells, which endogenously express high levels of surface CD39. The results showed that the anti-CD39 mAb exhibited superior cell binding affinity compared to the two comparative antibodies. Furthermore, anti-CD39 mAb demonstrated greater efficacy in inhibiting ATP hydrolysis by membrane CD39 and soluble CD39 when compared to the comparative antibodies. In vitro functional assays using human PBMCs demonstrated that inhibiting CD39 enhances T cell proliferation. The anti-CD39 mAb showed superior efficacy in promoting proliferation in CD4+ cells compared to the competitor antibody. Similarly, in CD8+ cells, anti-CD39 mAb exhibited more efficacy compared to competitor 1 antibody. In conclusion, our data indicates that anti-CD39 mAb exhibits excellent characteristics, including superior antigen binding affinity, inhibitory efficacy of ATP hydrolysis and promotion of T cell proliferation. In the future, we will validate the efficacy and stability of this antibody in preclinical studies. The use of this antibody is expected to contribute to immune-based cancer therapy by inhibiting the generation of adenosine in the tumor microenvironment. Citation Format: Seonmi Yu, Wanki Park, Jaeho Song, Sooyoung Kim, Bumchan Park. Discovery and in vitro efficacy validation of anti-CD39 monoclonal antibody for improving the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB348.

First description of adenosine production by Gnomoniopsis smithogilvyi, causal agent of chestnut brown rot

Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) is the main causal agent of chestnut brown rot on sweet chestnut worldwide. The rotting of nuts leads to alterations in the organoleptic qualities and decreased fruit production, resulting in significant economic losses. In 2021, there was an important outbreak of chestnut rot in southern Galicia (Spanish northwest). The profile of secondary metabolites from G. smithogilvyi was studied, especially to determine its capability for producing mycotoxins, as happens with other rotting fungi, due to the possible consequences on the safety of chestnut consumption. Secondary metabolites produced by isolates of G. smithogilvyi growing in potato dextrose agar (PDA) medium were identified using liquid chromatography coupled with high-resolution mass spectrometry. Three metabolites with interesting pharmacological and phyto-toxicological properties were identified based on their exact mass and fragmentation patterns, namely adenosine, oxasetin, and phytosphingosine. The capacity of G. smithogilvyi to produce adenosine in PDA cultures was assessed, finding concentrations ranging from 176 to 834 µg/kg. Similarly, the production of mycotoxins was ruled out, indicating that the consumption of chestnuts with necrotic lesions does not pose a health risk to the consumer in terms of mycotoxins.

Abstract 2728: Discovery of a multispecific CD73/PD-1 targeting drug Fc-conjugate (DFC), which improves tumor reduction compared to PD-1 monotherapy in a humanized mouse model

Abstract Background: The approval of immune checkpoint therapies has revolutionized cancer treatment and established immuno-therapy as an additional treatment option. However, while anti-PD-(L)1 therapies have demonstrated durable responses, only a small subset of patients respond. One mechanism of tumor escape is via the release of immunosuppressive metabolites such as adenosine. CD73, an extracellular enzyme expressed on immune cells and some tumors, catalyzes the conversion of AMP to adenosine, the rate limiting step in adenosine production. As a strategy to improve response rates we developed a first in class CD73/PD-1 targeting DFC, comprised of a multivalent conjugate of a small molecule CD73 inhibitor stably linked to a proprietary human IgG1 Fc-fusion with a PD-1 inhibitor peptide. This multispecific DFC has the potential for differentiation and increased therapeutic potency compared to approved PD-(L)1 inhibitors. Methods: Binding of CD73/PD-1-001 to biotinylated hPD-1 was determined using ELISA and to human CD8+ T cells by flow cytometry. CD73 inhibition was measured in cell-free and cell-based assays. Efficacy was determined in a humanized mouse model optimized for PD-1 activity against a transgenic MC-38 colon cell line expressing hPD-L1 (Genoway, France). Tumor volumes were recorded and statistical analysis was conducted by t-test (Mann-Whitney) or two-way ANOVA. Results: In vitro, CD73/PD-1-001 demonstrated potent activity against both checkpoint targets. CD73/PD-1-001 bound to hPD-1 with an IC50 of < 1 nM and demonstrated functional inhibition of CD73 with an EC50 of <10 nM, which is on par with respective single agent molecules. Importantly, addition of the CD73 targeting moiety resulted in no significant loss in PD-1 binding relative to PD-1-001, a DFC containing only the PD-l inhibitor peptide. Efficacy was determined in a humanized mouse model against a transgenic MC-38 cell line. Both CD73/PD-1-001 and PD-1-001 DFCs were highly potent in this model with tumor growth inhibition (TGI) of >70% at dose concentrations of 3 mg/kg or greater (p≤0.01). However, at the lowest test concentration of 1 mg/kg, only CD73/PD-1-001 retained significant potency with a TGI of 56.0 % (p ≤ 0.05). Tumor reduction in the 1 mg/kg PD-1-001 group did not reach statistical significance (36.5%, p > 0.05). Conclusions: This work demonstrates that a multispecific CD73/PD-1 targeting DFC, is a potent inhibitor of tumor growth in a humanized mouse model. Furthermore, CD73/PD-1-001 was more potent than PD-1 monotherapy, demonstrating the therapeutic benefit of targeting two important checkpoints with a single agent molecule. Collectively, preclinical testing of a CD73/PD-1 DFC has validated the benefits of targeting the PD-(L)1 axis in combination with adenosine suppression, resulting in improved anti-tumor effects. Citation Format: James Levin, Simon Döhrmann, Nicholas Dedeic, Amanda Almaguer, Doug Zuill, Elizabeth Abelovski, Jason N. Cole, Jeffrey B. Locke, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Leslie W. Tari. Discovery of a multispecific CD73/PD-1 targeting drug Fc-conjugate (DFC), which improves tumor reduction compared to PD-1 monotherapy in a humanized mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2728.

Abstract 2668: CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway

Abstract Background CD73 (5’-nucleotidase or NT5E) is a cell surface enzyme responsible for the rate limiting step in adenosine production; the conversion of adenosine monophosphate (AMP) into adenosine. Adenosine contributes to immune evasion by solid tumors by concentrating immunosuppressive adenosine in the tumor microenvironment. CBO421, a Drug Fc-Conjugate (DFC), comprises a novel small molecule CD73 inhibitor stably conjugated to an immune-silent human IgG1 Fc, combining the strengths of small molecule inhibitors and CD73-targeting monoclonal antibodies (mAbs) for potential best-in-class efficacy. Methods CBO421’s binding affinity to human CD73 and Fc receptors was assessed by surface plasmon resonance. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using a commercial kit. Immunophenotyping and binding to cancer cells was measured by flow cytometry. Inhibition of soluble and membrane-bound CD73 on human PBMCs and mouse cancer cells was determined by measuring free phosphate levels. Functional activity was measured using PBMC rescue assays. In vivo efficacy of CBO421 monotherapy was evaluated in a syngeneic mouse model. Results CBO421 exhibited a binding affinity to human CD73 (KD = 0.8 nM) that was comparable to or greater to the affinity observed with small molecule inhibitors (AB680, OP-5244) and CD73-targeting mAbs (oleclumab, mupadolimab biosimilars). As expected, human FcRn binding of CBO421 was comparable to the unconjugated Fc and the full-length wild-type IgG1 control mAb. CBO421 binding to human Fc gamma receptors and ADCC activity was significantly reduced or abolished compared to the control mAb, such that it poses a minimal risk for undesired autoimmune activity. CBO421 potently inhibited soluble CD73 and CD73 expressed on human MDA-MB-231 and mouse EMT6 cancer cells. Immunophenotyping revealed that CD73 expression was primarily observed on CD8+ T and B cells from human PBMCs. In functional PBMC rescue assays, CBO421 demonstrated potent reactivation of AMP- or ATP-suppressed CD4+ or CD8+ T cells at levels comparable to small molecule inhibitors and greater than anti-CD73 mAb comparators. CBO421 monotherapy demonstrated a dose-dependent reduction in tumor volume in a syngeneic mouse model using MC38, a colorectal cancer cell line. Conclusions CBO421 exhibited strong binding and potent inhibition of both soluble and membrane-bound CD73, differentiating it from mAb CD73 inhibitors. The proprietary immune-silent Fc of CBO421 did not affect FcRn binding but substantially reduced Fc gamma receptor binding, leading to the elimination of ADCC activity to prevent undesired immune cell depletion in host tissues. CBO421 demonstrated high potency in functional cell-based assays and robust antitumor efficacy in a syngeneic mouse model. Currently, CBO421 is being advanced as a clinical development candidate for the treatment of solid cancers. Citation Format: Amanda Almaguer, Doug Zuill, Simon Döhrmann, James Levin, Nicholas Dedeic, Elizabeth Abelovski, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Jeffrey B. Locke, Jason N. Cole, Leslie W. Tari. CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2668.