Enhanced Effector Functions Due to Antibody Defucosylation Depend on the Effector Cell Fcγ Receptor Profile This information is current as Christine W. Bruggeman, Gillian Dekkers, Arthur E. H. of September 24, 2021. Bentlage, Louise W. Treffers, Sietse Q. Nagelkerke, Suzanne Lissenberg-Thunnissen, Carolien A. M. Koeleman, Manfred Wuhrer, Timo K. van den Berg, Theo Rispens, Gestur Vidarsson and Taco W. Kuijpers J Immunol published online 31 May 2017 Downloaded from http://www.jimmunol.org/content/early/2017/05/31/jimmun ol.1700116 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 31, 2017, doi:10.4049/jimmunol.1700116 The Journal of Immunology Enhanced Effector Functions Due to Antibody Defucosylation Depend on the Effector Cell Fcg Receptor Profile Christine W. Bruggeman,* Gillian Dekkers,† Arthur E. H. Bentlage,† Louise W. Treffers,* Sietse Q. Nagelkerke,* Suzanne Lissenberg-Thunnissen,† Carolien A. M. Koeleman,‡ Manfred Wuhrer,‡ Timo K. van den Berg,* Theo Rispens,x Gestur Vidarsson,† and Taco W. Kuijpers*,{ Abs of the IgG isotype are glycosylated in their Fc domain at a conserved asparagine at position 297. Removal of the core fucose of this glycan greatly increases the affinity for FcgRIII, resulting in enhanced FcgRIII-mediated effector functions. Normal plasma IgG contains ∼94% fucosylated Abs, but alloantibodies against, for example, Rhesus D (RhD) and platelet Ags frequently have reduced fucosylation that enhances their pathogenicity. The increased FcgRIII-mediated effector functions have been put to use in Downloaded from various afucosylated therapeutic Abs in anticancer treatment. To test the functional consequences of Ab fucosylation, we produced V-gene–matched recombinant anti-RhD IgG Abs of the four different subclasses (IgG1–4) with and without core fucose (i.e., 20% fucose remaining). Binding to all human FcgR types and their functional isoforms was assessed with surface plasmon resonance. All hypofucosylated anti-RhD IgGs of all IgG subclasses indeed showed enhanced binding affinity for isolated FcgRIII isoforms, without affecting binding affinity to other FcgRs. In contrast, when testing hypofucosylated anti-RhD Abs with FcgRIIIa- expressing NK cells, a 12- and 7-fold increased erythrocyte lysis was observed with the IgG1 and IgG3, respectively, but no http://www.jimmunol.org/ increase with IgG2 and IgG4 anti-RhD Abs. Notably, none of the hypofucosylated IgGs enhanced effector function of macro- phages, which, in contrast to NK cells, express a complex set of FcgRs, including FcgRIIIa. Our data suggest that the beneficial effects of afucosylated biologicals for clinical use can be particularly anticipated when there is a substantial involvement of FcgRIIIa-expressing cells, such as NK cells. The Journal of Immunology, 2017, 199: 000–000. mmunoglobulin G is the most abundant class of Abs in human through an ITAM. This motif is found either within the cyto- plasma, consisting of four subclasses: IgG1, IgG2, IgG3, and plasmic tail of FcgRIIa/c or in the cytoplasmic tail of the common IgG4. Because the four subclasses differ in the structure of g-chain associated with the other activating FcgRs. Alternatively, I by guest on September 24, 2021 their constant regions (Fc domain), recognized by FcgRs and FcgRIIb contains an ITIM and FcgRIIIb lacks an intracellular sig- complement component C1q, they have different effector func- naling motif (3, 4). FcgRs bind IgGs of the four subclasses with tions. This includes Ab-dependent cellular cytotoxicity (ADCC) different affinities. Moreover, polymorphisms in FcgRs influence the and complement-dependent cytotoxicity (1, 2). binding affinities, most notably for FcgRIIa (FCGR2A-His131Arg, The family of FcgRs consists of the high-affinity receptor FcgRI with higher affinity for the 131His variant) and for FcgRIIIa and the low-to-medium affinity receptors FcgRII and FcgRIII. All (FCGR3A-Val158Phe, with higher affinity for the 158Val variant). FcgRs, except for FcgRIIb and FcgRIIIb, are activating receptors Polymorphisms in FcgRIIIb (e.g., FCGR3B-NA1NA2) do not affect affinity for IgG (3). All four IgG subclasses contain a conserved asparagine at po- *Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, sition 297 to which a glycan is attached. This biantennary glycan Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, the Netherlands; †Department of Experimental Immunohematology, Sanquin Research consists of a core structure of N-acetylglucosamine and mannose and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, residues and can be variably extended with galactose, sialic acid, 1066 CX Amsterdam, the Netherlands; ‡Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC Leiden, the Netherlands; xDepartment of core fucose, and a bisecting N-acetylglucosamine (5). The com- 297 Immunopathology, Sanquin Research and Landsteiner Laboratory, Academic Medi- position of the Asn glycan can influence the quaternary struc- cal Center, University of Amsterdam, 1066 CX Amsterdam, the Netherlands; and ture of the IgG and thereby the interactions with FcgRs (2, 6–8). {Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, 1100 DD Amsterdam, the Netherlands Furthermore, there is a direct carbohydrate–carbohydrate inter- 297 ORCIDs: 0002-9127-356X (G.D.); 0000-0002-0814-4995 (M.W.); 0000-0001-5621- action between the Fc glycan at Asn and the FcgR glycan on 162 003X (G.V.). FcgRIII at Asn, which affects the binding affinity (6, 9, 10). Received for publication January 23, 2017. Accepted for publication May 4, 2017. The glycoform of FcgRIII may change the interaction with IgGs, This work was supported by Program on Prevention Outcomes Practices Grant but this is beyond the scope of this study. PPOP-12-001 titled “Sweet IVIg: a blend of different tastes.” Hypofucosylation of IgG1 has been reported to result in a Address correspondence and reprint requests to Christine W. Bruggeman, Depart- significantly increased binding to FcgRIIIa and FcgRIIIb (4, 6, 9, ment of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Aca- 11–13). This results in enhanced effector functions, most notably demic Medical Center, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, the Netherlands. E-mail address: [email protected] increased ADCC by freshly isolated PBMCs, purified peripheral Abbreviations used in this article: ADCC, Ab-dependent cellular cytotoxicity; AIHA, blood monocytes, or NK cells through FcgRIIIa (11, 14–16). The autoimmune hemolytic anemia; 2FF, 2-deoxy-2-fluoro-L-fucose; ITP, immune throm- increased affinity to the GPI-linked FcgRIIIb seems to adversely bocytopenia; Ig; RhD, rhesus D; SPR, surface plasmon resonance; TNBS, affect the phagocytosis function of neutrophils (14, 17). In hu- trinitrobenzene sulfonic acid; TNP, 2,4,6-trinitrophenol. mans, hypofucosylated Abs can arise, as they have been described Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 in alloimmune responses against Rhesus D (RhD) and platelet www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700116 2 EFFECTOR FUNCTIONS UPON Ab DEFUCOSYLATION Ags, as well as in elite controllers of HIV infection (18). The IgG2 (EEQFNSTFR). Tryptic digestion of IgG3 resulted in a glycopeptide degree of fucosylation correlates with FcgRIIIa-mediated ADCC bearing a peptide sequence identical to that of IgG4, as well as a mis- such that Ab afucosylation enhances pathogenicity (14, 19). cleaved glycopeptide (LREEQFNSTYR). Using the three-dimensional Max Xtractor software, intensity values With this in mind, it is not surprising that several nonfucosylated were extracted for each peak within a manually specified m/z window and therapeutic mAbs have been put to therapeutic use, especially fo- retention time window. The background-subtracted peak intensity of the cusing on cancer treatment (4, 20–24). Furthermore, there are in- first three isotopic peaks in both 2+ and 3+ charge state was summed. For dications that glycan composition plays a role in the working the two types of IgG3 glycopeptides, the intensity of both was summed. mechanisms of other Ig therapies, such as IVIg, a product of IgG The values were subsequently normalized by dividing by the total intensity of all glycopeptides, yielding percentage data for each IgG subclass. These pooled from the plasma of thousands of donors. IVIg is used as data were used to calculate (a)fucosylation levels, that is, the percentage of replacement therapy in primary immunodeficiencies and as an N-glycans carrying a core fucose. immune-modulating agent in various autoimmune and immune