Peripheral blood from anonymous, healthy volunteers was obtained with informed, written consent in accordance with Dutch regulations. This study was approved by the Sanquin Ethical Advisory Board in accordance with the Declaration of Helsinki.

Heparinized blood samples were used for isolation of peripheral blood mononuclear cells (PBMCs) or RBCs. NK cell isolation was only performed with blood from well-genotyped donors who do not express FcγRIIc (32) to exclude any possible effects of this receptor. Serum was obtained by allowing blood without anticoagulants to coagulate for 1 h at room temperature (RT) and collecting the supernatant after centrifugation at 950 × g for 10 min. Serum of three different volunteers was combined to create a serum pool.

Escherichia coli strain DH5α was used for recombinant DNA work. Restriction endonucleases, DNA modification enzymes were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Oligonucleotides were obtained from Geneart (Thermo Fisher Scientific) or Integrated DNA Technologies (Coralville, IA, USA).

Variable (V) genes for anti-human RhD (anti-D clone 19A10) heavy and light chain were sequenced from a single human B cell from a hyper immunized donor (33). A single-gene vector containing anti-D or anti-TNP IgG1 heavy- and kappa light-chain-encoding sequences were cloned as described previously by Kruijsen et al. (34) into a pEE14.4 (Lonza, Basel, Switzerland) expression vector. For both anti-TNP and anti-D IgG, a single expression vector was generated. In brief, the codon-optimized V gene for both heavy and light chain, including 5′-HindIII and 3′-NheI or 5′-HindIII and 3′-XhoI restriction sites respectively, Kozak sequence, and HAVT20-leader sequence, were designed and ordered from Geneart (Thermo Fisher Scientific). The HindIII-NheI or HindIII-XhoI fragments for the codon-optimized heavy or light chain were ligated into γ or κ constant region flanking 3′-EcoRI restriction site, respectively. The HindIII–EcoRI fragment for the codon-optimized light chain was ligated into pEE14.4 (Lonza), and the HindIII–EcoRI fragment for the heavy chain was ligated into pEE6.4 (Lonza). A single-gene vector encoding IgG1 was subsequently generated by ligation of the BamHI–NotI fragment from pEE6.4 (including a cytomegalovirus promoter), IgG1 heavy chain, and poly (A) into the light-chain-encoding pEE14.4 vector.

IgG1 production in human embryonic kidney (HEK) F cells and purification using protein A affinity chromatography was performed as described previously by Kruijssen et al. (34) Glyco-engineering of IgG1 was optimized as described by Dekkers et al. (31) In short, to decrease either fucosylation or galactosylation, 0.4 mM 2-deoxy-2-fluoro-l-fucose (2FF) (Carbosynth, Berkshire, United Kingdom) or 1 mM 2-deoxy-2-fluoro-d-galactose (2FG) (Carbosynth), respectively, was added to the cell suspension 4 h post transfection. To increase bisecting GlcNAc, 1% pEE6.4 + GNTIII encoding mannosyl (beta-1,4-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase (GNTIII) enzyme was co-transfected with 99% IgG1-κ HC + LC vector. To increase galactose, 1% pEE6.4 + B4GALT1 encoding β-1,4-galactosyltransferase 1 (B4GALT1) enzyme was co-transfected with 99% IgG1 vector and 5 mM d-galactose (Sigma Aldrich, Saint Louis, MO, USA) was added to the cell suspension 1 h before transfection. To increase sialylation, the level of galactosylation must also be elevated as sialic acid is the terminal sugar group with galactose residues as substrate. Thus, 1% pEE6.4 + B4GALT1 and 2.5% pEE14.4 + STGALT encoding β-galactoside alpha-2,6-sialyltransferase 1 (ST6GALT) were both co-transfected 96.5% IgG1 vector and 5 mM d-galactose was added to the cell suspension 1 h before transfection. To further increase sialylation, in vitro sialylation (ivs) was performed on the purified in vivo sialylated IgG created using the previous method. Recombinant human α-2,6-sialyltransferase (Roche, Basel, Switzerland) and cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-NANA) (Roche) were incubated at 37°C for 24 h with purified IgG1 with already in vivo enhanced galactose and sialic acid (as described above), after incubation samples were re-purified with protein A, as described previously (31, 34).

Immunoglobulin G-Fc glycan composition of produced IgG1 was determined by mass spectrometry as described previously by Dekkers et al. (31) Trypsin-digested glycopeptide samples were analyzed by nanoLC–ESI–QTOF–MS. The separation was performed on an RSLCnano Ultimate 3000 system (Thermofisher, Breda, the Netherlands) with a gradient pump, loading pump and an autosampler. 250 nl of sample was injected and washed on a Dionex Acclaim PepMap100 C18 trap column (5 mm × 300 µm i.d.; Thermofisher) for 1 min with 0.1% TFA at a flow rate of 25 µl/min. The sample was then separated on an Ascentis Express C18 nanoLC analytic column (50 mm × 75 µm i.d.; 2.7-µm fused core particles; Supelco, Bellefonte, PA) with a flow rate of 0.9 µl/min using linear gradient as described in Ref (30). The resulting co-elution of the different glycoforms of the IgG1-Fc glycosylation site warrants fair comparison by ensuring identical ionization conditions for the various glycopeptide species. The LC was coupled to the MS detector via a CaptiveSpray source with a NanoBooster (Bruker Daltonics, Bremen, Germany). The latter enriched the N₂ flow (3 l/min) with CH₃CN (pressure 0.2 bar), resulting in increased sensitivity. The samples were ionized in positive ion mode at 1,100 V. The Maxis Impact quadrupole-TOF–MS (micrOTOF-Q, Bruker Daltonics) was used as detector. MS1 spectra were collected at a frequency of 1 Hz with a scan range of m/z 550–1,800. The mass spectrometric data were calibrated internally in DataAnalysis 4.0 (Bruker Daltonics) using a list of known IgG glycopeptide masses. MSConvert (Proteowizard 3.0) (35) was used to convert the data files to mzXML format, and an in-house alignment tool (36) was used to align the retention times of the data files. The highest intensity of selected peaks (within an m/z window of ±0.2 and within a time window of ±15 s surrounding the retention time) was extracted using the in-house developed 3D Max Xtractor software tool. If above a signal:background ratio of 3, the background-subtracted area of the first three isotopic peaks of each glycopeptide in both 2+, 3+, and 4+ charge state were summed, and this summed value was then divided by the total summed value of all IgG1 glycopeptides to arrive at a percentage for each glycopeptide. From these percentages, we calculated several derived traits using the following formulas: fucosylation (H3N3F1 + H4N3F1 + H5N3F1 + H6N3F1 + G0F + G1F + G2F + H6N4F1 + G0FN + G1FN + G2FN + H6N5F1 + H4N3F1S1 + H5N3F1S1 + H6N3F1S1 + G1FS + G2FS + H6N4F1S1 + G2FS2 + G1FNS + G2FNS + H6N5F1S1 + G2FNS2), bisection (H6N4F1 + G0FN + G1FN + G2FN + H6N5F1 + H6N4F1S1 + G1FNS + G2FNS + H6N5F1S1 + G2FNS2 + H6N4 + G0N + G1N + G2N + H6N5 + H6N4S1 + G1NS + G2NS + H6N5S1 + G2NS2), galactosylation [(H4N3F1 + H5N3F1 + G1F + H6N4F1 + G1FN + H6N5F1 + H4N3F1S1 + H5N3F1S1 + H6N3F1S1 + G1FS + H6N4F1S1 + G1FNS + H6N5F1S1 + H4N3 + H5N3 + H6N3 + G1 + H6N4 + G1N + H6N5 + H4N3S1 + H5N3S1 + H6N3S1 + G1S + H6N4S1 + G1NS + H6N5S1) * 0.5 + G2F + G2FN + G2FS + G2FS2 + G2FNS + G2FNS2 + G2 + G2N + G2S + G2S2 + G2NS + G2NS2], sialylation [(H4N3F1S1 + H5N3F1S1 + H6N3F1S1 + G1FS + G2FS + H6N4F1S1 + G1FNS + G2FNS + H6N5F1S1 + H4N3S1 + H5N3S1 + H6N3S1 + G1S + G2S + H6N4S1 + G1NS + G2NS + H6N5S1) * 0.5 + G2FS2 + G2FNS2 + G2S2 + G2NS2], hybrid-types (H5N3F1 + H6N3F1 + H6N4F1 + H6N5F1 + H5N3F1S1 + H6N3F1S1 + H6N4F1S1 + H6N5F1S1 + H5N3 + H6N3 + H6N4 + H6N5 + H5N3S1 + H6N3S1 + H6N4S1 + H6N5S1), and high-mannose (H5N2 + H6N2 + H7N2 + H8N2 + H9N2). For some of the minor hybrid-type glycans, it could not be determined conclusively whether a galactose or a bisecting N-acetylglucosamine was present, so an educated guess was made based on structural knowledge (for instance, since the hybrid glycan H6N4F1 is elevated in GNTIII-co-transfected HEK cell-derived IgG samples, it is likely to be a bisected species rather than triantennary).

Protein A purified IgG was analyzed for monomeric and dimeric IgG on a Superdex 200 10/300 gel filtration column (30 cm, 24 ml, 17-15175-01, GE Healthcare, Little Chalfont, United Kingdom) connected to an Äkta explorer (GE Healthcare) HPLC system at RT with a flow rate of 0.5 ml/min and PBS as running buffer. Elution profiles were obtained by recording the absorbance at 215 nm.

Human FcγR constructs [FcγRIa (HIS tag), FcγRIIa (131His, Biotinylated, and 131Arg, Biotinylated), FcγRIIb (Biotinylated), FcγRIIIa (158Phe, Biotinylated, and 158Val, Biotinylated) and FcγRIIIb (NA2, HIS tag)] for surface plasmon resonance (SPR) analysis were obtained from Sino biological (Beijing, China). To further include all human FcγRs, a fusion Fc–FcγR construct composed of the extracellular domain of the FcγRIIIb in both allotypes followed by a Fc domain was created. To create the fusion Fc–FcγRIIIb constructs the amino acid code of the extracellular domain of either FcγRIIIb of NA1 allotype or FcγRIIIb NA2 allotype (37) (NCBI reference sequence NP_000561.3), and IgG2 Fc domain, composed of a human IgA1a hinge, human IgG2 Fc CH2 and CH3 domains including mutations deleting the Fc-glycan (N297A) and introducing a C-terminal biotinylation tag (BirA) were reverse translated and codon optimized at Geneart. DNA was ordered (Integrated DNA technologies, Coralville, IA, USA) and cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) expression vector using flanking HindIII and EcoRV restriction sites. A model of the construct and sequences are displayed in Figures S7A,B in Supplementary Material. The construct was produced and purified as described previously (31). After purification the protein was site-specifically biotinylated on the BirA tag using BirA enzyme as described by Rodenko et al. (38). For biotinylation of 1 µM FcγR protein 0.00657 µM BirA ligase was used. After biotinylation overnight at 25°C, the FcγR sample was buffer-exchanged and subsequently concentrated in PBS pH 7.4 using Amicon Ultra centrifugal filter units (MWCO 30 kDa) (Merck, Millipore, Darmstadt, Germany). The quality of the Fc-Fusion receptors was confirmed by comparing the binding of normally glycosylated IgG1 to the acquired his-tagged receptor (Sino-biological) and in-house made Fc-Fusion of the same allotype (NA2) (Figures S7C,D in Supplementary Material).

Surface plasmon resonance measurement were performed as described by Dekkers et al. (39). All biotinylated FcγR were spotted using a Continuous Flow Microspotter (Wasatch Microfluidics, Salt Lake City, UT, USA) onto a single SensEye G-streptavidin sensor (Ssens, Enschede, Netherlands) allowing for binding affinity measurements of each antibody to all FcγR simultaneously on the IBIS MX96 (IBIS Technologies, Enschede, Netherlands) as described by de Lau et al. (40). The biotinylated FcγRs were spotted in threefold dilutions, ranging from 100 to 3 nM for FcγRIIb and fusion FcγRIIIb-IgG2-Fc. All the other FcγRs were spotted in threefold dilutions, ranging from 30 to 1 nM in PBS 0.0075% Tween-80 (Amresco), pH 7.4. The IgGs were then injected over the IBIS at 1.5 dilution series starting at 5.9 nM until 506.25 nM or 0.9 nM until 2,000 nM, when necessary, in PBS in 0.075% Tween-80. For FcγRI affinity and FcγRIIIb control measurements, his-tagged FcγRI or FcγRIIIb was used. Biotinylated anti-His-tagged antibody (Genscript Piscataway, NJ, USA) was spotted in threefold dilutions, ranging from 30 to 1 nM. Before every IgG injection, 50 nM his-tagged FcγR was injected. The IgGs were then injected over the IBIS at threefold dilution series starting at 0.41 nM until 100 nM for FcγRI and 94 nM until 3,000 nM for FcγRIIIb. Regeneration after every sample was carried out with acid buffer (10 mM Gly–HCl, pH 2.4). Calculation of the dissociation constant (K_D) was done using an equilibrium analysis by linear intrapolation to Rmax = 500 (41). Analysis and calculation of all binding data were carried out with Scrubber software version 2 (Biologic Software, Campbell, ACT, Australia) and Microsoft Office Excel 2013.

NK cells were isolated from Ficoll-Plaque™-Plus (GE Healthcare) gradient obtained PBMCs by a CD56 magnetic-activated cell separation isolation kit (Miltenyi Biotec, Leiden, The Netherlands), according to manufacturer’s description. D + RBCs were isolated and labeled with radioactive chromium (100 μCi ⁵¹Cr, PerkinElmer, Waltham, MA, USA) at 10⁹ cells/ml. An amount of 10⁵ erythrocytes were incubated with NK cells for 2 h at 37°C in a 2:1 ratio in Iscove’s modified dulbecco’s medium (IMDM, Gibco, Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS, Bodinco, Alkmaar, The Netherlands) and anti-D IgG1-glycoforms at a total volume of 100 µl. To determine 100% lysis, 2.5% saponine (Fluka, Sigma Aldrich) was added to RBC in control wells and spontaneous lysis (sp) was determined by incubation of RBC without NK cells. Supernatants were collected and released ⁵¹Cr was quantified in a Packard Cobra II Auto-Gamma Counter Model D5005 (PerkinElmer). Percentage cytotoxicity was determined by the following formula: ADCC(%)=counts sample−counts spcounts 100%−counts sp×100, each value consisted of at least three individual sample wells.

A 2.4-mM 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Sigma-Aldrich) solution was added to 20 mg human serum albumin (HSA) diluted to 20 mg/ml (Sanquin, Amsterdam, The Netherlands) in 0.2 M NA₂HPO₄ (Merck, Millipore) and incubated 30 min at RT. To remove unbound TNBS, the solution was dialyzed (1:2,000) using a dialysis cassette (Thermo Fisher Scientific Slide-A-Lyzer G2 cassette, 10K MWCO) for 1.5 h at RT against PBS and additionally overnight at 4°C to obtain HSA-TNP.

To coat, maxisorp plates (Thermo Scientific, Nunc flat-bottom 96-well plate) were incubated o/n at RT with 20 µg/ml HSA-TNP in PBS. The plates were washed 5× with PBS + 0.1% tween-20 (Sigma-Aldrich) (wash buffer) using an ELISA washer (Biotek, 405 LSRS). All following washing steps were done similarly. The IgG samples were diluted in 100 µl PBS/plx [PBS + 0.1% poloxamer (Sigma-Aldrich, poloxamer 407)] per well and incubated for 1.5 h at RT. The plates were washed and incubated with 100 µl 1:35 serum pool in VB^+/+/plx {veronalbuffer [3 mM Barbital (Sigma-Aldrich), 1.8 mM Sodium-Barbital (Sigma Aldrich), 0.146 M NaCl (Fagron, Capelle aan den Ijssel, The Netherlands), pH 7.4] + 10 mM CaCl₂ (Merck) + 2 mM MgCl₂ (Merck) + 0.1% poloxamer} for 1 h at RT. When C1q was blocked, 10 min prior to addition of serum to the ELISA plate, anti-C1q-85 blocking antibody (42) was added to the VB^+/+/plx + 1:35 serum solution in a 1:2 molar ratio of C1q:anti-C1q-85 with final concentration of 8.57 µg/ml anti-C1q-85. The plates were washed and 100 µl with either 2 µg/ml biotinylated anti-C1q-2 (42), 0.5 µg/ml biotinylated anti-C4-10 (43), 0.6 µg/ml biotinylated anti-C3-19 (44), or 1 µg/ml HRP labeled anti-human IgG (Sanquin, Peliclass) in PBS/plx was added to respectively detect C1q, C4b, C3b, or IgG deposition and incubated for 1 h at RT. The plates were washed, C1q, C4b, and C3 plates were incubated with 100 µl 0.2 µg/ml strep-poly HRP (Sanquin, Peliclass) (C1q) or 0.25 µg/ml strep-HRP (Sigma-Aldrich) (C4b and C3b) in PBS/plx for 1 h at RT. The plates were washed and developed for 5–10 min using 100 µl TMB mix composed of 0.11 M NaAc (pH 5.5) (Merck), 0.1 mg/ml 3,3′,5,5′-Tetramethylbenzidine (Merck) and 0.003% H₂O₂ (Merck) and the reaction was stopped with the addition of 100 µl 2 M H₂SO₄ (Merck). The optical density (OD) was measured at A450 nm using a plate reader (Biotek, Synergy 2, Winooski, VT, USA).

The results were analyzed with a parallel line assay in Microsoft Office Excel (45). We assessed the potency of the glycoforms relative to a standard, an independently titrated unmodified IgG1; these values were expressed as percentages relative to the unmodified glycoform.

Fifty microliters of washed, packed, D⁺ RBCs obtained from heparinized blood were mixed with 350 µl 0.313 mM TNBS in 0.15 M Na₂HPO₄, pH 8.8 and incubated for 10 min at RT. TNPylated RBCs were centrifuged for 2 min at 350 × g and washed two times with PBS. RBC were resuspended into VBG^+/+ [VB^+/+ + 0.05% w/v gelatin (Sigma-Aldrich)]. Anti-TNP IgG1 was serially diluted in VBG^−/− (3 mM Barbital, 1.8 mM Sodium-Barbital, 0.146 M NaCl, pH 7.4, 0.05% w/v gelatin). In round bottom plates to a final volume of 100 µl we combined the diluted IgG1, 10% serum, ~4.5 × 10⁶ RBC, and a glass bead (2 mm, Merck) to ensure mixing of the solution during incubation (1:1 final ratio VBG^−/−:VBG^+/+). This amount of RBC was taken to ensure the 100% absorbance between 1.8 and 2.2 delta (Δ) A412–A690 nm. The plates were incubated for 90 min at 37°C while shaking at 150 rpm (Orbital incubator S150, 16 mm shaking diameter). After incubation, 1.25% w/v saponine was supplemented to the 100% control wells, 100 µl VBG^−/− was added to all wells and the plates were centrifuged for 2 min at 350 × g. Subsequently, 150 µl of supernatant was transferred into a separate plate and OD was measured at Δ A412–A690 nm using a plate reader. The percentage of lysed cells was calculated as follows: Lysis(%)=OD sample-OD spontaneousOD 100-OD spontaneous×100. In GraphPad Prism, we calculated the half maximal effective concentration (EC₅₀) for each replicate of the different glycoforms using a non-linear fit for normalized response with a variable slope and combined these to an average EC₅₀.

Statistical analyses were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, CA, USA). The level of significance was set at p < 0.05 using two-tailed tests.

Human IgG1, produced in HEK cells, shows complex-type bi-antennary glycans similar to IgG from normal human plasma (Figure 1A) (31, 46). More specifically, without any modification (“Unmodified,” box labeled “U” in the x-axis legend, Figures 1B–E) HEK-derived IgG1 N-glycans feature high fucosylation, low bisection, intermediate-level galactosylation, and low sialylation (Figures 1B–E). We previously developed six glyco-engineering tools which can be implemented upon protein production, as we recently described (31). These were aimed to decrease fucosylation, increase bisection, decrease or increase galactosylation, or increase sialylation. In the present study, these tools were combined and used in all possible combinations during the transient transfection in HEK cells, which resulted in the anticipated glycosylation changes and allowed us to produce the 20 major glycoforms present in human serum. Only minor unanticipated effects (Figures 1B–E), were observed. A slight increase in galactosylation upon overexpression of beta 1,4-N-acetylglucosaminyltransferase III (GntIII) to increase bisection (e.g., 28 to 36% upon GntIII expression)—but this was only observed in samples with low starting-levels of galactosylation (Figures 1C,D). Some of the tools caused a minor increase (<21%) in high-mannose or hybrid glycan species (Figure S1 and Table S1 in Supplementary Material) (31). Using the glyco-engineering tools the most extreme levels were reached for fucose and galactose (Figures 1B,D), bisection was increased up to 60%, and sialylation never reached over half of what was possible by the underlying galactose (~40%). The level of sialylation was, therefore, further increased using in vitro sialylation as described before (up to ~70%) (Figure 1E; Table 1) (31, 47, 48). All in all, this resulted in 20 combinations and markedly different glycoforms. All 20 glycoforms were produced as two panels of IgG1, specific for the RhD (anti-D) antigen or 2,4,6-trinitrophenyl hapten (anti-TNP) (34), with both panels showing highly comparable glycosylation patterns depending of the glyco-engineering tools applied (Table 1; Table S1 in Supplementary Material). To avoid any possible confounding effects of Fab glycosylation on IgG function, we used anti-D and anti-TNP with variable domains sequences devoid of N-linked glycosylation sites.

We next used the IBIS MX96 biosensor system, as described in Dekkers et al. (39), capable of analyzing the binding of up to 48 different receptor ligand interactions in parallel by SPR, to probe the affinity of all IgG1-glycoforms to all human FcγRs and their allotypes affecting IgG binding (Table S2 in Supplementary Material) (49). The antibodies used for these experiments (anti-D) showed no signs of dimers or multimers (Figure S2 in Supplementary Material). The binding affinities of unmodified IgG1 to the different receptors resembled those reported earlier (Table 2) (49). We considered significant changes in the apparent K_D of more than twofold from unmodified IgG to be potentially meaningful changes and within the scope of the SPR method, using a simplified 1:1 Langmuir model that does not fully represent the actual interaction which is more complicated (39). No significant effects of glycan changes above twofold were seen on the binding to FcγRIa, FcγRIIa (neither the H131- or the R131-allotype), or FcγRIIb/c (Figures 2A–D). However, marked changes were seen for all FcγRIII-isoforms. Reduction of fucose resulted in enhanced binding to all FcγRIII species by approximately 10- to 20-fold depending on the type- and allotype (Figures 2E–H), as reported (4, 5). Importantly, addition of galactose consistently enhanced binding of hypo-fucosylated IgG1 for all FcγRIIIa allotypes, doubling the effect of hypo-fucosylation alone (Figures 2E,F). This effect was also seen for allotypes of FcγRIIIb, but less strong and only for IgG1 that was bisected in addition to hypo-fucosylated (Figures 2G,H). Further sialylation of low-fucosylated galactosylated IgG1 had little additional effect on the binding to FcγRIII, except for hypo-fucosylated and bisected IgG1 for both allotypes of FcγRIIIa and FcγRIIIb NA2, where sialylation cause a significant decrease in binding. Taken together, glycan changes in the IgG-Fc only affect binding to FcγRIIIa and FcγRIIIb, with a major effect of hypo-fucosylation increasing binding to FcγRIIIa/b that was boosted by galactosylation. Bisection only appeared to indirectly affect binding when occurring in conjunction with sialylation, causing a slight decreased binding to FcγRIIIa/b to otherwise hypo-fucosylated and galactosylated IgG.

We next tested the efficacy of these anti-D IgG1 antibodies to mediate ADCC against RBC. Curiously, no NK cell-mediated induction of ADCC was seen with any fucosylated IgG1 at any concentration tested (Figures 3A,B; Figure S3 in Supplementary Material). Only hypo-fucosylated IgG1 induced ADCC in variable degrees depending on the glycosylation (Figures 3A,B). The observed level of ADCC were in line with the binding results obtained by SPR for each of the FcγRIIIa allotypes (Figure 3C), confirming the essential role of both hypo-fucosylation and elevated galactosylation for increased FcγRIIIa-binding and effector functions. Again, sialic acid had a minor but significant negative effect, especially for the bisected, hypo-fucosylated, and galactosylated IgG1 (Figures 3A,B). Remarkably, the well-known allotypic differences in affinity were confirmed by our SPR experiments, but not by the functional NK cell-mediated ADCC.

We then tested the effect of IgG-Fc glycosylation on C1q binding and subsequent complement activation, using the anti-TNP panel of IgG1 antibodies as anti-D does not fix complement. The efficiency of C1q binding to TNP-lated human serum albumin (TNP-HSA) and subsequent C4b deposition was titrated by serial dilution (Figure S4 in Supplementary Material). All glycovariants of anti-TNP bound TNP-HSA equally well (Figure S4A in Supplementary Material), but C1q binding and C4b deposition differed profoundly for the different glycoforms (Figure S4B in Supplementary Material). The relative C1q binding and C4b deposition were then calculated (Figures 4A,B, respectively). Both data sets suggested that elevated galactosylation and sialylation positively influenced complement activity. This activity was fully depended on the classical pathway with no influence of the mannan-binding lectin- or the alternative pathway, as C4b and C3b deposition, were completely blocked by an anti-C1q blocking antibody (Figure S5 in Supplementary Material). We then determined if this also translates into more efficient complement-dependent cytotoxicity (CDC) by analyzing complement-dependent lysis of TNP-labeled RBC (Figure 4C; Figure S6 in Supplementary Material). The level of C1q binding of each glycoform correlated well with the C4b deposition (Figure 5A) and with the obtained EC₅₀ of CDC (Figure 5B). The level of galactosylation of each glycoform also showed a direct relationship with the efficacy of C1q binding, and EC₅₀ (Figures 5C,D). In conclusion, the degree of galactosylation, but also sialylation of the IgG1-Fc N-glycan directly steers the antibody’s efficacy to stimulate complement deposition and CDC.