Extracellular adenosine, produced through the activity of ecto-5′-nucleotidase CD73, elicits potent immunosuppressive effects, and its upregulation in tumor cells as well as in stromal and immune cell subsets within the tumor microenvironment is hypothesized to represent an important resistance mechanism to current cancer immunotherapies. Soluble CD73 (sCD73) enzymatic activity measured in patient serum or plasma at a baseline is reported to have prognostic as well as predictive relevance, with higher sCD73 activity associating with poor overall and progression-free survival in melanoma patients undergoing anti-PD1 monoclonal antibody treatment. Here, we report a novel NMR-based method that measures the ex-vivo kinetics of sCD73 activity with high specificity and reproducibility and is suitable for future high-throughput implementation. Unlike the existing assays, this method has the advantage of directly and simultaneously measuring the concentration of both the CD73 substrate and product with minimal sample manipulation or special reagents. We establish the utility of the assay for measuring the activity of sCD73 in human serum and show a strong linear correlation between sCD73 protein levels and enzyme activity. Together with our finding that sCD73 appears to be the predominant activity for the generation of adenosine in human blood, our results demonstrate a link between activity and protein levels that will inform future clinical application.

中文翻译：

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基于核磁共振的体外测定血清中可溶性CD73活性的方法

通过ecto-5'-核苷酸酶CD73的活性产生的细胞外腺苷引起有效的免疫抑制作用，并假设其在肿瘤细胞以及肿瘤微环境中的基质细胞和免疫细胞亚群中的上调代表了对电流的重要抵抗机制癌症免疫疗法。据报道在基线时在患者血清或血浆中测得的可溶性CD73（sCD73）酶活性具有预后和预测相关性，在接受抗PD1单克隆抗体治疗的黑素瘤患者中，较高的sCD73活性与较差的总体生存率和无进展生存率相关。在这里，我们报告了一种新颖的基于NMR的方法，该方法以高特异性和可再现性测量sCD73活性的离体动力学，适用于未来的高通量实施。与现有的检测方法不同，此方法的优点是可以用最少的样品操作或特殊试剂直接并同时测量CD73底物和产物的浓度。我们建立了测定人类血清中sCD73活性的测定工具，并显示了sCD73蛋白水平和酶活性之间的强线性相关性。连同我们的发现sCD73似乎是人血中腺苷生成的主要活性一起，我们的结果证明了活性与蛋白质水平之间的联系将为将来的临床应用提供参考。我们建立了测定人类血清中sCD73活性的测定工具，并显示了sCD73蛋白水平和酶活性之间的强线性相关性。连同我们的发现sCD73似乎是人血中腺苷生成的主要活性一起，我们的结果证明了活性与蛋白质水平之间的联系将为将来的临床应用提供参考。我们建立了测定人类血清中sCD73活性的测定工具，并显示了sCD73蛋白水平和酶活性之间的强线性相关性。连同我们的发现sCD73似乎是人血中腺苷生成的主要活性一起，我们的结果证明了活性与蛋白质水平之间的联系将为将来的临床应用提供参考。