Low‐affinity FcγR interactions can decide the fate of novel human IgG‐sensitised red blood cells and platelets

Abstract

G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1-sensitised counterparts. However, non-destructive splenic retention of G1Δnab-coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab-sensitised RBCs did not cause FcγRI-mediated monocyte activation, FcγRIIIa-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low-affinity binding to this receptor class. Additional contacts via P-selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII-dependent activation by G1Δnab.These results emphasise the physiological relevance of low-affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab-coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab-sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions.

Human platelet IgG Fc receptor FcγRIIA in immunity and thrombosis

“…IV.3, a murine IgG 2b mAb specific for human FcγRIIA , was used to isolate FcγRIIA from platelets . Contrary reports exist as to whether IV.3 mAb crossreacts with FcγRIIB , with one explanation being that the antibody could bind to FcγRIIB via its IgG 2b ‐Fc fragment instead of its F(ab)‐specific IV.3 portion . As FcγRIIB is not expressed on platelets, IV.3 mAb has become a fundamental tool for studies of platelet FcγRIIA signaling pathways and function.…”

Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening

“…By contrast to the Fab region, the Fc region is rarely profiled, but the Fc binding affinity for the FcγR receptors on the surface of phagocytes is essential for mounting an effective immune response [9,10]. A potential proxy for the FcγR is the affinity with bacteria evasion proteins, protein A, protein G and the hybrid protein A/G [11,12].…”

Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

“…For FcγRI, the cell line was B2KA (S. Gorman and G. Hale, unpublished) and CHO cells expressing FcγRIIIb of allotypes NA1 and NA2 [21] were provided by J. Bux. CHO cell lines expressing FcγRIIIa of allotypes 158F and 158V as GPI-anchored receptors or the various FcγRII molecules as transmembrane proteins with native cytoplasmic domains have been constructed [22] , [23] . Continued and uniform expression of the appropriate FcγR was confirmed in each antibody binding assay by staining a sample of the cells for the receptor.…”

“…ADCC, macrophage adhesion and phagocytosis and monocyte activation assays were carried out using human cells as described previously [23] . NK cell activation was assessed using a method adapted from [24] .…”