Insertion of N-Terminal Hinge Glycosylation Enhances Interactions of the Fc Region of Human IgG1 Monomers with Glycan-Dependent Receptors and Blocks Hemagglutination by the Influenza Virus

In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn297, either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein–Asn297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn221 sequon bound influenza virus hemagglutinin and disrupted influenza A–mediated agglutination of human erythrocytes.

M ultiple lines of evidence have shown that glycosylation is critical to driving either the anti-or proinflammatory capability of IgG (1). Glycosylation of the only available carbohydrate attachment site (Asn 297 ) in the Fc is essential for interactions with type 1 receptors (Fcg) and type 2 receptors (glycan dependent) but also for driving interactions with the complement cascade (2)(3)(4)(5).
In humans, infusion of Fc fragments is sufficient to ameliorate idiopathic thrombocytopenic purpura in children, demonstrating the therapeutic use of the Fc in vivo (6). These anti-inflammatory properties of the Fc are lost after deglycosylation of IgG, and a population of IgG-bearing sialylated Fcs has been identified as making a significant contribution to the control of inflammation in animal models (7,8). Higher levels of sialylation also leads to longer serum retention times (9,10), and studies in humans and mice have shown that influx and efflux of IgG into the CNS is glycan and sialic acid dependent (11)(12)(13)(14)(15)(16).
Consequently, the efficacy of sialylated Fc has generated an incentive to modify the existing glycans on Asn 297 , either by chemical means or through mutagenesis programs in the Fc protein backbone that disrupt the protein-Asn 297 -carbohydrate interface (17)(18)(19). However, chemical modification of pre-existing glycans is expensive and reliant on a sustainable source of human Fc, whereas mutagenesis approaches on the Fc, or expression in glycosidasedeficient/transgenic cell lines, have yielded little improvement in Asn 297 sialylation to the levels required for significant enhancements in the affinity of binding to FcgRs (18,19). Recently, coadministration of two glycosyltransferase Fc-fusion proteins has been shown to convert endogenous IgG into sialylated antiinflammatory IgGs that attenuate autoimmune disease in animal models in a platelet-dependent manner (20). Although in vivo enzymatic sialylation may circumvent many technical issues concerned with chemical or mutagenic approaches to generating sialylated IgG, it may not be appropriate in all clinical settings, for example in neurologic diseases (e.g., neuromyelitis optica) in which the target site is mostly devoid of platelets and in which two different Fc fusions would need to traverse the blood-brain barrier simultaneously. This approach also runs the risk of off-target glycan modifications and known immunogenicity of long-term administration of Fc fusions (21).
Mutagenesis studies to date have also been limited in two further respects. Side-chain changes have typically been restricted to alanine or serine, and functionality studies have mostly been confined to FcgR-binding studies (22,23). It is therefore of academic interest and potential clinical value to explore more thoroughly how the introduction of additional N-glycan sites into the Fc might affect changes in binding to FcgR and other atypical Fc glycan receptors, including sialic acid-binding Ig-type lectin (Siglecs) and C-type lectins.
We recently published two complementary approaches that radically increase the sialic acid content of the Fc (24) first by insertion of the 18-aa tailpiece from IgM onto the C terminus of the IgG1-Fc into which a cysteine-to-alanine substitution is made at Cys 575 and second by the addition of an extra N-glycan to the N terminus at position Asn 221 . This approach resulted in both multimeric and monomeric molecules that are .75% sialylated (compared with 2% for the IgG-Fc control) that bind to sialic acid-dependent receptors, including Siglec-1 and myelinassociated glycoprotein (MAG) (24), which are clinically implicated in the control of neuropathology (15,25). As many pathogens rely on glycans to infect host cells, these reagents may also be useful as inhibitors of infection (26).
The human IgG1-Fc typically does not bind glycan receptors because the glycan attached to Asn 297 is largely buried within the cavity formed by the CH2-CH3 homodimer (27,28). The location and content of glycans attached at Asn 297 also modulates the affinity of the Fc for binding to the classical FcgRs through conformational changes imparted to the FcgR-binding region located in the lower hinge (29). In this article, we show that these limitations to Asn 297 -directed receptor binding can be overcome through a program of mutagenesis aimed at disrupting disulfide bonding while enhancing N-linked glycosylation within the IgG1 Fc (Figs. 1, 2).

Production of mutants
The generation of glycan mutants in all combinations has been described previously for the hexa-Fc that contains cysteines at both positions 309 and 575 (24). To make the new mutants described in Fig. 1 in which Cys 575 was mutated to alanine, PCR overlap extension mutagenesis was used with a pair of internal mismatched primers 59-ACCCTGCTTGCTCAACTCT-39 / 39-GGCCAGCTAGCTCAGTAGGCGGTGCCAGC-59 for each plasmid vector coding for a designated glycan modification. The parental plasmids used for these new PCR reactions have been described previously (24). The resulting C575A mutants were then further modified to remove Cys 309 using primer pair 59-TCACCGTCTTGCACCAGGACT-39 / 39-AGTCCTGGTG-CAAGACGGTGA-59 to create the panel of double cysteine knockouts described in Fig. 2. To verify incorporation of the desired mutation and to check for PCR-induced errors, the open reading frames of the new mutants were sequenced on both strands using previously described flanking primers (24). CHO-K1 cells (European Collection of Authenticated Cell Cultures) were transfected with plasmid using FuGene (Promega), and Fc-secreting cells were cloned, expanded, and the proteins purified as previously described (2,30).

Receptor and complement binding assays
Methods describing the binding of mutants to tetrameric human dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN; Elicityl), Siglec-1, Siglec-4, and Siglec-3 (Stratech Scientific) have all been described previously (2,30). The same ELISA protocol was used for Siglec-2, CD23, dec-1, dec-2, clec-4a, clec-4d, MBL, and MMR (Stratech Scientific or Bio-Techne). Binding of C1q and C5b-9 have been described previously (2,30). ELISAs were used to investigate binding of Fc glycan mutants to human FcgRI, FcgRIIA, FcgRIIB, FcgRIIIA, and FcgRIIIB (Bio-Techne). Receptors were coated down on ELISA plates (Nunc) in carbonate buffer (pH 9) (Sigma-Aldrich) at 2 mg/ml overnight at 4˚C, unless otherwise specified. The plates were blocked in PBS/0.1% Tween-20 containing 5% dried skimmed milk. Plates were washed three times in PBS/0.1% Tween-20 before adding Fc mutant proteins at the indicated concentrations and left at 4˚C overnight. Plates were washed as above and incubated for 2 h with 1:500 dilution of an alkaline phosphatase-conjugated goat F(ab9) 2 anti-human IgG (The Jackson Laboratory). Binding of the  Fig. 1 in which Cys 309 and Leucine 310 is additionally changed to leucine and histidine as found in the native IgG1 Fc sequence to create the C309L/C575A panel of mutants. Red stars indicate the hinge Asn 221 , the Cg2 Asn 297 , and the tailpiece Asn 563 glycan sites. secondary detecting Fab' 2 anti-human Fc was checked by direct ELISA to every mutant to ensure there were no potential biases in the detection of binding of different mutants to different receptors (Supplemental Fig. 1A). Plates were washed and developed with 100 ml/well of a SIGMAFAST p-nitrophenyl phosphate solution (Sigma-Aldrich). Plates were read at 405 nm, and data were plotted with GraphPad Prism.

Binding to HA
ELISA plates were coated with 5 mg/ml recombinant HA from different influenza A and B viruses (BEI Resources) or native influenza A New Caledonia H1N1 virus (2B Scientific) in carbonate buffer (pH 9) and left at 4˚C overnight. Plates were washed five times with TSM buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl 2 , 2 mM MgCl 2 ) prior to blocking for 2 h in 150 ml/well of TSM buffer containing 5% BSA. After washing as before, 100 ml of Fc fragments at 30 mg/ml in TSM buffer was added in triplicate wells. Fc fragments were allowed to bind overnight at 4˚C. Plates were washed five times with excess TSM buffer prior to the addition of 100 ml/well of alkaline phosphatase-conjugated F(ab9) 2 goat anti-human IgG1 Fcg fragment-specific detection Ab diluted 1 in 500 in TSM buffer. Glycosylated Fc fragments that bound to the glycan receptors were left to bind the conjugated Ab for 1 h at room temperature on a rocking platform. Plates were washed as above and developed for 10 min with 100 ml/well of p-Nitrophenyl phosphate. Plates were read at 405 nm using a LT-4500 Microplate Absorbance Reader (Labtech), and the data were plotted with GraphPad Prism.

Hemagglutination inhibition assay
To determine the optimal virus-to-erythrocyte ratio, 2-fold virus stock (2B Scientific) dilutions were prepared in U-shaped 96-well plates (Thermo Fisher Scientific). The same volume of a 1% human O + RBC suspension (Innovative Research) was added to each well and incubated at room temperature for 60 min until erythrocyte pellets had formed in the negative control. After quantifying the optimal virus-to-erythrocyte concentration (4HA units), serial 2-fold dilutions of Fc, control IVIG (GAMMAGARD, Baxter Healthcare), or polyclonal goat anti-influenza H1N1 (Bio-Rad Laboratories) were prepared, starting at a concentration of 2 mM, and mixed with 50 ml of the optimal virus dilution. After a 30 min incubation at 4˚C, 50 ml of the human erythrocyte suspension was added to all wells and plates incubated at room temperature for 1 h, after which erythrocyte pellets could be observed in the positive controls. with folding intermediates (blue arrows) that are different to those formed by the hexa-Fc control. The C575A and N297A/C575A mutants run as monomers, with dimers and trimers also seen. Removal of Asn 563 favors multimerization in the presence of Cys 309 but the absence of Cys 575 . The addition of a N-X-(T/S) glycan sequon to generate N-terminally glycosylated hinges (the D221N series of mutants) did not affect multimerization but increased the molecular mass of all mutants. (B) The same mutants as in (A) but run under reducing conditions. (C) The same mutants as in (A) but stained with Coomassie reagent. The decreasing molecular masses seen in the Fc represent sequential loss of N-linked glycans. The N297A/N563A/C575A mutant has the smallest molecular mass because it has no glycans attached to the Fc, and D221N/C575A has the largest molecular mass because it has three glycans attached. The types of glycans attached at Asn 221 , Asn 297 , and Asn 563 for all mutants are shown in Fig. 9 and Supplemental Figs. 2-4. (D) Substitution of Cys 309 with leucine onto the mutants shown in (A) to create the double cysteine knockouts, which run as monomers. Differing molecular masses are seen with C309L/N297A/C575A monomers, which may represent differential glycosylation of Asn 563 . (E) The same mutants as in (D) but run under reducing conditions. (F) Coomassie-stained gel of (D). All proteins were run under either nonreducing or reducing conditions at 2 mg protein per lane on a 4-8% acrylamide gradient gel, transferred to nitrocellulose, and blotted with anti-human IgG Fc (Sigma-Aldrich). N-glycomic analysis N-glycomic analysis was based on previously developed protocol with some modifications (31). Briefly, the N-glycans from 50 mg of each sample were released by incubation with New England BioLabs Rapid PNGase F and isolated from peptides using Sep-Pak C18 cartridges (Waters). The released N-glycans were permethylated, prior to MALDI mass spectrometry analysis. Data were acquired using a 4800 MALDI-TOF/TOF mass spectrometer (Applied Biosystems) in the positive ion mode. The data were analyzed using Data Explorer (Applied Biosystems) and GlycoWorkbench (32). The proposed assignments for the selected peaks were based on composition together with knowledge of the biosynthetic pathways.

Binding to FcgRs by Biacore
Binding to FcgRs was carried out using a Biacore T200 biosensor (GE Healthcare). Recombinantly expressed FcgRs (R&D systems and Sino Biological) were captured via their histidine tags onto CM5 chips precoupled with ∼9000 reflective units anti-His Ab (GE Healthcare) using standard amine chemistry. Fc mutants were injected over captured receptors at a flow rate of 20 ml/min, and association and dissociation were monitored over indicated time scales before regeneration with two injections of glycine (pH 1.5) and recalibration of the sensor surface with running buffer (10 mM HEPES, 150 mM NaCl [pH 7]). Assays were visualized with Biacore T200 evaluation software v 2.0.1.

Disulfide bonding and glycosylation influence the multimerization states of hexa-Fc
To determine the contribution of two N-linked glycosylation sites (Asn 297 and Asn 563 ) and two cysteine residues (Cys 309 and Cys 575 ) in the multimerization of hexa-Fc (2), we created two panels of glycosylation-and cysteine-deficient mutants by site-directed mutagenesis, using the previously described hexa-Fc as the template Because we had previously shown that removal of the tailpiece glycan (Asn 563 ) in hexa-Fc led to the formation of dodecamers (24), we reasoned that a similar mutation introduced into the C575A mutants would also lead to enhanced dodecamer formation. Surprisingly, removal of Asn 563 , as in N563A/C575A, N297A/N563A/C575A, D221N/N563A/C575A, and D221N/ N297A/N563A/C575A, led to the formation of a laddering pattern of different molecular masses from ∼50 to .500 kDa (Fig. 3A, red arrows, 3C), representing monomers, dimers, trimers, tetramers, pentamers, hexamers, etc. Weaker bands between these species may represent 25 kDa folding intermediates that include Fc halfmers (Fig. 3A, blue arrows). All proteins in which the tailpiece Asn 563 glycan was substituted for alanine run as multimers in solution when examined by SEC-HPLC (Supplemental Fig. 1C).
By running these mutants under reducing conditions, we were able to determine the relative sizes and occupancy of the glycans attached at each position, showing that the Asn 221 and Asn 563 glycans are larger than that at Asn 297 and that fully aglycosylated null mutants such as N297A/N563A/C575A are ∼10 kDa lighter than either hexa-Fc or C575A glycan-competent molecules (Fig. 3B).
As Cys 309 is present in these mutants (Figs. 1, 3A-C), the ladders may arise through disulfide bond formation between the only freely available sulfhydryl at Cys 309 in two adjacent monomers. We reasoned that the loss of the tailpiece glycan in these four N563A mutants allows the hydrophobic amino acid residues (Val 564 , Leu 566 and Ile 567 ) also located in the tailpiece to cluster, thereby permitting disulfide bonding at Cys 309 .
To test the hypothesis that Cys 309 was indeed responsible for the laddering seen with the N563A-deficient mutants, we generated a second panel of C575A mutants in which Cys 309 /Leu 310 are mutated to Leu 309 /His 310 as found in the wild-type IgG1 Fc sequence (Fig. 2). We also generated the mutant CL309-310LH (C309L) in which the tailpiece Cys 575 was still present. This mutant ran similarly to hexa-Fc under nonreducing conditions, albeit with the presence of intermediates (Fig. 3D, blue arrows) that were notably absent in hexa-Fc, showing that Cys 309 stabilizes the quaternary structure in the presence of Cys 575 .
Importantly, the loss of Cys 309 also resulted in the loss of the ladders previously seen in the Cys 309 -competent mutants (Fig. 3D, 3F versus 3A, 3C), with all the double cysteine mutants now running principally as monomers by SDS-PAGE. The C309L/ N297A/C575A mutant runs as four different monomeric species (Fig. 3D) that resolve as two bands under reduction (Fig. 3E). These bands may represent glycan variants arising at Asn 563 . Given that these variants are absent in the C309L/C575A mutant, we conclude that the presence of Asn 297 glycan also controls glycosylation efficiency at Asn 563 . To a degree, the presence of the Asn 221 glycan also limits the occurrence of these Asn 563 glycoforms because under reduction, only a single band is seen in the D221N/C309L/N297A/C575A mutant (Fig. 3E).
Although the panel of double cysteine knockouts run mostly as monomers on SDS-PAGE (Fig. 3D, 3F), the double cysteine knockouts containing the N563A substitution run as a mixture of monomers and multimers in solution (Supplemental Fig. 1C). Thus, removal of the bulky Asn 563 glycan exposes hydrophobic amino acid residues in the tailpiece that facilitate noncovalent interactions in solution that would not otherwise readily occur in the presence of the sugar.
The Asn 297 and Asn 563 glycans are critical for the interactions of mutants with glycan receptors, and their absence can be compensated by the presence of Asn 221 To determine which N-linked glycan in the double cysteine knockout mutants (Fig. 2) contributes to receptor binding, we investigated their interaction with soluble recombinant glycan receptors by ELISA (Fig. 4, Table I). In stark contrast to the IgG1-Fc control, mutants in which both Asn 297 and Asn 563 are present (e.g., C309L/C575A) bound all 12 glycan receptors investigated (Fig. 4). Removal of the tailpiece glycan Asn 563 , as in C309L/N563A/C575A or C309L/ N297A/N563A/C575A, abolished binding to these same receptors, showing that Asn 563 is required for glycan receptor binding.
Removal of the glycan at Asn 297 , as in C309L/N297A/C575A, also abolished binding to all glycan receptors with the exception of Siglec-1. Taken together, the data show that both Asn 563 and Asn 297 are required for the broad glycan receptor binding seen with the C309L/C575A mutant ( Fig. 4 and Table I). Table I Figs. 2-4). This is in agreement with our previous work in which we demonstrated in fully cysteine-competent multimers that Asn 221 is .75% terminally sialylated (24).
The C309L mutant that can form cysteine-linked multimers because of the retention of Cys 575 in the tailpiece (Fig. 3D, 3F and Supplemental Fig. 1C) was unable to bind to any glycan receptors with the exception of CD23 (Fig. 4). Thus, the Asn 563 glycans are only available for binding when attached to lower valency molecules and are buried within multimers that form either through Cys 309 -driven covalent bridging or by noncovalent clustering through multiple hydrophobic amino acids located in the tailpiece (e.g., C309L/N563A/C575A).
We next investigated binding of the panel of C575A mutants in which Cys 309 is still present (Fig. 1) and that we had shown to have the tendency to form dimers and laddered multimers (Fig. 3A, 3C and Supplemental Fig. 1C). This panel of molecules, in which disulfide bonding mediated by Cys 309 could still occur, bound less well to all the glycan receptors investigated (Fig. 5). With the sole exception of Siglec-1, the presence of the Asn 221 glycan was unable to improve binding, in contrast to the double cysteine knockouts. We conclude that N-glycans at all three attachment sites (Asn 221 , Asn 297 , and Asn 563 ) are more predisposed to binding to glycan receptors when expressed on monomers and that the presence of Asn 221 as the only glycan is sufficient to impart this broad specificity of binding, as exemplified by D221N/N297A/N563A/C575A and D221N/C309L/N297A/N563A/C575A (Figs. 4, 5).
We observed that the aglycosylated mutant N297A/N563A/ C575A had a propensity to bind glycan receptors (Fig. 5). We do not have a simple answer for this observation, although the lack of binding by its counterpart C309L/N297A/N563A/C575A in which Cys 309 is absent suggests that it may be glycan independent and a consequence of increased avidity interactions through multimerization (compare Fig. 3A v 3D).

Glycan receptor binding is critically dependent on the presence of N-linked glycans
To be certain that glycan receptor binding was dependent on the presence of N-linked carbohydrates, and more specifically sialic acid, these sugars were removed from the triglycan D221N/C309L/C575A mutant using either PNGase F or neuraminidase (Supplemental Fig. 1B). As expected, the D221N/C309L/C575A mutant treated with PNGase F was unable to bind any of the receptors investigated, whereas treatment with neuraminidase inhibited binding to the sialic acid-dependent receptors (Supplemental Fig. 1B).
Asn 221 -based monomers show differential binding to low-affinity human FcgRs Given the remarkable binding to glycan receptors seen with some of the glycan-modified mutants, we tested the impact that this extra glycosylation conferred on binding to the classical human FcgRs (Fig. 6, Table II). The presence of Asn 221 , for example in the D221N/C309L/N297A/N563A/C575A mutant, imparted improved binding to FcgRIIB (CD32B) even in the FIGURE 5. Binding of C575A mutants to glycan receptors. Proteins with a predisposition to multimerize via Cys 309 interactions (as shown in Fig. 3A, 3C, and Supplemental Fig. 1C) are less able to engage glycan receptors than their equivalent mutants in which Cys 309 was changed to leucine (Fig. 3D, 3F). With the exception of Siglec-1, the insertion of Asn 221 into mutants that tend to form multimers had no effect on, or was detrimental to, binding of glycan receptors. Error bars represent SD around the mean value; n = 2 independent experiments.  Fig. 3A, 3C) binds strongly to FcgRI and FcgRIIIA as is also seen with C309L/N563A/C575A that carries the same N563A mutation. The D221N/N563A/C575A mutant shows enhanced binding to FcgRI and FcgRIIIA. In multimers, the presence of Asn 221 constrains interactions with FcgRIIB that are enhanced when Asn 221 is attached to monomers (E). No improvement in binding was observed to FcgRIIA or FcgRIIIB for any of the mutants tested (data not shown). Error bars represent SD around the mean value; n = 2 independent experiments. absence of both Asn 297 and Asn 563 when compared with the IgG1-Fc and controls in which Asn 221 was absent (Fig. 6, for FcgRIIB compare filled symbols versus unfilled symbols). However, the presence of Asn 221 did not improve binding to FcgRIIIA (compare D221N/C309L/N563A/C575A and C309L/N563A/C575A), although binding of both mutants was considerably stronger than the IgG1-Fc monomer control (Figs. 6, 7B). We hypothesize that the enhanced binding observed with the N563A-deficient mutants is a consequence of increased tailpiece-mediated assembly by all the Asn 563 -deficient proteins (Supplemental Fig. 1C). Improved binding to FcgRI was also observed with these two mutants against the IgG1-Fc control (Figs. 6, 7A), although no improvements were seen with respect to either FcgRIIA or FcgRIIIB for any of the mutants tested.
Both the double cysteine knockouts, C309L/N563A/C575A and D221N/C309L/N563A/C575A, that form multimers in solution and bound FcgRI and FcgRIIIA (Val 176 ) strongly in ELISAs were tested for binding FcgRs receptors by surface plasmon resonance analysis (Fig. 7). Both mutants displayed slower apparent off rates compared with the control Fc monomer, consistent with avidity effects either through binding to multiple immobilized FcgRs molecules or rebinding effects (Fig. 7). The loss of Asn 297 in the C309L/N297A/C575A and D221N/C309L/N297A/C575A mutants resulted in molecules that were unable to bind FcgRs, as previously shown by ELISA (Figs. 6, 7).
We next investigated binding of the multimers formed through Cys 309 (Figs. 1, 3A, 3C). In multimers, the presence of Asn 221 reduced binding to all FcgRs (Fig. 6, Table II), whereas binding to the glycan receptors, although lower than that seen with monomers, was retained (Fig. 5). Multimers in which Asn 563 and Cys 575 are both mutated to alanine, as in N563A/C575A, bound very strongly to FcgRI and FcgRIIIA, with improved binding to FcgRIIB when compared with either the hexa-Fc or IgG1-Fc controls (Fig. 6). The aglycosylated multimer N297A/N563A/C575A bound very well to the inhibitory FcgRIIB receptor while retaining binding to FcgRI (Fig. 6).

Asn 221 -based monomers and multimers exhibit complex sialylation patterns
The structure of the N-glycan on the Fc of IgG Abs has been shown to influence multiple receptor interactions. For example, the interaction of IVIG with glycan receptors has been attributed to direct and/or indirect effects of N-glycan sialic acid on the Fc (29,34,35). Therefore, we investigated the nature of the Nglycans on the two panels of glycosylation-and cysteine-deficient mutants by MALDI-TOF mass spectrometry-based glycomic analysis (Fig. 9, Supplemental Fig. 2-4).
In both samples, the spectra demonstrate a higher level of N-glycan processing with enhanced levels of biantennary galactosylation and sialylation. In addition, larger tri-and tetraantennary complex N-glycans are also observed, which can be fully sialylated (for example, peaks at m/z 3776 and 4587). Therefore, the glycomic analysis revealed that both Asn-221 and Asn-575 contained larger, more highly processed N-glycans that are not observed on the IgG1-Fc control ( Fig. 9 and Supplemental  Figs. 2-4). As predicted, no glycans could be detected on the glycosylation-deficient double mutants (N297A/N563A/C575A and C309L/N297A/N563A/C575A).

The Asn 221 glycan imparts enhanced binding to influenza HA
To determine if any of the hypersialylated Fc mutants possessed biologically useful properties, we investigated their binding to HA, a prototypic viral sialic acid-binding ligand (Fig. 10A, 10B). We used clinically available IVIG as a positive control because IVIG is known to contain high concentrations of IgG Abs against a diverse range of influenza HAs (36).
As expected, IVIG bound strongly to recombinant HA from both influenza A and B viruses (Fig. 10A, 10B). With the exception of the aglycosylated mutants (C309L/N297A/N563A/C575A and N297A/N563A/C575A) and the IgG1-Fc control, all the glycanmodified Fc fragments bound recombinant HA from both group A and B viruses. Binding was also reflected in the abundance of sialylated N-glycans of the mutant proteins (Supplemental Figs. 2-4).  Fig. 3 and Supplemental Fig. 1C), an accurate determination of interaction kinetics is not possible. Binding to FcgRs from R&D Systems is illustrated, although binding with receptors sourced from Sino Biological gave nearly identical results.
Thus, mutants containing Asn 221 bound more strongly than their equivalents in which Asn 221 was absent (Fig. 10A, 10B).

Asn 221 -containing mutants inhibit hemagglutination by influenza
To test if the binding to HA has any functional relevance, we used the World Health Organization-based hemagglutination inhibition protocol to quantify influenza-specific inhibitory titers of the mutants that bound the native virus strongly (Fig. 10C). Both D221N/C309L/N297A/C575A and D221N/C575A prevented hemagglutination by New Caledonia/20/99 virus (H1N1) at concentrations as low as 0.1 mM and were demonstrably more effective than molar equivalents of either IVIG or anti-H1N1 polyclonal IgG.
In contrast, the equivalent molecules that lack Asn 221 (i.e., C309L/N297A/C575A and C575A) failed to inhibit hemagglutination although partial inhibition was observed with the C575A mutant at the highest concentrations in some experiments (Fig. 10C). Hence, receptor binding of influenza A viruses is competed out only by mutants in which Asn 221 and Asn 563 are present. That both mutants run entirely as monomers by SEC-HPLC (Supplemental Fig. 1C) shows that the disposition of the glycans at the N terminus and C terminus of the Fc are more favorably orientated for binding native viral HA in monomers than multimers.

Discussion
Many groups have postulated that multivalent Fc constructs have potential for the treatment of immune conditions involving pathogenic Abs (2,5,37,38), and a recent study has shown that hexavalent Fcs can block FcgRs leading to their downmodulation and prolonged disruption of FcgR effector functions both in vitro and in vivo (39,40). Hexameric Fcs have also been shown to inhibit platelet phagocytosis in mouse models of idiopathic thrombocytopenic purpura (33,39,41).
Although disulfide-bonded hexameric Fcs may provide exciting new treatment approaches to control autoimmune diseases, they are more difficult to manufacture than smaller simpler Fc molecules. Their beneficial effects must also be carefully balanced with the acute risk of proinflammatory responses observed upon FcgR crosslinking and the increased risk from infection or cancers due to long-term immune suppression. These potential drawbacks with multimeric Fcs led us to investigate if complex monomers may be developed that retain the advantages of multimers (e.g., highavidity binding to low-affinity receptors) but that are also more readily manufactured to scale.
Furthermore, reported Fc mutations or glycan modifications have mostly focused on the conserved Asn 297 glycan that is largely buried within the Fc (4, 17-20, 27, 28), and thus monomeric IgG1 is unable to interact with a broad range of glycan receptors (Fig. 11A). Although Siglec-2 (35), DC-SIGN (2,54,55), DCIR (34), and FcRL5 (2,56) have all recently been shown to be ligands for IVIG, these interactions may also stem from specific Fabmediated binding (57). Thus, glycosylation of intact IgG is known to be critically important, but the relative contribution of the Fc, Fab, and/or their attached glycans, together with the identity of the salient receptors involved in IVIG efficacy, remain controversial.
The observed binding to CD22 was particularly surprising as this receptor prefers a-2,6 linked neuraminic acid and not a-2,3 linkages attached by CHO-K1 cells, although proximity-labeling experiments have recently shown that glycan-independent interactions of CD22/Siglec-2 with Ig in the BCR is possible (61).
The insertion of multiple glycan sites into the Fc, in particular at Asn 221 , enables new receptor interactions that are not possible with solely Asn 297 -directed approaches (Fig. 11A). For example, we generated the di-glycan D221N/C309L/N297A/C575A mutant that displayed marked binding to Siglec-1 and Siglec-4 (MAG), both receptors being clinically implicated in the control of neuropathy (15,25). This mutant showed no observable binding to either FcgRs or complement proteins (Tables II, III) yet was highly effective at blocking hemagglutination by influenza A virus (Fig. 10C).
As glycan-mediated binding is essential for the influenza virus to infect cells of the respiratory tract, mutations in HA that lead to loss of receptor binding are unlikely to survive any neutralizing Abs induced during an immune response (Fig. 11B). Modeling of the D221N/C575A mutant shows that the distance from the N-terminal to the C-terminal tips of the Fc is ∼60Å (Fig. 11B), which is the same distance between the sialic acid-binding domains on the HA trimer (65). The Asn 221 and Asn 563 sugars located at the tips of the Fc are not constrained by their location within the Fc, as with Asn 297 , and would therefore be expected to be highly mobile and flexible with respect to searching out the HA-binding pocket.
Alternative anti-influenza therapeutic strategies are urgently needed. The use of IVIG during the 2009 and 1918 pandemics reduced mortality from influenza by 26 and 50%, respectively (66,67), and a recent randomized, placebo-controlled study suggests these figures may be improved by enhancing influenza-specific Abs in IVIG (Flu-IVIG) preparations (36). As Flu-IVIG is manufactured in advance of future epidemics, there may be modest or no neutralizing activity against emerging strains. Combinations of Flu-IVIG or neuraminidase inhibitor drugs with Fc sialic acidbinding domain blockers may enhance the efficacy of Flu-IVIG or neuraminidase inhibitor-based medicines. Neither the D221N/ C575A nor D221N/C309L/N297A/C575A mutants that inhibited   Table II) and would thus not be expected to interfere with FcgRIIIA-dependent Ab-dependent cellular cytotoxicity toward influenza-infected cells by neutralizing IgG present in Flu-IVIG.
As well as direct HA binding, the molecules may shield sialic acid receptor binding sites on epithelial cells or act as decoy receptors through receptor mimicry, thereby preventing binding of the virus to epithelial target cells. Similarly, being rich in sialic acid, the molecules may also act as decoy substrates for neuraminidase. Intranasal delivery of Fc fragments may therefore be feasible, as Fc-fused IL-7 can provide long-lasting prophylaxis against lethal influenza virus after intranasal delivery (68). We have previously shown that Fc multimers can bind the neonatal Fc receptor (FcRn) (69). Thus, binding to the FcRn may act to increase the residence time of Fc blockers delivered to the lung (70,71).
A potential drawback to the hypersialylation approach with respect to blocking HA may be the susceptibility of Fc glycans to viral neuraminidase. Although neuraminidase from Clostridium perfringens could catalyze the hydrolysis of sialic acid residues from our soluble Fc fragments and thus block interactions with glycan receptors (Supplemental Fig. 1B), it remains to be tested if HA-bound Fcs are susceptible to catalysis by the influenza neuraminidase. We believe that metabolic oligosaccharide engineering with alkyne sialic acids could create neuraminidase-resistant Fc blockers (72).
In another example, multiple mutants were shown to bind DEC-205 (Figs. 4, 5, Table I), the major endocytic receptor expressed by dendritic cells, which suggests that these constructs may be useful for the targeted delivery of Ags in vaccines. Current approaches to deliver Ag to DEC-205 rely on DEC-205-specific delivery, often with Ags fused to anti-DEC-205 mAbs (73)(74)(75), whereas approaches that target multiple dendritic cell receptors, including DEC-205, may make for more effective Ag delivery.
As summarized in Tables I-III, we identified the following: 1) mutant Fc molecules that are capable of binding C1q and activating complement but that show little or no detectable interaction with either FcgRs or glycan receptors; 2) molecules with enhanced activation of complement, improved binding to FcgRs, and little engagement of glycan receptors; 3) molecules with enhanced binding to C1q but little C5b-9 deposition that retain interaction with both Fcg and glycan receptors; and 4) monomeric molecules with enhanced binding to a subset of sialic acid-dependent glycan receptors, in particular Siglec-1, Siglec-4, and HA, with little or no interaction with either FcgRs or complement.
Consequently, by adding or removing glycosylation and/or disulfide-bonding sites within our original hexameric Fc platform (2,5,24), new repertoires of desirable binding attributes can be made. These molecules may be useful in the control of other pathogens, including Newcastle disease virus, group B streptococci, Streptococcus pneumoniae, and Mycoplasma genitalium, in which sialic acid-dependent interactions are also crucially important (79).