Structure-Function Analysis of the FCRL5-IgG1 Fc Complex Reveals an Unappreciated Effect of Fc-Silent Antibodies on B...

Human Fc receptor-like 5 (FCRL5) is a low-affinity IgG Fc receptor expressed on various B cell subsets and a potential therapeutic target. We discovered that commonly used Fc-silencing mutations, designed to prevent interactions between the Fcγ receptors on immune cells and the Fc domain of therapeutic IgG, do not prevent binding to FCRL5. As a result, unintended interactions between Fc-silent therapeutic IgG and human B cells may occur. We isolated a well-expressed variant of the Fc-binding portion of human FCRL5 by directed evolution and used structural modeling to guide the engineering of a human IgG1 Fc variant with approximately 100-fold higher affinity for FCRL5, enabling us to produce FCRL5:Fc complexes in solution. Native mass spectrometry, size exclusion chromatography, and the crystal structure of the FCRL5-IgG1 Fc complex solved at 3.4 Å indicate that the two proteins bind in a 1:1 stoichiometry. Furthermore, the structure revealed that FCRL5 binds to IgG1 Fc in a manner completely distinct from that of previously characterized Fc-binding proteins, such as Fcγ receptors, explaining why most Fc-silencing mutations do not disrupt FCRL5 binding. We demonstrate that selective cross-linking of FCRL5 with the B cell receptor (BCR) in cis, using Fc-engineered antibodies with either physiological or enhanced FCRL5 affinity, inhibits Ca²⁺ flux in FCRL5-expressing B cells. We compare this effect with the selective co-ligation of FcγRIIb with the BCR. Our work demonstrates that FCRL5 interacts with human IgG Fc in a distinctive manner and that engagement of FCRL5 by Fc-silent therapeutic IgG could influence B cell function.