Enhanced Effector Functions Due to 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

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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-–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 ; 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 Human FcgR constructs diseases, among which immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) (25–30). Although the Human FcgRconstructsFcgRIa (his tag, 10256-H08H-100), FcgRIIa 131 131 modes of action of IVIg as an immune-modulating agent are still ( His, biotinylated, 10374-H27H1-B-50 and Arg, biotinylated, 10374- largely unresolved (27), one of the proposed working mechanisms H27H-B-50), FcgRIIb (biotinylated, 10259-H27H-B-50), and FcgRIIIa (158Phe, biotinylated, 10389-H27H-B-50, and 158Val, biotinylated, 10389- is that IVIg saturates FcgRs on splenic macrophages and thereby H27H1-B-50) for SPR analysis were obtained from Sino Biological inhibits the phagocytosis of platelets or RBCs opsonized by auto- (Beijing, China). Fusion FcgRIIIb-IgG2-Fc constructs composed of the (in case of ITP and AIHA, respectively) (27, 29, 31, 32). extracellular domain of the FcgRIIIb of NA1 or NA2 allotype followed by

Because the degree of Ab fucosylation alters the binding affinity to an Fc domain were cloned, produced, and site specifically biotinylated as Downloaded from FcgRIII, it may also affect the working mechanisms of IVIg. described by (G. Dekkers, L. Treffers, R. Plomp, A.E. Bentlage, M. De Boer, C.A. Koeleman, S.L. Lissenberg-Thunnissen, R. Visser, M. Brouwer, To study the functional extent and significance of changes in core J.Y. Mok, H. Matlung, T.K. van den Berg, W.J. van Esch, T.W. Kuijpers, fucosylation of human IgG, we generated a series of anti-RhD mAbs D. Wouters, T. Rispens, M. Wuhrer, and G. Vidarsson, manuscript in preparation). of all four IgG subclasses with and without high levels of fucose. Binding to all human recombinant FcgRs isoforms was assessed Biosensor affinity measurements

using surface plasmon resonance (SPR), and the impact was assessed Using a Continuous Flow Microspotter (Wasatch Microfluidics, Salt Lake http://www.jimmunol.org/ using primary human NK cells and monocyte-derived macrophages. City, UT), biotinylated FcgRs were spotted onto a single SensEye G– streptavidin sensor (Ssens, Enschede, the Netherlands) for binding affinity measurements of each Ab to any of the spotted FcgRs in a parallel manner Materials and Methods on the IBIS MX96 (IBIS Technologies, Enschede, the Netherlands) (39). Human samples The biotinylated FcgRs were spotted in 3-fold dilutions, ranging from 100 to 3 nM for FcgRIIb and fusion FcgRIIIb-IgG2-Fc. All of the other FcgRs Peripheral blood from healthy volunteers was obtained in heparinized tubes. were spotted in 3-fold dilutions, ranging from 30 to 1 nM in PBS 0.0075% For NK cell isolation we only used PBMCs from FCGR-genotyped donors Tween 80 (Amresco, Solon, OH), pH 7.4. The IgGs were then injected that have two copies of FCGR3-158F and do not have a FCGR2C-ORF over the IBIS at 23 dilution series starting at 0.98 nM up to 2000 nM in allele to exclude FcgRIIc as a confounding factor (33). Genotyping was PBS 0.075% Tween 80. performed as described before (34, 35). For FcgRI affinity measurements, we used his-tagged FcgRI. Bio- by guest on September 24, 2021 The study was approved by the Medical Ethics Committee of the Ac- tinylated anti–his-tagged Ab (GenScript, Piscataway, NJ) was spotted in ademic Medical Center and was performed in accordance with the Dec- 3-fold dilutions, ranging from 30 to 1 nM. Prior to the IgG injection, laration of Helsinki. 50 nM his-tagged FcgR was injected. The IgGs were then injected over the IBIS at 3-fold dilution series starting at 0.41 nM up to 100 nM as the Ab production highest concentration because of the inherently higher affinity. Anti-RhD H chain variable domain of clone 19A10 (36) was cloned into Regeneration of the IBIS MX96 was carried out after every sample with pEE6.4 (Lonza) expression vector containing either IgG1, 2, 3, or 4 con- acid buffer (10 mM Gly-HCl, 0.075% Tween 80, pH 2.5). Calculation of KD stant domains. IgG constant domains with flanking 39 NheI and 59 EcoRI was done as described previously (40). Analysis and calculation of all restriction sites were designed and ordered at Mr. Gene and cloned as binding data were carried out with Scrubber software version 2 (Biologic described previously (37). Anti-RhD k L chain was cloned into pEE14.4 Software, Campbell, ACT, Australia). (Lonza) expression vector. A combined vector encoding anti–2,4,6-trini- NK cell ADCC trophenol (TNP) IgG1 H chain and k L chain was cloned into pEE14.4 vector as previously described (37). NK cells were isolated from PBMCs by a CD56 MACS isolation All IgGs were produced by transient transfection of HEK-freestyle cells (Miltenyi Biotec), according to the manufacturer’s description. (Thermo Fisher Scientific) (37). To block fucosylation, 400 mM 2-deoxy-2- Erythrocytes of RhD-positive donors were isolated and labeled with ra- fluoro-L-fucose (2FF) was added to the cell suspension (14, 38). After 5 d, dioactive chromium (51Cr). The cells were subsequently opsonized with anti- IgG-containing supernatant from these cells was harvested by spinning RhD mAb in different subclasses and glycovariants at 10 mg/ml for 30 min twice at maximum speed and subsequent filtration with a 0.45-mm filter. at 37˚C, after which excess Ab was washed away. Erythrocytes were in- cubated with NK cells for 2 h at 37˚C in a 1:1 ratio. Afterwards, cytotoxicity Ab isolation was quantified by counting the released chromium with a gamma counter. IgG was isolated from cell supernatant with affinity chromatography col- Target cell staining and preparation umns HiTrap A HP (GE Healthcare) for IgG1, 2, and 4 and HiTrap protein G HP (GE Healthcare) for IgG3 on A¨ KTA prime (GE Healthcare) The amount of Ab deposition on the erythrocyte surface was determined by according to standard procedures. Ab fractions were concentrated using staining with goat anti-human IgG and analysis by flow cytometry. protein concentrators, 9-kDa molecular mass cut-off (Pierce; Thermo To control for the amount of Ab deposition on the red cell surface, we Fisher Scientific) and dialyzed against PBS using Slide-A-Lyzer dialysis varied Ag density by loading the erythrocytes with different concentrations of cassettes, 10-kDa molecular mass cut-off (Thermo Fisher Scientific). the TNP target trinitrobenzene sulfonic acid (TNBS) (0.05–5 mM) (Sigma- Aldrich), after which the erythrocytes were opsonized with anti-TNP with or Mass spectrometry analysis of Ab glycan without fucose at a concentration of 10 mg/ml. The amount of Ab deposition was subsequently determined by staining with goat anti-human IgG. IgG was analyzed by nanoscale liquid chromatography coupled to elec- trospray ionization quadrupole time-of-flight mass spectrometry on an Culture of monocyte-derived macrophages UltiMate 3000 RSLCnano (Thermo Fisher Scientific) coupled to a maXis Impact micrOTOF (Bruker Daltonics, Bremen, Germany), as described Monocytes were isolated and cultured into monocyte-derived macrophages, previously (38). The IgG1 glycopeptides (peptide sequence EEQYN- as described previously (41). In short, monocytes were isolated using a STYR) were eluted first, followed by IgG4 (EEQFNSTYR) and lastly CD14 MACS isolation kit and cultured for 9 d in IMDM, supplemented The Journal of Immunology 3 with 10% FCS, glutamine, and antibiotics, containing either 10 ng/ml GM- FcgRs and their most relevant allotypic variants (FcgRI, CSF or 50 ng/ml M-CSF. FcgRIIa-131His, FcgRIIa-131Arg, FcgRIIb/c, FcgRIIIa-158Val, 158 Phagocytosis by monocyte-derived macrophages FcgRIIIa- Phe, FcgRIIIb-NA1, and FcgRIIIa-NA2). We found similar binding affinities for normal (highly fucosylated) IgG as Phagocytosis assays with monocyte-derived macrophages were performed as described previously (41). RhD-positive erythrocytes were isolated and previously reported (Fig. 2) (3). IgG1 and IgG3 bound all FcgR stained with CFSE (Life Technologies). The cells were opsonized at an variants, IgG2 only bound FcgRIIa, and IgG4 bound FcgRI, optimal dose of human polyclonal anti-RhD Abs (1.56 IU/ml, RheDQuin; FcgRIIa, FcgRIIb/c, and FcgRIIIa, but not FcgRIIIb. IgG defu- Sanquin, Amsterdam, the Netherlands) or anti-RhD mAbs of different cosylation increased the binding affinity to FcgRIIIa and subclasses with and without core fucose at 10 mg/ml for 30 min at 37˚C, or FcgRIIIb, with the extent depending on the IgG subclass, but it did left unopsonized. In other experiments, the level of Ab deposition was controlled by labeling the RBCs with TNBS and subsequently opsonizing not change the binding to any of the other FcgRs (Fig. 2). with anti-TNP mAb, as described above. After washing excess Ab away, We confirmed that the affinity of low-fucosylated IgG1 to the cells were added to the monocyte-derived macrophages in a ratio of FcgRIIIa was 5- and 14-fold increased compared with high- 10:1. After incubation, the nonphagocytized RBCs were lysed and the fucosylated IgG1 for the FcgRIIIa-158Val and the FcgRIIIa-158 percentage of phagocytosis was determined by flow cytometry. Phagocytosis of anti-RhD–opsonized erythrocytes was inhibited by add- Phe variant (Fig. 2). Similarly for IgG3, an 11- and 22-fold in- ing anti-TNP Abs with or without fucose (as described above), 5 min prior to crease in binding upon hypofucosylation of IgG3 was observed the addition of erythrocytes, at concentrations of 0.1, 1.0, or 10 mg/ml. for FcgRIIIa-158Val and FcgRIIIa-158Phe, respectively. Hypo- Flow cytometry fucosylation of IgG1 and IgG3 also increased the binding to FcgRIIIb 7- to 8-fold, irrespective of the NA1/NA2 allotype. The For determining surface expression on NK cells and monocyte-derived mac- affinity of IgG2 to both allotypes of FcgRIIIa became significant Downloaded from rophages by flow cytometry, the following Abs were used: PE-Cy7–labeled anti- CD3 (clone SK7; BD Pharmingen), allophycocyanin-labeled anti-CD56 (clone and quantifiable upon hypofucosylation, but not to FcgRIIIb. B159; BD Pharmingen), FITC-labeled anti-CD64 (clone 10.1; BD Pharmin- Hypofucosylation of IgG4 also affected its binding to FcgRIIIa, gen), anti-CD32a (clone IV.3; Stemcell Technologies), anti-CD32b/c (clone which went from observable but not reliably quantifiable to true 2B6; a gift from MacroGenics, Rockville, MD), and anti-CD16 (clone 3G8; BD reproducible binding. Upon hypofucosylation, a significant and Pharmingen). Samples were measured by FACSCanto II (BD Biosciences). reliable binding of IgG4 was also observed to FcgRIIIb of both

Statistical analysis allotypes (Fig. 2). http://www.jimmunol.org/ Statistical analysis was performed using GraphPad Prism 6.07. For com- Hypofucosylated IgG1 and IgG3 exert enhanced NK cell parison of IgG-mediated phagocytosis, a Mann–Whitney U test was used. effector functions For comparison of expression levels, cytotoxicity, or blocking studies, testing was performed with unpaired t tests. Because afucosylated Abs have an increased affinity for FcgRIIIa (4, 11, 13), we performed NK cell ADCC assays to investigate the Results importance of Ab hypofucosylation in a biological FcgRIIIa Abs with low Fc fucosylation were produced by adding 2FF to system. We used primary NK cells from donors expressing the culture medium FcgRIIIa as the only IgG receptor on their as confirmed by flow cytometry (Fig. 3A). by guest on September 24, 2021 To investigate the effects of Ab fucosylation, we produced IgG Although both normally fucosylated and hypofucosylated anti- against the RhD blood group in the four different subclasses with RhD led to similar deposition on erythrocytes (Fig. 3B), only and without core fucose. We added 2FF, a competitive inhibitor of hypofucosylated Abs led to a strong increase in NK cell ADCC fucosyl transferase, to the culture medium of the anti-RhD Ab- toward erythrocytes (Fig. 3C). The NK cell ADCC of anti-RhD producing HEK cells. This resulted in a decrease in fucosylation IgG1-opsonized erythrocytes was entirely FcgRIIIa-dependent, as from an average of 90% for all IgG subclasses to 26, 13, 29, and anti–FcgRIII-F(ab9) fragments, but not isotype F(ab9) frag- 60% for IgG1, IgG2, IgG3, and IgG4, respectively (Fig. 1). For 2 2 ments, completely blocked ADCC (Fig. 3D). Similar to IgG1 anti- IgG4, the reduction in fucosylation was consistently less efficient than for the other subclasses. RhD, IgG3 variants induced increased NK cell–mediated ADCC when hypofucosylated, but IgG2 and IgG4 did not (Fig. 3E–G). Hypofucosylated Abs have a higher binding affinity to FcgRIII Normally, FcgR-expressing cells carry out their function in the We subsequently determined the binding affinity of the mAbs of presence of competing irrelevant IgG, the concentration of which different IgG subclasses with high and low fucose for all human is increased in patients treated with IVIg, which most likely contributes to the effector mechanism of IVIg. We therefore tested whether hypofucosylation of aspecific IgG blocks NK cell–mediated ADCC more efficiently than does fucosylated IgG, using irrelevant recombinant anti-hapten (TNP) IgG1. Only hypofucosylated IgG1 Abs induced a significant inhibition of anti-RhD IgG1-mediated erythrocyte ADCC (Fig. 3H). Hypofucosylated IgGs do not influence macrophage effector functions In vivo, FcgR-expressing effector cells other than NK cells are also targeted by IgG-mediated immune responses. We therefore investigated the effect of Ab fucosylation with human macro- phages, which express a broader spectrum of FcgRs than NK cells FIGURE 1. Ab fucosylation is reduced by 2FF. 2FF was added to the and are considered relevant in immune-mediated clearance reac- culture medium of Ab-producing HEK cells. IgG Fc glycosylation of the tions by tissue-resident macrophages in liver and spleen. anti-RhD Abs was determined by mass spectrometry on the glycopeptide We compared human GM-CSF– and M-CSF–cultured macro- level. Data shown are the results of the anti-RhD Abs used in the func- phages, representing the so-called M1 and M2 type of pro- and tional experiments. anti-inflammatory macrophages (41). These macrophages differ in 4 EFFECTOR FUNCTIONS UPON Ab DEFUCOSYLATION Downloaded from http://www.jimmunol.org/

FIGURE 2. Binding of fucosylated and hypofucosylated (+2FF) anti-RhD IgG subclasses to human FcgRs by SPR. All FcgRs were site-specifically biotinylated at the C terminus and equipped on individual spots on SPR array. Binding of each purified IgG was then assessed by flowing different by guest on September 24, 2021 concentrations of IgG over the array, obtaining each sensorgrams in real time to all FcgRs and allotypic variants simultaneously. All IgG subclasses were

flowed over the chip at 0.41–100 nM for FcgRI and 0.98–2000 nM for all other FcgRs. Binding affinity in KD of each measurement is indicated in the top left of each graph in nanomolar as a representative of at least three independent experiments. n.b., nonbinding; n.c., noncalculable; RU, response unit. their relative expression of FcgRI, with GM-CSF–cultured mac- not observe any difference in erythrocyte phagocytosis with re- rophages expressing more of this receptor. The anti-inflammatory spect to the fucosylation state of the opsonizing mAb (Fig. 5B). M-CSF–cultured macrophages express significantly more FcgRIIa We then tested whether hypofucosylated IgG1 was superior or than the GM-CSF–cultured macrophages (Fig. 4A, 4B). Both cell not in blocking the phagocytosis of Ab-opsonized erythrocytes by types have considerably less FcgRIIIa expression than NK cells. macrophages. In this setting we used a clinically applied polyclonal GM-CSF–cultured macrophages phagocytize significantly more anti-RhD product purified from plasma (RheDQuin; Sanquin) for anti-RhD IgG3-opsonized RBCs than IgG1-opsonized cells; IgG4- Ab deposition (Fig. 5C). As expected from previous experiments opsonized RBCs were phagocytized to an even lower extent, (41), IgG Abs can block phagocytosis by competing in IgG whereas IgG2-opsonized cells were not phagocytized at all. binding to the FcgRs in a dose-dependent manner. Again under M-CSF–cultured macrophages phagocytized anti-RhD IgG1-, IgG3-, our conditions of macrophage phagocytosis, uptake of anti-RhD– and IgG4-opsonized erythrocytes to a similar extent, whereas IgG2- opsonized erythrocytes was blocked irrespective of the fucosyla- opsonized cells were again not phagocytized. Upon glycoengineer- tion status of the blocking IgG (Fig. 5D). ing, all four anti-RhD IgG subclasses generated as hypofucosylated Collectively, our data demonstrate that the fucosylation state of isoforms showed identical opsonizing capacity of the erythrocytes. IgG greatly impacts the binding affinity to FcgRIIIa, but whether No increase in phagocytosis was observed upon hypofucosylation of this affinity change results in an enhanced effector function de- any IgG subclass (Fig. 4C). pends on the effector cell type and its level of FcgRIII expression. To test whether the Ag density and corresponding level of opsonizing Ab binding would contribute to the difference between Discussion myeloid and NK cell effector cells, we made use of our mAb In the present study we investigated the functional effects of Ab against TNP. Erythrocytes were TNP haptenized to different de- hypofucosylation in different cellular systems. Whereas .90% of grees and opsonized with either normally fucosylated or hypo- IgG normally contains fucose, we reduced this level to ,30% fucosylated anti-TNP IgG1 mAb (Fig. 5A). When performing upon applying a well-characterized bioengineering method (14, phagocytosis assays with these erythrocytes now expressing a 38). Human NK cells, which express FcgRIIIa as their only IgG graded level of TNP, we found a strong positive correlation be- receptor, showed increased affinity of hypofucosylated IgG1 and tween the level of TNP-labeling, opsonization efficiency, and the IgG3 for FcgRIIIa, whereas no such increased ADCC was ob- percentage of uptake by macrophages (p , 0.0001). Still, we did served with IgG2 and IgG4. Even though a gain of function of The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. Defucosylation of IgG1 and IgG3 significantly affects NK cell–mediated ADCC. (A) Gating strategy for genotype-selected NK cells (dot plots) that express FcgRIIIa, but not other FcgRs (histograms). Relevant isotype control background binding is shown in gray shading. Bar graph represents mean and SEM of n =5.(B) The level of erythrocyte opsonization with 10 mg/ml anti-RhD IgG1 and IgG1 12FF is similar. Anti-RhD opsonization was visualized with goat anti-human Ig and analyzed by flow cytometry. (C) NK cell ADCC with anti-D IgG1-opsonized erythrocytes. Erythrocytes were labeled with 51Cr and subsequently opsonized with anti-RhD fucosylated or defucosylated (+2FF) IgG1 or left unopsonized. (D) NK cell ADCC depends on FcgRIIIa. NK cell–mediated ADCC of anti-RhD IgG1-opsonized erythrocytes, either with or without (+2FF) fucose, were compared in the presence of

CD16 F(ab9)2 to block FcgRIIIa. (E–G) NK cell ADCC with opsonized erythrocytes as in (C) but now with (E) IgG2, (F) IgG3, and (G) IgG4. (H) NK cell ADCC with erythrocytes opsonized with defucosylated IgG1 anti-RhD (10 mg/ml) can only be blocked with defucosylated (+2FF) nonspecific anti-TNP blocking Abs. Only FCGR2/3-genotyped donors not expressing FcgRIIc and homozygous for FCGR3-158F were used. Data represent means and SEM of 6 (B), 10 (C), 4 (D), 9 (E), 6 (F), 6 (G), and 4 (H) experiments. DMFI, median fluorescence intensity corrected for the proper isotype control. 6 EFFECTOR FUNCTIONS UPON Ab DEFUCOSYLATION Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Monocyte-derived macrophages do not show elevated erythrocyte phagocytosis with defucosylated IgG. (A)FcgR expression levels of FcgRI, FcgRIIa, FcgRIIb, and FcgRIIIa on monocyte-derived macrophages, cultured for 9 d with GM-CSF (upper panels) or M-CSF (lower panels). Gray shading represents isotype control background binding to the cells. (B) Bar graphs showing the median fluorescence intensity, corrected for the proper isotype control, of FcgRI (n = 37), FcgRIIa (n = 9), FcgRIIb (n = 22), and FcgRIII (n = 37) of GM-CSF (open bars) and M-CSF (filled bars) cultured macrophages and SEM. For staining of FcgRIIb, only individuals without FCGR2C-ORF alleles were used (33). (C) Phagocytosis of erythrocytes opsonized with Abs of different subclasses with and without (+2FF) fucose (shown on x-axis) by monocyte-derived macrophages cultured with GM-CSF (left) and M-CSF (right). Only individuals not expressing FcgRIIc were used. The percentage of positive macrophages is shown, corrected for unopsonized erythrocytes (n = 10–26). *p , 0.05, **p , 0.01, ****p , 0.0001. ns, not significant. The Journal of Immunology 7 Downloaded from

FIGURE 5. Macrophages expressing various FcgRs do not show a different response to fucosylated and defucosylated Abs. (A) Ab deposition on erythrocytes after the erythrocytes were incubated with different concentrations of TNBS and opsonized with 10 mg/ml anti-TNP IgG1 and IgG1 12FF. After opsonization, the cells were stained with goat anti-human Ig and analyzed by flow cytometry to measure the amount of Ab deposition. The median fluorescence intensity is shown on the y-axis (n = 2–5). (B) Phagocytosis of TNPylated erythrocytes opsonized with anti-TNP with (black) and without fucose (red) by monocyte-derived macrophages cultured with GM-CSF (left) and M-CSF (right). The median fluorescence intensity as a measure of Ab deposition on erythrocytes is shown on the x-axis. The percentage of positive macrophages, normalized to the amount of macrophages that phagocytized http://www.jimmunol.org/ anti-D RheDQuin-opsonized erythrocytes, is shown on the y-axis (n = 9–10). (C) Ab deposition on erythrocytes after the erythrocytes were opsonized with RheDQuin (polyclonal anti-RhD). The cells were stained with goat anti-human Ig and analyzed by flow cytometry. The median fluorescence intensity is shown on the y-axis (n = 54). (D) Phagocytosis of anti-RhD RheDQuin opsonized erythrocytes upon the addition of anti-TNP (black) and anti-TNP 12FF (gray) nonspecific blocking Abs, relative to the percentage of positive macrophages in unblocked macrophages that phagocytized anti-RhD RheDQuin- opsonized erythrocytes. The concentration of blocking Ab is depicted on the x-axis (n = 4). **p , 0.01. ns, not significant.

IgG2 and IgG4 has been previously reported upon fucose removal cytosis of erythrocytes (41). IgG1-, IgG3-, and IgG4-opsonized (1, 42), and was observed in our study with isolated recombinant erythrocytes were phagocytized equally well, whereas IgG2-opsonized by guest on September 24, 2021 FcgRs tested in an SPR array, the enhanced binding affinity of cells were again not ingested. This is partly surprising, as IgG2 nonfucosylated IgG2 and IgG4 must be considered insufficient to has reasonable affinity to FcgRIIa (2). IgG1, IgG3, and IgG4 Abs exert any biological effect under our conditions. In other cellular bind FcgRIIa with similar affinity as IgG2, but unlike IgG2, they systems, hypofucosylated IgG2 and IgG4 may increase ADCC, as coengage FcgRI with high affinity, explaining their phagocytosis was shown by Niwa et al. (1), who reported enhanced ADCC of capacity (3). Daudi cells opsonized with anti-CD20. Ab hypofucosylation did not affect macrophage effector function. Besides the increased binding affinity to FcgRIIIa and FcgRIIIb This could be explained by the fact that macrophages express a upon Ab defucosylation, no effect to other FcgRs is known. There variety of FcgRs, including FcgRI and FcgRIIa (41). Apparently, the is equivalent binding of defucosylated and fucosylated IgG1 to contribution of FcgRIIIa to the phagocytosis process is too small to the neonatal Fc receptor (11), implying that defucosylated Abs be affected by Ab defucosylation in our phagocytosis assay. have a half-life comparable to fucosylated Abs. Finally, fucose We also tested the impact of IgG fucosylation on macrophage removal also does not change the binding to C1q and have equal effector function in a different approach, as Ag-specific Abs can also complement-dependent cytotoxicity as fucosylated Abs (1, 11) compete for FcgR-binding with the overwhelming IgG concentra- (G. Dekkers et al., manuscript in preparation). tion found in plasma (41, 43). In this study, we showed that adding To compare the enhanced effector function of NK cells with an irrelevant IgG mAb to macrophages prior to the addition of anti- hypofucosylated IgG, we also studied human macrophages. We RhD–opsonized erythrocytes inhibited phagocytosis in a dose- previously reported that GM-CSF–cultured macrophages mainly dependent manner, similar to the addition of IVIg (41). Although phagocytize via FcgRI, but also, albeit less, through FcgRIIIa the precise working mechanism of IVIg is still debated (32), one (41). Consistent with IgG1 and IgG3 being the major IgG sub- hypothesis is that IVIg saturates FcgRs on splenic and/or liver classes recognized by both FcgRI and FcgRIIIa, we found that macrophages, hence inhibiting the phagocytosis of platelets or IgG1 and IgG3 showed significant phagocytosis capacity, whereas RBCs opsonized by autoantibodies (in the case of ITP and AIHA, IgG2-opsonized erythrocytes were not phagocytized, in accor- respectively) (27, 29, 31, 32). Our studies indicate that irrelevant dance with IgG2 not being recognized by FcgRI. IgG4 is recog- anti-TNP mAbs inhibit phagocytosis of opsonized red cells by nized by FcgRI, resulting in less phagocytosis of IgG4-opsonized macrophages irrespective the level of fucosylation. If saturation of RBCs compared with IgG1- and IgG3-opsonized cells. In accor- FcgRs were indeed the major working mechanism of IVIg, defu- dance with the major role of FcgRI in this system (41) and the cosylation alone would not alter the efficacy of IVIg treatment. main effect of hypofucosylation being restricted to FcgRIII, no Recently, nonfucosylated mAbs have been generated for clinical role for IgG hypofucosylation was found using macrophages. use, because the increased affinity for FcgRIIIa is thought to im- Unlike GM-CSF–cultured macrophages, M-CSF–cultured macro- prove effector functions against cancer (20, 21, 44, 45). In this phages mostly use FcgRIIa and FcgRI for IgG-mediated phago- study, we show that this approach may represent an oversimplified 8 EFFECTOR FUNCTIONS UPON Ab DEFUCOSYLATION strategy and will definitely not work in all clinical approaches glycosylation of HIV-specific antibodies impacts antiviral activity. J. Clin. Invest. 123: 2183–2192. equally well. We suggest that the clinical efficacy of nonfucosylated 19. Kapur, R., L. Della Valle, M. Sonneveld, A. Hipgrave Ederveen, R. Visser, Abs will also depend on the cell type predominating in a certain P. Ligthart, M. de Haas, M. Wuhrer, C. E. van der Schoot, and G. Vidarsson. biological effect in vivo. In processes in which NK cells play a 2014. Low anti-RhD IgG-Fc-fucosylation in pregnancy: a new variable predicting severity in haemolytic disease of the fetus and newborn. Br.J.Haematol.166: 936–945. predominant role, it would be beneficial to defucosylate therapeutic 20. Jefferis, R. 2009. Recombinant antibody therapeutics: the impact of glycosyla- Abs. However, in a more complex cellular system, where multiple tion on mechanisms of action. Trends Pharmacol. Sci. 30: 356–362. FcgRs contribute to the cellular effector function, no difference 21. Yamane-Ohnuki, N., and M. Satoh. 2009. Production of therapeutic antibodies with controlled fucosylation. MAbs 1: 230–236. may be expected between fucosylated and defucosylated Abs. 22. Jefferis, R. 2009. Glycosylation as a strategy to improve antibody-based thera- In sum, alteration of the 297Asn glycan of IgG Abs may enhance peutics. Nat. Rev. Drug Discov. 8: 226–234. the clinical response depending on the FcgR expression pattern of 23. Suzuki, E., R. Niwa, S. Saji, M. Muta, M. Hirose, S. Iida, Y. Shiotsu, M. Satoh, K. Shitara, M. Kondo, and M. Toi. 2007. A nonfucosylated anti-HER2 antibody the effector cell type that predominates. Alternatively, altered levels augments antibody-dependent cellular cytotoxicity in breast cancer patients. of IgG fucosylation may be only one of the alterations that could be Clin. Cancer Res. 13: 1875–1882. 24. Satoh, M., S. Iida, and K. Shitara. 2006. Non-fucosylated therapeutic anti- used to afford enhanced binding activity and effector function be- bodies as next-generation therapeutic antibodies. Expert Opin. Biol. Ther. 6: 1161–1173. yond the FcgRIII expression, processes that we are currently 25. Flores, G., C. 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