Evidence of variable human Fcγ receptor-Fc affinities across differentially-complexed IgG

Abstract

Antibody-mediated effector functions are widely considered to unfold according to an associative model of IgG-Fcγ receptor (FcγR) interactions. The associative model presupposes that Fc receptors cannot discriminate antigen-bound IgG from free IgG in solution and have equivalent affinities for each. Therefore, the clustering of Fcγ receptors (FcγR) in the cell membrane, cross-activation of intracellular signaling domains, and the formation of the immune synapse are all the result of avid interactions between the Fc region of IgG and FcγRs that collectively overcome the individually weak, transient interactions between binding partners. Antibody allostery, specifically conformational allostery, is a competing model in which antigen-bound antibody molecules undergo a physical rearrangement that causes them to stand out from the background of free IgG by virtue of greater FcγR affinity. Various evidence exists in support of this model of antibody allostery, but it remains controversial. We report observations from multiplexed, label-free kinetic experiments in which the affinity values of FcγR were characterized for covalently immobilized, captured, and antigen-bound IgG. Across the strategies tested, receptors had greater affinity for the antigen-bound mode of IgG presentation. This phenomenon was observed across multiple FcγRs and generalized to multiple antigens, antibody specificities, and subclasses. Furthermore, the thermodynamic signatures of FcγR binding to free or immune-complexed IgG in solution differed when measured by an orthogonal label-free method, but the failure to recapitulate the trend in overall affinity leaves open questions as to what additional factors may be at play.

The hinge-engineered IgG1-IgG3 hybrid subclass IgGh47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies

“…While the binding characteristics of IgG1, IgG3, and IgGh 47 to human CD64 (FcgRI) were distinctly similar, the affinities of one IgGh 47 mAb (Ab77) differed compared to the other two (Ab11 and Ab36) for some mouse receptors, suggesting that the variable domains or the light chain constant domain might impact the interaction with various FcR. Clone-specific differences have been observed in other work 31 . Some antibody-FcR interactions (Supplementary Fig.…”

“…This is most likely due to the short time frame for the experiment. Furthermore, other factors such as Fab-interactions with the FcRn in vivo 49 , clone-specific differences 31 and changes to antibody FcR-affinity upon antigen-binding have all been shown to influence FcR affinity of mAbs. These factors further complicate the interpretation between the relationship of affinity determination and in vitro and in vivo outcome.…”

“…The absence of such data is a limitation of the study. Since clone-specific differences can exist 31 , it is difficult to estimate what role different affinities to mouse FcR played on the in vivo outcome. Human IgG1 and IgG3 Fc bind with similar relative affinity to murine FcR, reflecting their human counterparts 50 , but mouse phagocytes have different expressions of FcR 51 .…”