Glycine 236 in the Lower Hinge Region of Human IgG1 Differentiates FcγR from Complement Effector Function.

Maximilian Brinkhaus,A Robin Temming,Giso Brasser,Peter C Ligthart,Gestur Vidarsson,Arthur E H Bentlage,Steven W De Taeye,Ruben G J Douwes,Hans De Haard,Peter Verheesen,Matthias Tammes Buirs,Wim J E Van Esch,Juk Yee Mok,Theo Rispens,Jacoline Gerritsen

doi:10.4049/jimmunol.2000961

Abstract

Abs of the IgG isotype mediate effector functions like Ab-dependent cellular cytotoxicity and Ab-dependent cellular phagocytosis by Fc interactions with FcγRs and complement-dependent cytotoxicity upon IgG-Fc binding to C1q. In this study, we describe the crucial role of the highly conserved dual glycines at position 236-237 in the lower hinge region of human IgG, including the lack of one glycine as found in IgG2. We found several permutations in this region that either silence or largely abrogate FcγR binding and downstream FcγR effector functions, as demonstrated by surface plasmon resonance, Ab-dependent cellular phagocytosis, and Ab-dependent cellular cytotoxicity assays. Although the binding regions of FcγRs and C1q on the IgG-Fc largely overlap, IgG1 with a deletion of G236 only silences FcγR-mediated effector functions without affecting C1q-binding or activation. Several mutations resulted in only residual FcγRI binding with differing affinities that are either complement competent or silenced. Interestingly, we also found that IgG2, naturally only binding FcγRIIa, gains binding to FcγRI and FcγRIIIa after insertion of G236, highlighting the crucial importance of G236 in IgG for FcγR interaction. These mutants may become invaluable tools for FcγR-related research as well as for therapeutic purposes in which only complement-mediated functions are required without the involvement of FcγR.

Highlights

We recently reported that this process is negatively affected for IgG2 in comparison with IgG1 by the lack of G236 in IgG2, which may explain the relatively low placental transport rates of IgG2 compared with IgG1
To understanding the structural and functional importance of single amino acids, function-driven fragment crystallizable (Fc) engineering has become a field of interest in the context of Ab-based therapeutics [5, 56,57,58,59]
We recently found that the deletion of G236 from IgG1 decreased neonatal Fc receptor (FcRn)-dependent placental transport in our model to levels of IgG2, whereas the addition of G236 to IgG2 increased transport to levels of IgG1 [22]

Summary

Introduction

Raji cells were opsonized with a 3-fold dilution series of anti-CD20 Ab variants starting at a concentration of 10 mg/ml in RPMI 1640 (Life Technologies) containing 10% (v/v) FCS for 15 min at RT in a 96-well round-bottom plate (Corning Costar). A total of 1.1 3 105 RAMOS cells per well were opsonized with 3-fold dilution series of anti-CD20 Ab variants, starting at a concentration of 10 mg/ml in RPMI 1640 (Life Technologies) containing 10% (v/v) FCS for 15 min at RT in a total volume of 80 ml in a 96-well round-bottom plate (Corning Costar).

Results

Conclusion