A homogeneous SIRPα-CD47 cell-based, ligand-binding assay: Utility for small molecule drug development in immuno-oncology.

Autor: Burgess TL; Paradigm Shift Therapeutics LLC, Rockville, Maryland, United States of America., Amason JD; Paradigm Shift Therapeutics LLC, Rockville, Maryland, United States of America.; Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America., Rubin JS; Paradigm Shift Therapeutics LLC, Rockville, Maryland, United States of America., Duveau DY; Division of Preclinical Innovation, National Center for Advancing Translational Studies, National Institutes of Health, Rockville, Maryland, United States of America., Lamy L; Division of Preclinical Innovation, National Center for Advancing Translational Studies, National Institutes of Health, Rockville, Maryland, United States of America., Roberts DD; Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America., Farrell CL; Paradigm Shift Therapeutics LLC, Rockville, Maryland, United States of America., Inglese J; Division of Preclinical Innovation, National Center for Advancing Translational Studies, National Institutes of Health, Rockville, Maryland, United States of America., Thomas CJ; Division of Preclinical Innovation, National Center for Advancing Translational Studies, National Institutes of Health, Rockville, Maryland, United States of America., Miller TW; Paradigm Shift Therapeutics LLC, Rockville, Maryland, United States of America.; Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2020 Apr 02; Vol. 15 (4), pp. e0226661. Date of Electronic Publication: 2020 Apr 02 (Print Publication: 2020).
DOI: 10.1371/journal.pone.0226661
Abstrakt: CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRPα. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRPα receptors, that block the SIRPα-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRPα and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRPα variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. We also utilized the LSC assay to confirm published studies showing that the inhibition of amino-terminal pyroglutamate formation on CD47 using the glutaminyl cyclase inhibitor SEN177 disrupts SIRPα binding. The SIRPα-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRPα-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign.
Competing Interests: TWM, TLB, and CLF are co-owners of Paradigm Shift Therapeutics, a company focused on the development of CD47-targeting therapeutics for immune-oncology. JDA was an employee of, and JSR is a scientific advisor to, Paradigm Shift Therapeutics. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Databáze: MEDLINE
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