Avidity confers FcgR binding and immune effector function to aglycosylated immunoglobulin G1 † Thomas C. Nesspor*, T. Shantha Raju, Chen -Ni Chin, Omid Vafa and Randall J. Brezski* Immunoglobulin G (IgG) antibodies are an integral part of the adaptive immune response that provide a direct link between humoral and cellular components of the immune system. Insights into relationships between the structure and function of human IgGs have prompted molecular engineering efforts to enhance or eliminate specific properties, such as Fc-mediated immune effector functions. Human IgGs have an N-glycosylation site at Asn297, located in the second heavy chain constant region (CH2). The composition of the Fc glycan can have substantial impacts on Fc gamma receptor(FcgR) binding. The removal of the glycan through enzymatic deglycosylation or mutagenesis of the N-linked glycosylation site has been reported to “silence” FcgR-binding and effector functions, particularly with assays that mea- sure monomeric binding. However, interactions between IgGs and FcgRs are not limited to monomeric interactions but can be influenced by avidity, which takes into account the sum of multimeric interactions between antigen-engaged IgGs and FcgRs. We show here that under in vitro conditions, which allowed avidity binding, aglycosylated IgGs can bind to one of the FcgRs, FcgRI, and mediate effector functions. These studies highlight how the valency of a molecular in- teraction (monomeric binding versus avidity binding) can influence antibody/FcgR interactions such that avidity effects can translate very low intrinsic affinities into significant functional outcomes. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: glycosylation; imune effector function ; avidity; monoclonal antibodies; Fc gamma receptor INTRODUCTION The structure of an immunoglobulin G (IgG) antibody is character- ized by two Fab arms linked to a single Fc domain by a flexible hinge region. The Fab arms contain variable regions that bind to antigen, whereas the Fc domain can link IgGs to the cellular immune system via interactions with the Fcg family of receptors as well as by binding C1q, which leads to activation of the complement cascade. In addition, the Fc domain of IgGs can bind to the neonatal Fc receptor (FcRn), an interaction that contributes to the long circulating half-life of IgGs (Roopenian and Akilesh 2007). The bivalent nature of the Fab arms presents a potential for an IgG to bind to two antigen epitopes simultaneously. When the antigens are immobilized, such as on a cell surface, the engagement of two or more molecules results in a synergistically enhanced binding affinity, known as avidity. Such avidity can also be realized by simultaneous binding of an IgG to cell-surface antigen and cell-surface Fc gamma receptors (FcgRs), whether the FcgRs are expressed on the same cell as the antigen or on a neighboring cell. In humans, three classes of activating FcgRs have been identified, FcgRI (CD64), FcgRIIa (CD32a), and FcgRIIIa (CD16a). Each of the activating FcgRs has different binding affinities for the four human IgG isotypes consisting of IgG1, IgG2, IgG3, and IgG4 (Nimmerjahn and Ravetch 2008). FcgRI binds IgG1 and IgG3 with high affinity (10 8 –10 9 M -1 ) and is capable of binding to a mono- meric antibody. FcgRI binds to IgG4 with approximately 10-fold lower affinity than IgG1 (Woof et al, 1986), whereas IgG2 does not have appreciable binding to FcgRI (Bruhns et al., 2009). The FcgRIIa and FcgRIIIa classes bind to IgG1 and IgG3 with lower affinities (10 6 –10 7 M -1 ) and therefore need to engage multiple Fc domains avidly in the form of immune complexes to facilitate an interaction. IgG2 only shows appreciable affinity for the two isoforms of FcgRIIa, with higher binding to the FcgRIIa H131 than FcgRIIa R131 , whereas IgG4 binds weakly to FcgRIIa and FcgRIIIa compared with IgG1 and IgG3 (Bruhns et al., 2009). The Fc domain of IgG is a homodimer of two heavy chains, each containing two characteristic structural domains known as immunoglobulin folds. Many studies over the years have indicated that the lower hinge/CH2 region of IgG is critical for mediating interactions between IgG and FcgRs (reviewed by Brezski and Jordan, 2010). An N-linked glycosylation site is located at N297 within the CH2 domain proximal to the lower hinge (EU numbering; Edelman et al., 1969). The core structure of this sugar side chain consists of N-acetylglucosamine (GlcNac) and mannose, with additional modifications containing fucose, * Correspondence to: Thomas C. Nesspor and Randall J. Brezski, Biologics Re- search, Centocor R&D Inc., 145 King of Prussia Road, Radnor, PA 19087, USA. E-mail: tnesspor@its.jnj.com; rbrezski@its.jnj.com † This article is published as part of the Biologics and Molecular Recognition- Memorial Issue for Mike Brigham-Burke of the Journal of Molecular Recognition, edited by Gabriela A. Canziani (In Silico Molecular, LLC) and Jennifer F. Nemeth, Raymond Sweet, Bernie Scallon, Juan Carlos Almagro, David Knight, and Gary L. Gilliland (Centocor R&D, Inc.) T.C. Nesspor, T. Shantha Raju, C.-N. Chin, O. Vafa, R.J. Brezski Biologics Research, Centocor R&D Inc., 145 King of Prussia Road, Radnor, PA, 19087, USA Abbreviations: mAb, monoclonal antibody; FcgR, Fc gamma receptor Research Article Received: 2 November 2011, Accepted: 2 December 2011, Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jmr.2155 J. Mol. Recognit. 2012; 25: 147–154 Copyright © 2012 John Wiley & Sons, Ltd. 147