NMR-Based Assay for the Ex Vivo Determination of Soluble CD73 Activity in Serum

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.