The Carbohydrate at FcγRIIIa Asn-162

Abstract

FcgammaRIIIa plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. Non-fucosylated bisected IgGs bind this receptor with increased affinity and trigger FcgammaRIII-mediated effector functions more efficiently than native, fucosylated antibodies. In this study the contribution of the carbohydrates of both binding partners to the strength of the complex was analyzed. Glycoengineering of the antibody increased affinity for two polymorphic forms of soluble human FcgammaRIIIa (by up to 50-fold) but did not affect binding to the inhibitory FcgammaRIIb receptor. While the absence of carbohydrate at FcgammaRIIIa's Asn-162 increased affinity for native IgG, presumably due to the removal of steric hindrance caused by the bulky sugars, it unexpectedly reduced affinity for glycoengineered (GE) antibodies by over one order of magnitude, bringing the affinity down to the same level as for native IgG. We conclude that the high affinity between GE antibodies and FcgammaRIII is mediated by productive interactions formed between the receptor carbohydrate attached at Asn-162 and regions of the Fc that are only accessible when it is nonfucosylated. As FcgammaRIIIa and FcgammaRIIIb are the only human Fcgamma receptors glycosylated at this position, the proposed interactions explain the observed selective affinity increase of GE antibodies for only these receptors. Furthermore, we predict from our structural model that only one of the two Fc-fucose residues needs to be absent for increased binding affinity toward FcgammaRIII. This information can be exploited for the design of new antibodies with altered Fc receptor binding affinity and enhanced therapeutic potential.

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

“…, 19 Between the two variants, FcγRIIIa-V158 has a higher affinity to human IgG1. For example, under similar experimental conditions, FcγRIIIa-V158 demonstrated an approximately 10-fold higher affinity for IgG than FcγRIIIa-F158 20 . Cells expressing the FcγRIIIa-V158 allele mediate ADCC more effectively 19 .…”

“…Nonetheless, the authors did highlight that Asn162 is a potential glycosylation site of FcγRIII that is close to a binding site and a larger carbohydrate moiety attached to this site may influence the affinity to IgG 9 . Indeed, a subsequent study revealed that, compared to the unglycosylated form of FcγRIII (by mutating Asn162 to Gln162), the glycosylated FcγRIII (Asn162) showed reduced affinity for native (fucosylated) IgG antibodies, while antibodies with or without the core fucose showed a similar affinity for unglycosylated FcγRIII 20 . However, when fucose-free antibody binds glycosylated FcγRIII (Asn162), the affinity increased significantly.…”

“…However, when fucose-free antibody binds glycosylated FcγRIII (Asn162), the affinity increased significantly. The binding affinities of different glycoforms of IgG-FcγRIII pairs are in the following order: IgG-fucose-free/FcγRIIIa-Asn162 >> IgG-native glycan/FcγRIIIa-Gln162 > IgG-native glycan/FcγRIIIa-Asn162 20 . The authors concluded that the carbohydrate moieties of both FcγRIIIa and IgG are important for the interaction.…”

“…The authors concluded that the carbohydrate moieties of both FcγRIIIa and IgG are important for the interaction. An N -glycan needs to be attached to FcγRIIIa Asn162 and enhanced binding affinity can be achieved if the antibody is afucosylated 20 . In their proposed model, the fucose residue protrudes from the continuous surface of the Fc into open space, which prohibits close contact of the Fc receptor N -glycan core, thereby precluding additional productive interactions.…”

Epitope interactions of monoclonal antibodies targeting CD20 and their relationship to functional properties

“…[40][41][42] The binding of the Fc region of antibodies to FcγRIIIa is dependent on interactions between the carbohydrate moieties of both the FcγRIIIa and antibody. 43 Notably, ADCC activity does not differ between Type I and Type II anti-CD20 antibodies, 3 but antibodies such as GA101 have been engineered for enhanced affinity for FcγRIIIa leading to an increased ability to bind and recruit effector cells and hence a higher ADCC level. 27,44 The contribution of ADCC to the clinical activity of antibodies remains to be established.…”