Abstract 2185: Advancing macrophage-directed therapies: bioluminescent assays for macrophage effector function

Abstract Macrophages play a pivotal role in anti-cancer immunity through phagocytosis and presentation of tumor antigens to T cells. Antibody-dependent cellular phagocytosis (ADCP), mediated by macrophages and other myeloid cells, is a key mechanism-of-action (MoA) for antibody-based cancer immunotherapies. Strategies that enhance ADCP and other macrophage effector functions not only promote direct tumor destruction but also facilitate broader anti-tumor immunity. ADCP is initiated by the binding of antibody Fc domains to Fc gamma receptors (FcγRs), which triggers receptor crosslinking and signal transduction, leading to inflammatory cytokine production and phagocytosis. These effector functions are further regulated by immune checkpoints, and therapeutic interventions that block inhibitory checkpoints (e.g., SIRPα, ILT4, ILT2) or activate stimulatory pathways (e.g., TLR signaling) can enhance macrophage activity. However, current methods to measure macrophage functions are often low-throughput, labor-intensive, and highly variable. To address these challenges, we developed a suite of robust, cell-based bioluminescent bioassays to assess macrophage effector functions. These include: 1) ADCP: Using monocytic cells with endogenous FcγR expression and a FcγR-activation-dependent luminescent reporter. 2) Direct Phagocytosis Detection: Utilizing the HiBiT split luciferase system for sensitive and quantitative measurements. 3) Immune Checkpoint Modulation: Bioassays to measure SIRPα/CD47, ILT2/HLA-G, and ILT4/HLA-G interactions. 4) TLR Activation: Reporter bioassays for TLR activation. These assays are robust, easy-to-use, and pre-qualified according to ICH guidelines. Together, this comprehensive toolbox enables screening and the accelerated development of immunotherapies that target macrophage effector functions, paving the way for innovative cancer treatment strategies. Citation Format: Jonathan Mitchell, Denise Garvin, Rich Moravec, Julia Gilden, Adityarup Chakravorty, Jim Hartnett, Pete Stecha, Mei Cong, Jamison Grailer. Advancing macrophage-directed therapies: bioluminescent assays for macrophage effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2185.