Influence de la N-glycosylation des immunoglobulines G et des récepteurs Fc gamma sur leurs interactions

RÉSUMÉ: L'immunothérapie regroupe l’ensemble des traitements utilisant certaines composantes du système immunitaire (p.ex. des protéines ou des cellules) d'une personne pour traiter ou prévenir des maladies. Parmi ces composantes, les anticorps monoclonaux (AcM), issus d’un même clone de lymphocyte B et reconnaissant le même épitope, sont les plus largement utilisés à des fins thérapeutiques pour lutter contre des maladies telles que le cancer et les maladies auto-immunes. Il s’agit d’ailleurs du groupe de molécules thérapeutiques ayant la plus forte croissance et selon les prévisions, les AcM et leurs dérivés continueront de dominer le marché des biopharmaceutiques dans les prochaines années. L’engouement pour les AcM est principalement lié à leur rôle d’agent de liaison entre la reconnaissance spécifique d’un antigène et le déclenchement de la réponse immunitaire liée à diverses fonctions effectrices. En effet, les AcM thérapeutiques sont principalement basés sur l’immunoglobuline G1 (IgG1). Structurellement, celle-ci se compose de deux régions Fab exprimant un site de liaison spécifique à un antigène et une région Fc, portant deux sites hautement conservés de N-glycosylation et les sites d’interaction de plusieurs ligands dont les récepteurs Fcγ (FcγR). Le déclenchement de la réponse immunitaire via l’interaction de l’IgG avec les FcγR est le mode d’action majoritaire des AcM thérapeutiques. Ces récepteurs sont présents à la surface des cellules de l’immunité innée et permettent la neutralisation ou la destruction de pathogènes via l’activation de fonctions effectrices telles que la cytotoxicité à médiation cellulaire dépendante des anticorps (ADCC). Par ailleurs, la N-glycosylation des IgG1 est considérée comme un attribut de qualité critique puisqu’elle est susceptible d’affecter leurs propriétés pharmacocinétiques et pharmacodynamiques. En particulier, la N-glycosylation des IgG1 est essentielle pour l’interaction avec les FcγR et le glycoprofil de l’IgG1 influence son affinité pour les FcγR, les fonctions effectrices et in fine l’efficacité thérapeutique. Les travaux présentés ici approfondissent le rôle de la N-glycosylation des IgG et des FcγR sur leurs interactions à l’aide d’une technique biophysique dont le principe de détection est basé sur la résonance des plasmons de surface (SPR).----------ABSTRACT: Immunotherapy refers to all treatments using certain components of the immune system (e.g. proteins or cells) to treat or prevent diseases. Among these components, monoclonal antibodies (mAbs), derived from the same B lymphocyte clone and recognizing the same epitope, are the most widely used as treatment for cancer and autoimmune diseases. This is the fastest growing group of therapeutic molecules and according to the forecasts, mAbs and mAb-based products will continue to dominate the biopharmaceutical market in the coming years. Such interest for mAbs is mainly related to their role as a link between the specific recognition of an antigen and the triggering of the immune response related to various effector functions. Indeed, therapeutic mAbs are mainly based on immunoglobulin G1 (IgG1). Structurally, it consists of two Fab regions expressing an antigen-specific binding site and one Fc region, carrying two highly conserved N-glycosylation sites and the interaction sites of several ligands including the Fcγ receptors (FcγRs). The triggering of the immune response via the interaction of IgG with FcγR is the main mode of action of therapeutic mAbs. These receptors are present on the surface of immune cells and enable the neutralization or the destruction of pathogens through the activation of effector functions such as antibody dependent cell-mediated cytotoxicity (ADCC). Furthermore, the N-glycosylation of mAbs is considered to be a critical quality attribute since it may affect their pharmacokinetic and pharmacodynamic properties. In particular, the N-glycosylation of IgG1 is necessary for interaction with FcγRs and it is well-known that the IgG1 glycoprofile influences its affinity for FcγRs, effector functions and in fine its therapeutic efficacy. The work presented here is focused on the role of IgG1 and FcγRs N-glycosylation on their interactions using a biophysical technique whose detection is based on surface plasmon resonance (SPR)

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

The N-glycosylation profile of immunoglobulin G (IgG) is considered a critical quality attribute due to its impact on IgG-Fc gamma receptor (Fc\u3b3R) interactions, which subsequently affect antibody-dependent cell-based immune responses. In this study, we investigated the impact of the Fc\u3b3R capture method, as well as Fc\u3b3R 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 Fc\u3b3Rs at the biosensor surface. Coil-tagged Fc\u3b3R capture outperformed all other capture strategies applied to the SPR study of IgG-Fc\u3b3R 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 Fc\u3b3Rs 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 Fc\u3b3R 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.Peer reviewed: YesNRC publication: Ye

Impact of IgG1 N-glycosylation on their interaction with Fc gamma receptors

The effector functions of the IgGs are modulated by the N-glycosylation of their Fc region. Particularly, the absence of core fucosylation is known to increase the affinity of IgG1s for the Fcγ receptor IIIa expressed by immune cells, in turn translating in an improvement in the antibody-dependent cellular cytotoxicity. However, the impact of galactosylation and sialylation is still debated in the literature. In this study, we have investigated the influence of high and low levels of core fucosylation, terminal galactosylation and terminal α2,6-sialylation of the Fc N-glycans of trastuzumab on its affinity for the FcγRIIIa. A large panel of antibody glycoforms (i.e., highly α2,6-sialylated or galactosylated IgG1s, with high or low levels of core fucosylation) were generated and characterized, while their interactions with the FcγRs were analysed by a robust surface plasmon resonance-based assay as well as in a cell-based reporter bioassay. Overall, IgG1 glycoforms with reduced fucosylation display a stronger affinity for the FcγRIIIa. In addition, fucosylation, and the presence of terminal galactose and sialic acids are shown to increase the affinity for the FcγRIIIa as compared to the agalactosylated forms. These observations perfectly translate in the response observed in our reporter bioassay