Fc-Galactosylation of Human Immunoglobulin Gamma Isotypes Improves C1q Binding and Enhances Complement-Dependent Cytotoxicity

Binding of the complement component C1q to the CH2 domain of antigen-bound immunoglobulin gamma (IgG) activates the classical complement pathway and depends on its close proximity to Fc fragments of neighboring antibodies. IgG subclasses contain a highly conserved asparagine 297 (N)-linked biantennary glycan within their CH2 domains, the core structure of which can be extended with terminal galactose and sialic acid residues. To investigate whether Fc-glycosylation regulates effector functions of human IgG subclasses, we cloned the antigen-binding region of the CD20-specific monoclonal antibody rituximab into IgG isotype expression vectors. We found that Fc-galactosylation enhances the efficacy of CD20-targeting complement-fixing antibodies for C1q binding and complement-mediated tumor cell lysis. Increased efficacies were restricted to IgG1 and IgG3 subclasses indicating that Fc-galactosylation alone is not sufficient for IgG2 and IgG4 to acquire complement-fixing properties. Addition of terminal galactose to the N-glycan specifically improved binding of C1q without changing antigen- and FcγRIIIa-binding affinities of IgG isotypes. These data indicate that Fc galactosylation can be harnessed to enhance the complement-activating properties of IgG1 and IgG3 antibodies.

folding and post-translational quality control mechanisms and is essential for antibody-mediated effector functions (3,4). The biantennary core glycan structure, which is composed of two N-acetylglucosamines (GlcNAc) and three mannose residues, can be further decorated with fucose, bisecting GlcNAc and terminal GlcNAc, galactose, and sialic acid. The highest degree of variability among IgG antibodies stems from the presence or absence of galactose with around 40% containing one galactose, 20-40% two galactoses, and the remainder none (5)(6)(7). Presence or absence of distinct N-glycan residues such as fucose and sialic acid can dramatically alter pro-and antiinflammatory IgG activities. Removal of the fucose residue from human IgG1 increases antibody-dependent cell-mediated cytotoxicity (ADCC) through improved affinity for Fcγ receptor IIIa (FcγRIIIa) (8,9). We have previously shown that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-dependent cytotoxicity (CDC) (10). Here, we determined whether Fc galactosylation of human IgG isotypes (IgG1-4) regulates antibody effector functions. resUlTs generation of Degalactosylated and galactosylated igg isotypes Targeting cD20 To test the impact of Fc galactosylation of human IgG isotypes on target cell depletion, we first cloned the sequence encoding the heavy chain (HC) and light chain (LC) antigen-binding regions of the monoclonal CD20-targeting antibody rituximab into human IgG1-4 HC and kappa LC expression vectors. Plasmids were cotransfected in HKB11 cells and antibodies were purified from the supernatant using protein G columns. Next, we either treated the antibodies with recombinant galactosidase to completely remove galactose or with β-1,4-galactosyltransferase in the presence of uridine diphosphate (UDP-) galactose to obtain galactosylated antibodies. After repurification, antibodies were analyzed for purity and integrity by gel electrophoresis and immunoblotting with the galactose-specific Erythrina Cristagalli lectin to confirm the successful generation of glycovariants (Figure 1). FigUre 2 | Fc-galactosylation does not increase CD20 binding of rituximab-derived IgG isotypes. Target antigen recognition by galactosylated and degalactosylated human IgG1-4 isotypes specific for CD20 analyzed by flow cytometry via titration on CD20 + Raji cells. MFI, median fluorescence intensity; IgG, immunoglobulin gamma. addition or removal of igg-Fc galactose Does not affect antigen Binding Modifications in the Fc domain may change the structural properties of the antibody, potentially leading to changes in its antigen-binding region. To determine antigen-binding affinities of IgG isotype glycovariants, we titrated the antibodies on CD20 expressing human Raji-Burkitt's lymphoma cells and analyzed binding by flow cytometry (Figure 2). For each isotype, galactosylated and degalactosylated glycovariants did not differ in their antigen-binding characteristics.
igg-Fc galactosylation of igg1 and igg3 isotypes increases c1q Binding and complement-Dependent cytotoxicity The efficacy of IgG isotype-derived glycovariants to induce complement-dependent cytotoxicity (CDC) was determined in Burkitt's lymphoma-derived Raji cells in the presence of active complement (human serum). Rituximab-derived IgG3 glycovariants showed the highest efficacy for CDC, followed by IgG1 (Figure 3). Glycovariants of IgG2 and IgG4 isotypes did not induce CDC. Galactosylation increased CDC mediated by both IgG1 (26% reduction of EC50) and IgG3 (13% reduction of EC50) but did not provide IgG2 and IgG4 with de novo ability to lyse target cells (Figure 3). To investigate the mechanism by which Fc-galactosylation impacts CDC, we determined the C1q binding affinities and kinetics of galactosylated and degalactosylated antibody variants (10). Incubation of CD20-expressing Raji cells in the presence of human serum depleted for C5, an essential component of the complement cascade, which allows to analyze the binding of members of the complement cascade to target cells while preventing cell lysis (10), led to rapid binding of C1q (Figure 4). Fc-galactosylation substantially enhanced the antibodies' capacity to bind C1q for IgG1 and IgG3 isotypes (Figure 4). These data indicate that the addition of terminal galactose to the Fc-glycan enhances cell-depleting efficacies of human IgG1 and IgG3 isotypes through increased C1q binding.
Fc-galactosylation Does not increase igg:Fcγriiia-Binding affinities Rituximab depletes B cells through a combination of CDC and antibody-dependent cell-mediated cytotoxicity (ADCC), which requires antibody binding to the human-activating FcγRIIIa (CD16) (10,11). Absence of the IgG-Fc core fucose increases binding to FcγRIIIa (8,12), a finding increasingly being used to improve the efficacy of therapeutic antibodies (13)(14)(15). Two studies reported that Fc-galactosylation results in a slight, albeit not statistically significant increase in FcγRIIIa-binding affinity and ADCC activity (16,17). A more recent study confirmed that afucosylated glycoforms show higher binding affinities for FcγRIIIa, while Fc-galactosylation did not significantly impact FcγRIIIa binding (18). To investigate whether Fc-galactosylation of IgG isotype glycovariants increases binding affinities of IgG isotypes for FcγRIIIa, thereby potentially enhancing the antibodies' ability to induce ADCC, we titrated IgG glycovariants onto CHO-derived cell lines recombinantly expressing FcγRIIIa. FcγRIIIa binding was strongest for IgG1 and IgG3 isotypes FigUre 3 | Increased CDC of rituximab-derived immunoglobulin gamma 1 (IgG1) and IgG3 but not IgG2 and IgG4 upon Fc-galactosylation. Complementdependent lysis of CD20 + target cells in the presence of galactosylated or degalactosylated human anti-CD20 IgG isotypes. Exemplary lysis curves and EC50 values of three independent experiments are shown. Statistical analysis: paired two-tailed Student's t-test *p < 0.05, **p < 0.01.
( Figure 5). IgG-Fc galactosylation showed, however, no effect on FcγRIIIa-binding affinities for any of the IgG isotypes tested ( Figure 5). Thus, while increasing the antibodies' affinity for C1q binding and their efficacy to induce CDC, Fc-galactosylation did not change the affinity of human IgG isotypes for FcγRIIIa which, upon ligation, mediates ADCC.

DiscUssiOn
Initiation of the classical complement cascade trough binding of C1q is a potent proinflammatory mechanism by which IgG antibodies trigger immune responses during infection and its deregulation causes tissue damage in a wide array of human inflammatory, degenerative, and autoimmune diseases (19). Our study shows that Fc-galactosylation enhances the efficacy of complement-fixing IgG isotypes to induce CDC through improved binding of C1q.
The antibody-dependent classical complement pathway is initiated if the C1 complex, formed by the multimeric pattern recognition molecule C1q and the modular proteases C1r and C1s, docks on antigen-bound IgG (20). C1q binds to monomeric IgG with very low affinity but antigen-driven antibody clustering allows for the formation of IgG hexamers that bind C1q with high avidity and promote efficient complement activation (21). The N-glycan resides in the CH2 domain, which is required for C1q binding (22). Sites on the surface of human IgG1 that constitute the C1q-binding epicenter are conserved in human IgG isotypes that are deficient in C1q binding and it has therefore been suggested that the composition of the N-glycan might be critical for the antibodies' conformation and its ability to bind C1q (23).
Each biantennary oligosaccharide chain extends one arm toward the CH2-CH3 interface region and the other arm into the space between the CH2 domains, resulting in multiple interactions with the surface of the CH2 domain and the glycan of the opposing CH2 domain, respectively (4). Indeed, optimal C1 activation requires the presence of the Fc-glycan since C1q-mediated effector functions are compromised or lost in aglycosylated or deglycosylated IgGs (24)(25)(26). The effect of deglycosylation on reducing C1q binding has recently been attributed to its inhibition of IgG hexamerization via modulation of IgG Fc:Fc interactions rather than reduction of direct C1q-Fc-binding affinities (27). Based on these data and our results, we suggest that Fc-galactosylation modulates Fc:Fc interactions for antigen-bound IgG, thereby improving binding of C1q and increasing the antibodies' ability to induce classical complement activation and CDC.
We systematically investigated whether Fc-galactosylation facilitates C1q binding and CDC effector functions across all human IgG isotypes. For murine IgG2b and IgG1, it has been demonstrated that addition of terminal galactose increases binding of C1q (28). While presence of terminal galactose enhanced complement activation by CD20 targeting, C1q-fixing human IgG1 and IgG3 isotypes, IgG2 and IgG4 remained deficient in initiating the classical complement cascade indicating that Fc-galactosylation alone is not sufficient for IgG2 and IgG4 to acquire complement-fixing properties.
Rituximab and therapeutic monoclonal antibodies (mAbs) that target tumor cells via ADCC or CDC are approved for the treatment of various cancers (29). B-cell depletion by CD20targeting antibodies is also widely used for the treatment of autoimmune diseases (30). However, some patients do not sufficiently  respond to rituximab therapy (31,32) and improved versions of B cell depleting antibodies have been developed to increase ADCC activity and improve clinical efficacy (33,34). Our data indicate that Fc-galactosylation, in addition to the established effect of defucosylation on ADCC, increases target cell cytotoxicity through enhancing CDC. Fc-galactosylation specifically improved binding of C1q to human IgG1 and IgG3 without changing antigen-binding affinities. Our data therefore indicate that Fc-galactosylation should be harnessed in glycoengineering therapeutic antibodies for effective antibody-dependent complement activation and target cell killing.

cDc assay
Raji cells were used as CD20 + target cells to assess CDC. Accordingly, 7 × 10 4 Raji cells were cultivated in RPMI-1640 containing P/S (50 U/ml) in a humidified incubator (37°C, 5% CO2) in 96-well V-bottom plates. Cells were incubated with titrated concentrations of the respective galactosylated and degalactosylated anti-CD20 antibody isotypes. After 30 min, human serum complement was added to a final concentration of 5% and incubation was continued for another 12 h. Thereafter, cells were washed twice by adding 200 µl cold PBS and centrifugation at 400 × g and 4°C for 5 min. Cells were resuspended in cold PBS and TO-PRO3 stain (final concentration of 200 nM) was added to detect dead cells. All samples were analyzed on a BD FACSCanto-II using FACSDiva v6.1.3 software and FlowJo software v9.3.1 (Tree Star Inc.). Specific lysis was calculated as percent increase in dead cells compared to spontaneous lysis in the absence of an anti-CD20 antibody. EC50 values were calculated via a non-linear regression in GraphPad Prism 5.

Fcγr-Binding assay
Human FcγRIIIa-expressing CHO cells (35) (2,37,38). The N terminal region of the rituximab heavy chain was cloned into the MCS using AgeI and SalI as previously described (36). Recombinant monoclonal antibodies were expressed and purified as previously described (10,32,39). Briefly, heavy and light chain expression plasmids were cotransfected into a human B cell-epithelial cell fusion cell line (HKB11) supporting high-level recombinant protein expression (40) using calcium phosphate-mediated transfection. After 6 days, antibodies were purified from cell culture supernatants by binding to a protein G column and elution with 0.1 M glycine (pH 2) followed by neutralization with 1 M Tris pH 8.8 and dialysis to PBS. Antibody purity and integrity were confirmed by polyacrylamide gel electrophoresis, coomassie brilliant blue staining and Western blotting with isotype-specific monoclonal antibodies.

generation of galactosylated and Degalactosylated antibody glycovariants
Antibody glycovariants were generated as previously described (10,39). For removal of galactose, antibodies were dialyzed to 50 mM sodium phosphate buffer pH 6.0, 60 mU β1-4 galactosidase was added per mg of antibody and the reaction was incubated for 6 h at room temperature followed by 1 h at 37°C. To add galactose, antibodies were dialyzed to 0.2 M MES pH 6.5 followed by the addition of 10 mM MnCl2, UDP-galactose, 0.02% NaN3, and 5 µg recombinant β1-4 galactosyltransferase [produced inhouse (39)]. The reaction was incubated at 37°C for 48 h. Finally, all antibodies were centrifuged for 2 h at 4°C and >20,000 × g to remove aggregates, repurified by gravity-flow protein-G sepharose columns, and dialyzed to PBS. Successful degalactosylation and galactosylation were monitored by lectin-blotting using the galactose-specific lectin from Erythrina Cristagalli.