Impact of N-glycosylation on Fcγ receptor / IgG interactions: unravelling differences with an enhanced surface plasmon resonance biosensor assay based on coiled-coil interactions

Abstract

The N-glycosylation profile of immunoglobulin G (IgG) is considered a critical quality attribute due to its impact on IgG-Fc gamma receptor (FcγR) interactions, which subsequently affect antibody-dependent cell-based immune responses. In this study, we investigated the impact of the FcγR capture method, as well as FcγR N-glycosylation, on the kinetics of interaction with various glycoforms of trastuzumab (TZM) in a surface plasmon resonance (SPR) biosensor assay. More specifically, we developed a novel strategy based on coiled-coil interactions for the stable and oriented capture of coil-tagged FcγRs at the biosensor surface. Coil-tagged FcγR capture outperformed all other capture strategies applied to the SPR study of IgG-FcγR interactions, as the robustness and reproducibility of the assay and the shelf life of the biosensor chip were excellent (> 1,000 IgG injections with the same biosensor surface). Coil-tagged FcγRs displaying different N-glycosylation profiles were generated either by different expression systems, in vitro glycoengineering or by size-exclusion chromatography, and roughly characterized by lectin blotting. Of salient interest, the overlay of their kinetics of interaction with several TZM glycoforms revealed key differences on both association and dissociation kinetics, confirming a complex influence of the FcγR N-glycosylation and its inherent heterogeneity upon receptor interaction with mAbs. This work is thus an important step towards better understanding of the impact of glycosylation upon binding of IgGs, either natural or engineered, to their receptors.

Determination of the composition of heterogeneous binder solutions by surface plasmon resonance biosensing

“…Figure 3 showed good fits between the calculated and observed SPR responses. In order to evaluate the accuracy of the kinetic parameter (26) www.nature.com/scientificreports/ determination when the parameters are extracted from mixtures of analytes, we compared them to the kinetic parameters determined from single-analyte experiments. For comparison sake, the deviation (or relative errorsee Table 3) was calculated as follows for a given parameter k:…”

“…Rather, we here demonstrate that for mixtures of macromolecules that are hard to purify, one may determine the kinetic parameters of each individual binder without any further purification. For instance, it is well-known that immunoglobulin G (IgG) production in bioreactors typically results in a heterogeneous distribution of glycoforms of the same IgG 26 , in turn impacting the IgG interactions with their Fcγ receptors, and the IgG therapeutic efficacy [21][22][23][24][25][26][27] . Hence, an algorithm similar to ours could potentially uncover meaningful binding kinetics of different IgG glycoforms without having access to pure IgG glycoform solutions.…”

“…Producing N differently glycosylated samples can be achieved by varying the cell culture conditions or through the use of various cellular and protein engineering strategies 35 . The compositions could be determined via mass spectroscopy, as it has already been performed in [21][22][23][24][25][26][27] . Once the glycoform kinetics have been uncovered, unknown glycoform mixtures could then be characterized.…”

“…The SPR identification of the kinetic parameters for each individual analyte within a complex analyte mixture would also be of great interest to determine the individual kinetic parameters of various (bio)molecules that are hard to separate from each other. Such is the case, for instance, for the various glycoforms of therapeutic monoclonal antibodies produced in mammalian cell cultures, for which binding to their receptors is affected by their glycosylation state [21][22][23][24][25][26][27] . Hence, SPR data analysis techniques that can extract specific kinetic parameters for different glycoforms are required for the analysis of antibody SPR sensorgrams.…”

Choice of Host Cell Line Is Essential for the Functional Glycosylation of the Fc Region of Human IgG1 Inhibitors of Influenza B Viruses

“…Less is known about glycosylation of the receptors to which the IgG1-Fc binds. Although recent studies implicate an active role for Fcg receptor-associated carbohydrates in fine-tuning Ab-receptor interactions (23,24), aglycosylated FcgRs expressed in Escherichia coli still retain the ability to bind IgG (25)(26)(27), bringing into question the exact role of Fcg receptor glycosylation to IgG1-Fc binding. Similar arguments apply for most of the glycan receptors.…”