Conversion Of Adenosine Monophosphate Research Articles

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 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.

Abstract 6115: Development of functionally differentiating anti-CD73 antibodies for cancer therapy

Abstract Background: Although the discovery and development of first-generation of immune checkpoint inhibitors (towards PD1 and CTLA-4) was a major milestone for cancer therapy, current clinical response rates are still considered very limited. Combination treatments are predicted to improve on these current immunotherapies including the targeting of the adenosine pathway (CD39, CD73 or A2AR), which has shown a lot of promise in preclinical and early clinical studies. CD73 is an ecto-5′-nucleotidase which transforms adenosine monophosphate (AMP) to adenosine. Adenosine is a soluble immunosuppressive metabolite that can suppress natural killer cells and cytotoxic CD8+ T cells. Blockade of CD73-mediated conversion of AMP to adenosine may therefore recover anti-tumor immunity through preventing the enrichment of adenosine in the tumor microenvironment. Method: In this campaign, two humanized and Fc-silenced IgG antibodies were generated named 7002-01 and 7002-04. The target binding epitopes of these candidates were revealed by binning experiments through bio-layer interferometry. Cell binding experiments were tested on human and cynomolgus CD73 overexpression CHO cell lines by flow cytometry. Cellular CD73 enzyme inhibition experiments were tested using A375, MDA-MB-231, H2030 and BT549 tumor cell lines via the CellTiter-Glo method. Soluble CD73 enzymatic tests were carried out on patient sera or recombinant CD73 protein using a similar method. T cell proliferation assays were performed using PBMC. In vivo efficacy studies were tested in B-NDG B2M-KO mice that were injected subcutaneously with A375 tumor cells and human PBMC. Results: Two candidates, 7002-01 and 7002-04, were selected based on their functional activity, that recognize different non-overlapping binding epitopes on CD73. Both candidates selectively bind to and can inhibit the activities of both membrane-bound and soluble human CD73 to high levels and seem to maintain inhibition at high dose-ranges without a hook effect. Both candidates can potently rescue adenosine-mediated T cell regulation. Additionally, 7002-01 and 7002-04 have combination synergy or additive effects for CD73 inhibition on soluble CD73, tumor cell lines that express CD73, and PBMC. 7002-01 and 7002-04 have single-agent anti-tumor efficacy and combination synergy with anti-PD1 antibodies in mice. 7002-01 has a typical antibody-like PK profile when rhesus monkeys were administered with a single intravenous dose at 25 or 50 mg/kg. No drug-related toxicities have been observed in GLP toxicity studies with dosages at 50, 250, and 500 mg/kg (QW, 4 weeks). Conclusion: Highly differentiating anti-CD73 antibodies were discovered that show maximal inhibition of both membranous and soluble CD73 without hook effects at high concentrations. 7002-01 was chosen as the lead molecule for its better overall activity profile and should be entering clinical trials by early 2022. Citation Format: Zhenqing Zhang, Yunli Jia, Xiaoniu Miao, Weifeng Huang, Chao Wang, Zhijun Yuan, Wenchao Jiang, Zhiyuan Li, Liandi Chen, Andy Tsun. Development of functionally differentiating anti-CD73 antibodies for cancer therapy [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 6115.

Appraisal of novel azomethine-thioxoimidazolidinone conjugates as ecto-5'-nucleotidase inhibitors: synthesis and molecular docking studies.

Purinergic signaling is regulated by a group of extracellular enzymes called ectonucleotidases. One of its members i.e., ecto-5′-nucleotidase (h-e5′NT) is involved in the final step of the enzymatic hydrolysis cascade that is the conversion of adenosine monophosphate (AMP) to adenosine and therefore, involves the regulation of adenosine level in extracellular space. The overexpression of h-e5′NT has been observed in various pathological conditions such as hypoxia, inflammation and cancers, and led to various complications. Hence, the identification of a potent as well as selective inhibitor of h-e5′NT is of greater importance in therapeutic treatment of various diseases. Azomethine-thioxoimidazolidinone derivatives were studied for their inhibition potential against e5′NT enzyme along with cytotoxic potential against cancer cell lines possessing overexpression of e5′NT enzyme. The derivative (E)-3-((4-((3-methoxybenzyl)oxy)benzylidene)amino)-2-thioxoimidazolidin-4-one (4g) displayed selective and significant inhibition towards h-e5′NT with an IC50 value of 0.23 ± 0.08 μM. While two other derivatives i.e., (E)-3-(((5-bromothiophen-2-yl)methylene)amino)-2-thioxoimidazolidin-4-one (4b) and 2-thioxo-3-((3,4,5-trimethoxybenzylidene)amino)imidazolidin-4-one (4e), exhibited non-selective potent inhibitory behavior against both human and rat enzymes. Moreover, these derivatives (4b, 4e and 4g) were further investigated for their effect on the expression of h-e5′NT using quantitative real time polymerase chain reaction. Additionally, molecular docking and DFT studies were also performed to determine the putative binding mode of potent inhibitors within the enzyme active site. HOMO, LUMO, ΔE, and molecular electrostatic potential maps were computed by DFT and the charge transfer regions within the molecules were identified to find out the regions for electrophilic and nucleophilic attack.

Abstract 1877: IOA-237 - A potent anti-CD73 mAb demonstrates monotherapeutic and combination efficacy in solid tumor models

Abstract CD73, an ecto-5'-nucleotidase, is frequently overexpressed in the tumor microenvironment and can be found on tumor, stromal cells and immune cells. Its function is to catalyze the conversion of adenosine monophosphate (AMP) to adenosine and phosphate, both at the cell surface and as a soluble factor. CD73 has gained interest as a therapeutic target in cancer due to the immunosuppressive role of adenosine in the tumor microenvironment. IOA-237 is a fully human, phage derived monoclonal antibody fully cross-reactive to mouse and human and with high potency against soluble and cell surface CD73. This novel antibody demonstrates in vitro and in vivo characteristics that suggest a best-in-class potential. We demonstrate that IOA-237 is able to significantly reduce tumor outgrowth in multiple mouse models of solid tumors. In the low CD73 expressing CT26 mouse colorectal cancer model, monotherapeutic application of IOA-237 showed tumor outgrowth reduction compared with isotype control. Best-in-class potential was confirmed with head-to-head comparison against a clinical stage anti-CD73 mAb, MEDI9447. Furthermore, inhibition of CD73 by IOA-237 was combined with inhibitors of novel immuno-oncology targets to study complementary modes of action in mouse models of solid tumors. These data indicate the potential of IOA-237 in monotherapeutic and combination therapeutic approaches and warrants further development. Additional in vitro and in vivo efficacy studies are ongoing and non-clinical safety studies are being planned. Citation Format: Marcel A. Deken, Alan M. Carruthers, Karolina Niewola-Staszkowska, Gráinne Gernon, Hilary Sandig, Pritom Shah, Michael M. Lahn, Zoë Johnson. IOA-237 - A potent anti-CD73 mAb demonstrates monotherapeutic and combination efficacy in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1877.

Abstract 5660: Characterisation of novel CD73 antibodies as a therapeutic method of adenosine regulation

Abstract CD73 is a membrane-bound nucleotidase receptor which is frequently overexpressed in the tumour microenvironment and can be found on both tumour and immune cell types. Its function is to catalyse the conversion of adenosine monophosphate (AMP) to adenosine and phosphate and it has been proposed as a therapeutic target in cancer due to adenosines role in tumour immune suppression. Using multiple approaches, a series of novel CD73 antibodies have been characterised for their therapeutic potential and benchmarked against agents currently undergoing clinical evaluation Inhibition of CD73 activity was evaluated using an Amplex Red-based coupled adenosine assay against both human and mouse CD73, and kinetics of antibody binding were determined using BioLayer Interferometry. Cellular assays were then utilised to further evaluate the antibodies in vitro. The ability of the CD73 antibodies to internalise was evaluated using two different methods, a Fab-ZAP killing assay and the IncuCyteTM FabFluor internalisation assay. The antibodies have also undergone functional studies, investigating the ability of the CD73 antibodies to disrupt the production of adenosine in tumour cells. We demonstrate that the novel CD73 antibodies inhibit CD73 function by two different mechanisms; direct inhibition of enzyme activity and modulation of cell surface expression, both of a which have therapeutic potential to disrupt CD73-mediated adenosine production and therefore reduce antitumor immune responses. Ongoing studies are investigating the in vitro and in vivo efficacy of the clone selected for further development as a monotherapy and in combination with a novel small molecule inhibitor in murine models of cancer. Citation Format: Grainne Gernon, Suzanne Grooby, Louise Tonkin, Anja-Christina Bitterwolf, Lorna Stewart, Marcel Deken, Pritom Shah, Katherine Ewings, Zoe Johnson. Characterisation of novel CD73 antibodies as a therapeutic method of adenosine regulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5660.

Extracellular AMP Suppresses Endotoxemia-Induced Inflammation by Alleviating Neutrophil Activation

Intracellular adenosine monophosphate (AMP) is indispensable for cellular metabolic processes, and it is interconverted to ADP and/or ATP or activates AMP-activated protein kinase (AMPK). However, the specific biological function of extracellular AMP has not been identified. We evaluated the effect of extracellular AMP using in vivo and in vitro models of endotoxemia. We found that AMP inhibited inflammation and neutrophil activation in lipopolysaccharide (LPS)-induced endotoxemic mice. The effects of extracellular AMP were abolished by an adenosine 1 receptor (A1R) antagonist but were not influenced by inhibiting the conversion of AMP to adenosine (ADO), indicating that AMP inhibited inflammation by directly activating A1R. In addition, in vitro experiments using LPS-stimulated mouse neutrophils showed that AMP inhibited LPS-induced reactive oxygen species (ROS) production, degranulation, and cytokine production, while the effects were reversed by an A1R antagonist. Further research showed that AMP regulated LPS-stimulated neutrophil functions by inhibiting the p38 MAPK pathway. These findings were also confirmed in primary neutrophils derived from healthy human blood. Moreover, we collected serum samples from septic patients. We found that AMP levels were increased compared with those of healthy volunteers and that AMP levels were negatively correlated with disease severity. Together, these data provide evidence that extracellular AMP acts on A1R to suppress endotoxemia-induced inflammation by inhibiting neutrophil overactivation and that the p38 MAPK signaling pathway is involved.

Abstract B048: A Promising Pathway in Immuno-oncology: CD73-Adenosine axis highlighted by quantitative mass spectrometry imaging

Abstract Introduction: The CD73-Adenosine (CD73-ADO) axis constitutes one of the most promising pathways in immuno-oncology. CD73 catalyzes the conversion of adenosine monophosphate (AMP) to ADO. CD73 is believed to play a role in mediating the inhibitory function of regulatory B and T cells. It is widely expressed and up-regulated in many cancerous tissues. The presence of extracellular ADO within tumor microenvironment has been described as an immunosuppressive halo surrounding the tumor, permeating the tumor microenvironment and preventing antitumor immunity. In this study, the endogenous level of AMP and ADO were analyzed by quantitative mass spectrometry imaging (QMSI) and LC-MS/MS in CT26 tumor mouse model. Clones with different CD73 expression levels were selected (low, medium & high), which mimed different CD73-inhibitors efficacies. Then, ADO metabolic pathway was targeted in order to highlight the tumor microenvironment response linked to a mimic immunotherapy effect. Methods: Undifferentiated colon carcinoma CT26 cell line was implanted into C57BL/6 mice. Then, tumors were harvested and frozen at -80°C until use. 1,5-diaminonaphthalene (1,5-DAN) and 2,5-dihydroxybenzoic acid (2,5-DHB) matrices mixed to internal standards were sprayed onto tumor sections of 10 µm thickness with the automatic TM Sprayer (HTX Technologies, LLC) prior analysis. Data acquisition was performed using 7T MALDI-FTICR (SolariX XR, Bruker Daltonics) and analyses were set at 120µm spatial resolution in full scan positive and negative modes. Acquired data were treated with MultimagingTM software (ImaBiotech). All quantitative results were confirmed by LC-MS/MS analysis. Histological and immunohistochemistry staining anti-CD73 were applied on adjacent section. Results: A validation of CT26 mouse model occurred since an increase of ADO/AMP ratio of 77% was relative to the high CD73 expression. In other words, CT26 mouse models with high CD73 expression showed higher ADO/AMP ratio (2.1) than CT26 medium (1.7) and low (1.6) clones. Then, as an immunosuppressive halo, the impact of ADO expression on tumor microenvironment was also characterized using MSI analysis. A large number of metabolites that are implicated in this pathway were highlighted (ADP, IMP, GMP, Guanosine, Inosine, Hypoxanthine, Xanthine, Uric acid…) and differences were also observed. Conclusion: The combination of all these data extended our understanding on the relative contribution of ADO signaling in suppressing antitumor immunity. Because the development of immunotherapies such as CD73 inhibitor requires a deep understanding of the interplay between the immune system and cancer cells in the tumor microenvironment, these immune/tumor metabolites and nutrients can now be followed by QMSI as biomarkers of response to treatment towards enhancing immunotherapies efficacy. Citation Format: Lauranne Poncelet, Rima Ait-Belkacem, Bruno Gomes, Jonathan Stauber. A Promising Pathway in Immuno-oncology: CD73-Adenosine axis highlighted by quantitative mass spectrometry imaging [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B048. doi:10.1158/1535-7163.TARG-19-B048

CD73 Promotes Age-Dependent Accretion of Atherosclerosis.

CD73 is an ectonucleotidase which catalyzes the conversion of AMP (adenosine monophosphate) to adenosine. Adenosine has been shown to be anti-inflammatory and vasorelaxant. The impact of ectonucleotidases on age-dependent atherosclerosis remains unclear. Our aim was to investigate the role of CD73 in age-dependent accumulation of atherosclerosis. Approach and results: Mice doubly deficient in CD73 and ApoE (apolipoprotein E; (cd73-/-/apoE-/-) were generated, and the extent of aortic atherosclerotic plaque was compared with apoE-/- controls at 12, 20, 32, and 52 weeks. By 12 weeks of age, cd73-/-/apoE-/- mice exhibited a significant increase in plaque (1.4±0.5% of the total vessel surface versus 0.4±0.1% in apoE-/- controls, P<0.005). By 20 weeks of age, this difference disappeared (2.9±0.4% versus 3.3±0.7%). A significant reversal in phenotype emerged at 32 weeks (9.8±1.2% versus 18.3±1.4%; P<0.0001) and persisted at the 52 week timepoint (22.4±2.1% versus 37.0±2.1%; P<0.0001). The inflammatory response to aging was found to be comparable between cd73-/-/apoE-/- mice and apoE-/- controls. A reduction in lipolysis in CD73 competent mice was observed, even with similar plasma lipid levels (cd73-/-/apoE-/- versus apoE-/- at 12 weeks [16.2±0.7 versus 9.5±1.4 nmol glycerol/well], 32 weeks [24.1±1.5 versus 7.4±0.4 nmol/well], and 52 weeks [13.8±0.62 versus 12.7±2.0 nmol/well], P<0.001). At early time points, CD73 exerts a subtle antiatherosclerotic influence, but with age, the pattern reverses, and the presence of CD73 promoted suppression of lipid catabolism.

Abstract 5485: A promising pathway in immuno-oncology: CD73-adenosine axis highlighted by quantitative mass spectrometry imaging

Abstract Introduction The CD73-Adenosine (CD73-ADO) axis constitutes one of the most promising pathways in immuno-oncology. CD73 catalyzes the conversion of adenosine monophosphate (AMP) to ADO. CD73 is believed to play a role in mediating the inhibitory function of regulatory B and T cells. It is widely expressed and up-regulated in many cancerous tissues. The presence of extracellular ADO within tumor microenvironment has been described as an immunosuppressive halo surrounding the tumor, permeating the tumor microenvironment and preventing antitumor immunity. In this study, the endogenous level of AMP and ADO were analyzed by quantitative mass spectrometry imaging (QMSI) and LC-MS/MS in CT26 tumor mouse model. Clones with different CD73 expression levels were selected (low, medium &amp; high), which mimed different CD73-inhibitors efficacies. Then, ADO metabolic pathway was targeted in order to highlight the tumor microenvironment response linked to a mimic immunotherapy effect. Methods Undifferentiated colon carcinoma CT26 cell line was implanted into C57BL/6 mice. Then, tumors were harvested and frozen at -80°C until use. 1,5-diaminonaphthalene (1,5-DAN) and 2,5-dihydroxybenzoic acid (2,5-DHB) matrices mixed to internal standards were sprayed onto tumor sections of 10 µm thickness with the automatic TM Sprayer (HTX Technologies, LLC) prior analysis. Data acquisition was performed using 7T MALDI-FTICR (SolariX XR, Bruker Daltonics) and analyses were set at 120µm spatial resolution in full scan positive and negative modes. Acquired data were treated with MultimagingTM software (ImaBiotech). All quantitative results were confirmed by LC-MS/MS analysis. Histological and immunohistochemistry staining anti-CD73 were applied on adjacent section. Results A validation of CT26 mouse model occurred since an increase of ADO/AMP ratio of 77% was relative to the high CD73 expression. In other words, CT26 mouse models with high CD73 expression showed higher ADO/AMP ratio (2.1) than CT26 medium (1.7) and low (1.6) clones. Then, as an immunosuppressive halo, the impact of ADO expression on tumor microenvironment was also characterized using MSI analysis. A large number of metabolites that are implicated in this pathway were highlighted (ADP, IMP, GMP, Guanosine, Inosine, Hypoxanthine, Xanthine, Uric acid⋯) and differences were also observed. Conclusion The combination of all these data extended our understanding on the relative contribution of ADO signaling in suppressing antitumor immunity. Because the development of immunotherapies such as CD73 inhibitor requires a deep understanding of the interplay between the immune system and cancer cells in the tumor microenvironment, these immune/tumor metabolites and nutrients can now be followed by QMSI as biomarkers of response to treatment towards enhancing immunotherapies efficacy. Citation Format: Lauranne Poncelet, Rima Ait-Belkacem, Bruno Gomes, Stefan Linehan, Jonathan Stauber. A promising pathway in immuno-oncology: CD73-adenosine axis highlighted by quantitative mass spectrometry imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5485.

Abstract 146: Adenosine Production by Biomaterial-supported Mesenchymal Stromal Cells Reduces the Innate Inflammatory Response in Myocardial Ischemia/Reperfusion Injury

Rationale: During myocardial ischemia/reperfusion (MI/R) injury there is excessive release of immunogenic metabolites that activate cells of the innate immune system. These metabolites include adenine nucleotides such as adenosine triphosphate (ATP) and adenosine monophosphate (AMP) which up-regulate chemotaxis, migration, and effector function of early infiltrating inflammatory cells. These cells subsequently drive further tissue devitalization. Mesenchymal stromal cells (MSCs) are a potential treatment modality for MI/R due to their powerful anti-inflammatory capabilities; however, the manner in which they regulate the acute inflammatory milieu requires further elucidation. Surface membrane ecto-5’-nucleotidase CD73 may play a critical role in inflammatory regulation by converting pro-inflammatory AMP to anti-inflammatory adenosine (ADO). We hypothesized that the MSC-mediated conversion of AMP into ADO reduces inflammation in early MI/R, favoring a micro-environment that attenuates excessive innate immune cell activation and facilitates earlier cardiac recovery. Objective: To determine the contribution of MSC-mediated production of ADO in regulating the innate inflammatory response following MI/R. Methods and Results: Adult rats were subjected to 30 minutes of MI/R injury. MSCs were encapsulated within a hydrogel vehicle and implanted onto the myocardium. A subset of MSCs were treated with the CD73 inhibitor, α,β-methylene adenosine diphosphate, prior to implantation. Using liquid chromatography/mass spectrometry, we found MSCs increase myocardial ADO availability following injury via CD73 activity. We also demonstrated that MSCs reduce innate immune cell infiltration as measured by flow cytometry and hydrogen peroxide formation as measured by Amplex Red assay. These effects were also dependent on MSC-mediated CD73 activity. Furthermore, through echocardiography we found CD73 activity on MSCs was critical to optimal protection of cardiac function following MI/R injury. Conclusion: MSC-mediated conversion of AMP to ADO by CD73 ecto-5’-nucleotidase activity exerts a powerful anti-inflammatory effect following MI/R injury.

Metabolic Responses to Weight Lifting

Metabolic Responses to Weight Lifting

Soluble Ecto-5′-nucleotidase (5′-NT), Alkaline Phosphatase, and Adenosine Deaminase (ADA1) Activities in Neonatal Blood Favor Elevated Extracellular Adenosine

Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5'-nucleotidase (5'-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5'-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5'-NT enhanced Toll-like receptor-mediated TNF-α production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.

Abstract 4328: Phenotypic selection for identification of functional antibodies and development of high throughput screening assays.

Abstract Discovery of therapeutic antibodies requires the continued identification of tractable targets, and we have employed a phenotypic screening strategy to this end. In this study, antibodies from phage display libraries were screened for their abilities to inhibit MDA-MB-231 (‘triple negative’) breast cancer cells. The screening cascade included tests for specific cell binding, antibody internalization, and cytotoxicity as an antibody-drug conjugate. Targets of these antibodies then were identified using immunoprecipitation and mass spectrometry, and confirmed by showing diminished antibody binding after siRNA-mediated knockdown of the putative target gene. One target was identified as NT5E (also known as CD73), a 5’-ectonucleotidase. NT5E catalyzes the conversion of adenosine monophosphate to adenosine, and its function has been linked with immunity, angiogenesis, and cancer. To screen for additional inhibitors of this target, a novel, high-throughput ectonucleotidase assay was developed. NT5E activity is typically assayed either by high performance liquid chromatography or by quantification of free phosphate using malachite green, and neither of these methods is suitable for high-throughput screening. One inhibitory antibody was found to significantly slow growth of MDA-MB-231 xenograft tumors in mice. In summary, our phenotypic screening approach provides a mechanism for rapidly discovering and evaluating new antibody targets and is being used to accelerate the discovery of new cancer drugs. Citation Format: Carl Hay, Steven Rust, Erin Sult, Lori Clarke, Kim Rosenthal, Sandrine Guillard, David Lowne, Matt Flynn, Lutz Jermutus, Ralph Minter, Robert Hollingsworth, Kris Sachsenmeier. Phenotypic selection for identification of functional antibodies and development of high throughput screening assays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4328. doi:10.1158/1538-7445.AM2013-4328

Cardiac 5’-nucleotidase activity increases with age and inversely relates to recovery from ischemia

The metabolic basis for the enhanced tolerance of immature hearts to ischemia remains to be elucidated. Loss of high-energy phosphate nucleotides occurs during ischemia/reperfusion in mature (adult) hearts through the breakdown of adenosine triphosphate, diphosphate, and monophosphate (nondiffusible) to adenosine (freely diffusible). However, previous work has shown that after ischemia nondiffusible nucleotides are better retained by immature (neonatal) hearts than by mature hearts. The enzyme responsible for the conversion of adenosine monophosphate to adenosine is 5'-nucleotidase. We therefore hypothesized lower activity of this enzyme in neonatal than in adult myocardium. The purposes of this study were (1) to document 5'-nucleotidase activities in neonatal and adult rabbit myocardium and (2) to correlate differences of 5'-nucleotidase activity with functional recovery from ischemia. Neonatal (5- to 10-day-old) and adult (4- to 6-month-old) rabbit hearts were isolated and perfused (retrograde Langendorff). A left ventricular balloon measured functional parameters. Hearts were subjected to 20 minutes of global 37 degrees C ischemia and 10 minutes of reperfusion followed by freeze clamping. Tissue homogenates were assayed for 5'-nucleotidase by the linked formation of nicotinamide-adenine dinucleotide at 340 nm (Arkesteijn method). Postischemic recovery of developed pressure was 86% +/- 3% in neonates (n = 5) versus 38% +/- 3% in adults (n = 8) (mean +/- standard deviation) (p less than 0.01). 5'-Nucleotidase activity was 4400 +/- 1208 nmol/min/gm in neonates (n = 5) versus 13,938 +/- 830 nmol/min/gm in adults (n = 8) (mean +/- standard deviation) (p less than 0.01). We conclude that (1) 5'-nucleotidase activity is 68% lower in neonatal than in adult myocardium and (2) functional recovery after ischemia inversely relates to 5'-nucleotidase activity.

Studies on the receptor mediating cyclic AMP‐independent enhancement by adenosine of IgE‐dependent mediator release from rat mast cells

Adenosine produced a concentration-related enhancement of antigen-induced 5-hydroxytryptamine (5-HT) release from rat serosal mast cells. This potentiation was maximal following the simultaneous addition of adenosine with antigen. Enhancement of 5-HT release was accompanied by potentiation of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) response to challenge. The cyclic AMP response, which was antagonized by 8-phenyltheophylline, was characterized as an A2-purinoceptor-mediated effect by the use of 5'-N-ethylcarboxamideadenosine (NECA) and L-N6-phenylisopropyladenosine (L-PIA). Enhancement of 5-HT release, conversely, was not blocked by 8-phenyltheophylline suggesting it to be mediated by a cyclic AMP-independent mechanism. The effect of adenosine on 5-HT release was not reduced by the inhibition of the facilitated uptake of adenosine with dipyridamole, hexobendine or p-nitrobenzylthioguanosine, therefore, suggesting it to be mediated by a cell surface receptor. The receptor mediating enhancement of 5-HT does not appear to belong to the P2-purinoceptor subtype as adenosine was more potent than both adenosine monophosphate (AMP) and adenosine diphosphate (ADP) and alpha, beta-methylene ATP was inactive. Furthermore, the effects of AMP were blocked by alpha, beta-methylene ADP, which inhibits the conversion of AMP to adenosine. Adenosine, NECA, L- and D-PIA were all of equal potency in enhancing 5-HT release. Inosine and 3-deazaadenosine were also active. The rank order of potency of these adenosine analogues is not consistent with an effect at A1- or A2-purinoceptors. There appear to be two adenosine receptors on rat mast cells, an A2-purinoceptor which stimulates adenylate cyclase and a separate purinoceptor, stimulation of which produces enhancement of mediator release by an unknown mechanism. The effects mediated by these receptors appear to be independent of each other.

Metabolic intervention to affect myocardial recovery following ischemia.

Myocardial recovery during reperfusion following ischemia is critical to patient survival in a broad spectrum of clinical settings. Myocardial functional recovery following ischemia correlates well with recovery of myocardial adenosine triphosphate (ATP). Adenosine triphosphate recovery is uniformly incomplete during reperfusion following moderate ischemic injury and is therefore subject to manipulation by metabolic intervention. By definition ATP recovery is limited either by (1) energy availability and application in the phosphorylation of adenosine monophosphate (AMP) to ATP or (2) availability of AMP for this conversion. Experimental data suggest that substrate energy and the mechanisms required for its application in the creation of high energy phosphate bonds (AMP conversion to ATP) are more than adequate during reperfusion following moderate ischemic injury. Adenosine monophosphate availability, however, is inadequate following ischemia due to loss of diffusable adenine nucleotide purine metabolites. These purine precursors are necessary to fuel adenine nucleotide salvage pathways. Metabolic interventions that enhance AMP recovery rather than those that improve substrate energy availability during reperfusion are therefore recommended. The mechanisms of various metabolic interventions are discussed in this framework along with the rationale for or against their clinical application.

Exercising muscle does not produce hypoxanthine in adenylate deaminase deficiency.

The failure of forearm exercise to increase plasma hypoxanthine in subjects with adenylate deaminase deficiency confirms this enzyme's role in hypoxanthine production by normal forearm exercise. The conversion of adenosine monophosphate (AMP) to hypoxanthine may reflect an alternative method of adenosine triphosphate (ATP) regeneration in working muscle.

ENZYME DEFECT IN PRIMARY GOUT

The rate-limiting step in the degradation of adenine nucleotides in the liver is the conversion of adenosine monophosphate (A.M.P.) to inosine monophosphate by A.M.P. deaminase, which is normally 95% inhibited. When the inhibition is released, uric acid is formed in large excess, and the biosynthesis of purines is increased. We therefore propose that congenital hyperuricæmia is caused by the presence of an abnormal A.M.P. deaminase, which is less sensitive to its physiological inhibitors. Verification of the hypothesis depends upon the availability of liver tissue from patients with congenital hyperuricæmia for kinetic analysis of A.M.P. deaminase. A call for collaboration is addressed to the medical community.