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).

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.

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.

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 (13–15). 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 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.

Human serum complement (Cat. No.: A100; Lot No.: 035997) and C5-depleted human serum (Cat. No.: A501; Lot No.: 031795) were purchased from Quidel^® Corporation. Accutase was purchased from StemCell Technologies Inc. (Cat. No.: 07920), Gibco™ RPMI-1640, HEPES (Cat. No.: 52400025), penicillin–streptomycin (P/S; 5,000 U/ml; Cat. No.: 15070063), LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Cat. No.: L34957), LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Cat. No.: L10119), and TO-PRO-3 Stain (Cat. No.: T3605) were purchased from Invitrogen. Fetal calf serum (FCS) was purchased from Sigma-Aldrich (Cat. No.: F7524-500M; Lot No.: 051M3395) and from Bioswisstech (Cat. No.: S0615; Lot No.: 1047D). β1-4 galactosidase (Cat. No.: 345806-50MIU) and UDP-galactose (Cat. No.: 670111-50MG) were purchased from Merck Millipore Corporation, Calbiochem^®. Protein-G sepharose was purchased from GE healthcare (Cat. No.: 17-0618-01). Biotinylated Erythrina Cristagalli lectin (Cat. No.: B-1145) and Lens Culinaris Agglutinin (Cat. No.: B-1045) were purchased from Reactolab. Phosphate-buffered saline (PBS; 2.68 mM KCl, 8 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, pH adjusted to 7.3) was produced in-house.

Monoclonal mouse anti-human C1q (Cat. No.: A201) and biotinylated anti-human C1q (Cat. No.: A700) were purchased from Quidel^® Corporation. Polyclonal rabbit anti-mouse IgG (H + L)-Alexa Fluor 488 (Cat. No.: A-11059) was purchased from Invitrogen. PE-conjugated streptavidin (Cat. No.: 405203) was purchased from Biolegend.

Raji cells were used as CD20⁺ target cells to assess CDC. Accordingly, 7 × 10⁴ 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.

A total of 2 × 10⁵ Raji cells were incubated with 10 µg/ml with galactosylated and degalactosylated anti-CD20 antibody isotypes in RPMI-1640 containing 1% P/S (50 U/ml) and 1% C5-depleted human serum for 5, 15, 30, or 60 min in a humidified incubator (37°C, 5% CO₂). After incubation with the different anti-CD20 antibody isotypes, cells were washed twice with 200 µl cold PBS, and C1q binding was determined by adding biotinylated anti-C1q antibody (25 µg/ml in PBS) for 1 h on ice followed by washing twice with 200 µl cold PBS and incubation with PE-conjugated streptavidin (1:400 in PBS) and dead cell stain (LIVE/DEAD Fixable Aqua Dead Cell Stain Kit; 1:250 in PBS) for 20 min. After washing twice with 200 µl PBS, samples were analyzed on a BD FACSCanto-II using FACSDiva v6.1.3 software and FlowJo software v9.3.1 (Tree Star Inc.).

Raji cells were maintained in RPMI-1640 containing P/S (50 U/ml) and 10% FCS in a humidified incubator (37°C, 5% CO₂). 1.2 × 10⁵ Raji cells and anti-CD20 antibody isotypes and glycovariants were incubated for 25 min on ice. Dead cells were excluded using LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit. Cells were washed twice with 200 µl PBS and anti–mouse IgG (H + L)-Alexa Fluor 488 was added and incubated for 20 min on ice followed by two washing steps with 200 µl cold PBS. All samples were analyzed on a BD FACSCanto-II using FACSDiva v6.1.3 software and FlowJo software v9.3.1 (Tree Star Inc.).

Human FcγRIIIa-expressing CHO cells (35) were kindly provided by Falk Nimmerjahn (Department of Biology, University of Erlangen-Nürnberg, Germany). Cells were maintained in RPMI-1640 containing P/S (50 U/ml) and 10% FCS in a humidified incubator (37°C, 5% CO₂). To detect binding of anti-CD20 antibodies, cells were detached using accutase, washed with cold PBS, and 2 × 10⁵ cells were incubated on ice for 30 min with anti-CD20 antibodies. After incubation, cells were washed twice with 200 µl cold PBS and anti-mouse IgG (H + L)-Alexa Fluor 488 was added for 20 min on ice. All samples were washed twice with 200 µl cold PBS and analyzed on a BD FACSCanto-II using FACSDiva v6.1.3 software and FlowJo software v9.3.1 (Tree Star Inc.).

The DNA encoding for the N terminal region of the light chain (amino acids QIVLS until KLEIK) and heavy chain (amino acids QVQLQ until TVSAA) of rituximab (www.drugbank.ca; accession number DB00073) was synthesized by Invitrogen GeneArt gene synthesis. The N terminal region of the light chain was cloned into the multiple cloning site (MCS) of Igκ-AbVec (NCBI GenBank accession number FJ475056.1) using AgeI and BsiWI as previously described (36). To obtain human IgG-1, -2, -3 and -4 heavy chain expression vectors IgG-AbVec (NCBI GenBank accession number FJ475055.1) (36) was modified to encode for human IgG1 (allotype G1m17,1), IgG2 (allotype G2m), IgG3 (allotype G3m), and IgG4 (kindly provided by Lars Hangartner, the Scripps Research Institute, Department of Immunology and Microbial Science, La Jolla, CA, USA) (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.

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 in-house (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.

A p-value of 0.05 or less was defined as statistically significant. For all analyses, Prism software, version 5 (GraphPad Software, Inc.) was used. Specific statistical tests are indicated in the respective figure legends.