The Carbohydrate at FcγRIIIa Asn-162

Abstract

FcγRIIIa 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 FcγRIII-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 FcγRIIIa (by up to 50-fold) but did not affect binding to the inhibitory FcγRIIb receptor. While the absence of carbohydrate at FcγRIIIa’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 FcγRIII 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 FcγRIIIa and FcγRIIIb are the only human Fcγ 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 FcγRIII. This information can be exploited for the design of new antibodies with altered Fc receptor binding affinity and enhanced therapeutic potential. FcγRIIIa 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 FcγRIII-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 FcγRIIIa (by up to 50-fold) but did not affect binding to the inhibitory FcγRIIb receptor. While the absence of carbohydrate at FcγRIIIa’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 FcγRIII 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 FcγRIIIa and FcγRIIIb are the only human Fcγ 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 FcγRIII. This information can be exploited for the design of new antibodies with altered Fc receptor binding affinity and enhanced therapeutic potential. Antibodies provide a link between the humoral and the cellular immune system with IgG 2The abbreviations used are: IgG, immunoglobulin G; GE, glycoengineered; Fuc, fucose; GnT-III, β1,4-N-acetylglucosaminyltransferase III; FcγR, Fcγ receptor; mAb, monoclonal antibody; SPR, surface plasmon resonance; h, human; s, soluble; m, mouse. 2The abbreviations used are: IgG, immunoglobulin G; GE, glycoengineered; Fuc, fucose; GnT-III, β1,4-N-acetylglucosaminyltransferase III; FcγR, Fcγ receptor; mAb, monoclonal antibody; SPR, surface plasmon resonance; h, human; s, soluble; m, mouse. being the most abundant serum immunoglobulin. While the Fab regions of the antibody recognize antigens, the Fc part interacts with membrane-bound Fcγ receptors (FcγRs) that are differentially expressed by all immune competent cells. Receptor crosslinking by a multivalent antigen-antibody complex triggers degranulation, cytolysis or phagocytosis of the target cell, and transcriptional activation of cytokine-encoding genes (1Deo Y.M. Graziano R.F. Repp R. van de Winkel J.G. Immunol. Today. 1997; 18: 127-135Abstract Full Text PDF PubMed Scopus (219) Google Scholar). Recently, the importance of the activating receptor FcγRIIIa for the in vivo elimination of tumor cells in humans has been demonstrated. In follicular non-Hodgkin’s lymphoma patients, a relationship was discovered between the FcγRIIIa genotype and clinical and molecular responses to rituximab, an anti-CD20 chimeric antibody used against hematological malignancies (2Cartron G. Dacheux L. Salles G. Solal-Celigny P. Bardos P. Colombat P. Watier H. Blood. 2002; 99: 754-758Crossref PubMed Scopus (1603)