FcγRI (CD64) is the only Fcγ receptor capable of high-affinity binding to monomeric IgG and found on monocytes, macrophages, eosinophils, neutrophils, and dendritic cells. FcγRI contains three C2-type immunoglobulin (Ig) extracellular domains (EC1-3), while all other Fcγ receptors contain only two EC domains. For detection, several FcγRI-specific antibodies have been described. The most frequently used commercial antibody is clone 10.1, which is proposed to bind the membrane proximal domain EC3. Other anti-FcγRI antibodies include 197, m22/H22 and C09, but their exact binding domains are unknown. A clear overview of binding affinities and functional properties for all these antibodies is lacking. We identified the binding characteristics and functional properties of five anti-human FcγRI antibodies via flow cytometry, LigandTracer and luminol-based chemiluminescence assays. Subsequently we verified their domain specificity using chimeric FcγRI receptors in which EC1-EC3 were swapped with their murine counterparts. Surprisingly, all anti-FcγRI antibodies bind to EC1 of FcγRI, while swapping of EC3 had no effect on binding. Affinity measurements showed similar affinities amongst all antibodies, despite varying association and dissociation rates, except for clone 10.1, which has a > 100-fold lower affinity. These findings strengthen the notion that EC1 is critical for receptor folding, structural integrity, and high-affinity IgG recognition, reinforcing its importance in FcγRI and its potential implications for targeted therapies. By redefining the binding domain of anti-FcγRI antibodies, this study provides a more accurate framework for utilizing FcγRI as a biomarker and therapeutic target in immunotherapy and diagnostics.
Keywords: Antibodies; CD64; Fc receptor; FcγRI; IgG.
