Cell-Based Reporter Bioassays for Development of Fc-Functional and Fc-Silent SIRPa/CD47 Checkpoint Inhibitors
Mitchell, J. et al., Promega Corporation
AACR 2020 Abstract #3163
CD47, a membrane glycoprotein commonly overexpressed in human cancers, interacts with its cognate receptor SIRPα on myeloid cells to deliver a “don’t eat me” signal that inhibits phagocytosis. This SIRPα/CD47 interaction has emerged as a critical and potentially druggable immune checkpoint. Blockade of SIRPα/CD47 interaction promotes phagocytosis of tumor cells in vitro, and SIRPα/CD47 inhibitors exhibit anti-tumor activity, including synergistic activity with other tumor-targeted mAbs (e.g. rituximab), in vivo. These encouraging pre-clinical data have sparked widespread development of SIRPα/CD47 inhibitors and driven a need for robust functional assays to measure the activity of these drug candidates. Biological activity of SIRPα/CD47 inhibitors is typically assessed via in vitro phagocytosis assays based on imaging or flow cytometry. These assays often use primary monocyte-derived macrophages and are inherently prone to donor variability, are low-throughput, and difficult to implement in drug development settings. Additionally, many SIRPα/CD47 inhibitors under development are designed to minimize Fc function to enhance safety and are thus incapable of driving phagocytosis directly.
To overcome these limitations, we have developed a pair of reporter-based bioassays for measuring the biological activity of Fc-functional and Fc-silent SIRPα/CD47 inhibitors. These bioassays utilize a SIRPα-positive monocyte effector cell-line that expresses multiple Fc gamma receptors (FcγRs) and an FcγR-responsive NanoLuc luciferase reporter that is inhibited by SIRPα/CD47 interaction upon co-culture of SIRPα effector cells with CD47-positive target cells. Addition of CD47 inhibitors with Fc functional activity simultaneously disrupts the SIRPα/CD47 interaction and engages FcγRs on the SIRPα effector cells, resulting in NanoLuc reporter activation. To enable testing of Fc-silent inhibitors, we have engineered a second CD47 target cell-line which provides a constitutive activating stimulus to SIRPα effector cells that is restricted by SIRPα/CD47 interaction. Addition of Fc-silent SIRPα/CD47 blockers releases inhibition by SIRPα/CD47, resulting in NanoLuc reporter activation. These newly developed reporter bioassays provide a robust, high-throughput platform to facilitate discovery and development of diverse SIRPα/CD47 checkpoint inhibitors.
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