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. 2023 Jan-Dec;15(1):2231128.
doi: 10.1080/19420862.2023.2231128.

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

Andrew R Crowley  1 , Matthew R Mehlenbacher  2 , Mohammad M Sajadi  3   4 , Anthony L DeVico  3 , George K Lewis  3 , Margaret E Ackerman  1   2   5
Affiliations

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

Andrew R Crowley et al. MAbs. 2023 Jan-Dec.

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.

Keywords: Affinity; Fc receptor; allostery; antibody; immunoglobulin G.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Binding and dissociation isotherms demonstrating higher affinity binding of FcγR to antibody that is complexed with antigen as compared to printed or otherwise captured on the sensor surface.
Figure 1.
Antibody-FcγR association and dissociation profiles by SPR across assay configurations.
Dot plot showing the affinity of each receptor-antibody pair across repeated experiments. Antigen-complexed antibody often displays a distinct improvement in affinity for FcγR.
Figure 2.
Antibody affinity for FcγR across assay configurations.
Dot plot showing the affinity of each receptor-antibody pair across repeated experiments. Antibodies captured by Protein A or an anti-Fab capture reagent generally do not show differences in affinity for FcγR.
Figure 3.
Evaluation of an alternative Fc domain capture strategy.
Volcano plot depicting the statistical significance and change in affinity to FcγR for antibody bound to antigen or to Protein A. Antibodies bound to antigen show statistically significant improvements in affinity to each FcγR tested.
Figure 4.
Improved affinity for FcγR is unique to antigen-bound IgG.
Binding and dissociation isotherms demonstrating higher affinity binding and dramatically slower dissociation of antigen-complexed IgG to FcγR on biosensor tips as compared to IgG in solution.
Figure 5.
Antibody-FcγR association and dissociation profiles by BLI across assay configurations.
Bar graph of thermodynamic properties of IgG: FcγR complexes. Antigen-bound IgG exhibits decreased enthalpy.
Figure 6.
Thermodynamic properties as measured by isothermal titration calorimetry.
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