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NMR-Based Assay for the Ex Vivo Determination of Soluble CD73 Activity in Serum
Yan He1, Yue Zhao1, David M Nelson1
1Research and Early Development, Bristol Myers Squibb Company, P.O. Box 4000, Princeton, New Jersey 08540, United States.
Insights
A new NMR-based method accurately measures soluble CD73 (sCD73) activity in patient serum. This advancement links sCD73 enzyme activity to protein levels, aiding cancer immunotherapy research.
Area of Science:
- Biochemistry
- Immunology
- Oncology
Background:
- Extracellular adenosine, generated by ecto-5'-nucleotidase CD73, has immunosuppressive effects.
- Upregulation of CD73 in the tumor microenvironment is linked to resistance against cancer immunotherapies.
- Soluble CD73 (sCD73) activity in patient serum shows prognostic and predictive value in melanoma patients treated with anti-PD1 therapy.
Purpose of the Study:
- To develop and validate a novel Nuclear Magnetic Resonance (NMR)-based method for measuring ex-vivo soluble CD73 (sCD73) enzymatic activity.
- To establish a reproducible and specific assay suitable for high-throughput screening.
- To investigate the relationship between sCD73 protein levels and its enzymatic activity in human serum.
Main Methods:
- Development of a novel NMR-based assay for direct, simultaneous measurement of CD73 substrate and product concentrations.
- Validation of the assay using human serum samples.
- Correlation analysis between measured sCD73 protein levels and enzymatic activity.
Main Results:
- The NMR-based method demonstrated high specificity and reproducibility for measuring sCD73 activity.
- A strong linear correlation was observed between sCD73 protein levels and enzyme activity in human serum.
- sCD73 was identified as the predominant source of adenosine generation in human blood.
Conclusions:
- The developed NMR assay provides a robust tool for quantifying sCD73 activity.
- The established link between sCD73 protein levels and activity supports its clinical utility.
- This method has the potential to inform future clinical applications in cancer immunotherapy.
Abstract:
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.
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