Antibody fucosylation differentially impacts cytotoxicity mediated by NK and PMN effector cells

Glycosylation of the antibody Fc fragment is essential for Fc receptor-mediated activity. Carbohydrate heterogeneity is known to modulate the activity of effector cells in the blood, in which fucosylation particularly affects NK-cell mediated killing. Here, we investigated how the glycosylation profile of 2F8, a human IgG1 monoclonal antibody (mAb) against EGF-R in clinical development, impacted effector function. Various 2F8 batches differing in fucosylation, galactosylation and sialylation of the complex-type oligosaccharides in the Fc fragment were investigated. Our results confirmed that low fucose levels enhance MNC-mediated ADCC. In contrast, PMN were found to preferentially kill via high-fucosylated antibody. Whole blood ADCC assays, containing both types of effector cells, revealed little differences in tumor cell killing between both batches. Significantly, however, high-fucose antibody induced superior ADCC in blood from G-CSF-primed donors containing higher numbers of activated PMN. In conclusion, our data demonstrated for the first time that lack of fucose does not generally increase the ADCC activity of therapeutic antibodies, and that the impact of Fc glycosylation on ADCC is critically dependent on the recruited effector cell type. Assays effector and target cells a (E:T) ratio 80:1, unless otherwise PMN stimulated by GM-CSF (50 U/ml). and variation of antibody concentrations at a fixed E:T ratio (80:1). To define Fc receptors involved in (B) MNC- or (D) PMN-mediated target cell killing, ADCC assays were performed in the presence of F(ab´) 2 -fragments of Fc γ  receptor blocking antibodies AT10 (Fc γ RII), or 3G8 (Fc γ RIII), or control F(ab´) 2 -fragments (all at 10 μ g/ml). 2F8-C ( ), 2F8-H ( ),


Introduction
Monoclonal antibodies constitute a growing class of therapeutics -with major indications in oncology, infectious diseases and autoimmunity 1 . In oncology, antibody-mediated cellular cytotoxicity (ADCC) is considered a particularly relevant mechanism of action for therapeutic antibodies 2 . Evidence for this is mainly derived from studies with the CD20 antibody rituximab -the most intensively investigated antibody in this regard. For example, rituximab lost most of its therapeutic efficacy against xeno-transplanted human tumors in mice lacking activating Fc receptors by knock-out of the common FcRγ-chain 3 .
Syngeneic B-cell depletion by murine CD20 antibodies has furthermore been correlated with antibody isotypes and with their respective binding to activating, compared to inhibitory Fcγ receptors 4,5 . In patients, rituximab's therapeutic efficacy has been correlated with well-defined FcR polymorphisms affecting binding of human IgG and the ability to induce ADCC in vitro 6,7 . These and other observations stimulated studies exploring opportunities to improve antibodies' capacity to trigger ADCC 8,9 . This can be achieved by increasing antibody binding to activating cellular Fc receptors such as NK-cell expressed FcγRIIIa, and by decreasing binding to the inhibitory FcγRIIb isoform. At least two different methodologies have been established: one modifying the protein structure of the antibody Fc region by mutating the respective cDNAs 10,11 , while the other is based on technologies altering the glycosylation profile of antibodies [12][13][14][15] .
Post-translational modifications such as glycosylation are increasingly recognized to alter the functional activity of biopharmaceuticals 16,17 . For antibodies, glycosylation of Asn297 in the CH2 domain has long been recognized to be critical for complement activation 18 and Fc receptor binding 19,20 . More detailed analyses revealed that lack of fucose at this glycosylation site selectively improved binding to FcγRIIIa, while binding to a number of other Fcγ receptors appeared to be unaffected by this modification [12][13][14] . Crystallographic studies demonstrated that sugar moieties on antibody Fc formed only minor interactions with the amino acids of FcγRIIIa 21 , suggesting that the sugars act indirectly by conferring subtle conformational alterations in a limited region of Fc and possibly by decreasing the mobility of the CH 2 domain 22,23 . Further functional analyses revealed enhanced ADCC by isolated mononuclear effector cells for lowfucosylated compared to high-fucosylated antibodies [12][13][14]24 . More recent studies suggested that in addition to Fc fucosylation also sialylation affected FcR binding and antibody function 25,26 . Antibody galactosylation, on the other hand, was demonstrated to impact complement activation via the lectin pathway 27 , but not Fc receptor mediated functions 28 . However, to the best of our knowledge, none of the previous studies addressed the impact of antibody glycosylation on PMN function. PMN constitute the first line of defense against invading bacteria 29 and may significantly contribute to tumor rejection 30 -at least for some antibodies such as those against the epidermal growth factor receptor (EGF-R) 31,32 .
EGF-R is a tyrosine kinase receptor, which is expressed on common solid cancers such as colon, lung, head and neck as well as select non-epithelial tumors such as glioblastomas 33 . Since activation of this receptor is associated with accelerated tumor cell proliferation and progression to a more malignant tumor phenotype, EGF-R constitutes an attractive molecule for targeted therapies. Consequently, several EGF-R-directed monoclonal antibodies have been developed for clinical applications, two of which have obtained FDA approval 34 . In this manuscript, we describe effector functions of glycosylation variants of a fully human IgG1 EGF-R antibody 35 . These variants bound comparably to EGF-R, inhibited EGF-R phosphorylation and efficiently blocked proliferation of EGF-R expressing tumor cells. Only fucosylation and not sialylation or galacotosylation was found to impact ADCC. As expected, lowfucosylated variants were more efficient in binding to FcγRIIIa and in the recruitment of MNC as effector cells for ADCC. Notably, however, highfucosylated batches were more effective in triggering PMN, which mediated tumor cell lysis via FcγRII and which significantly contributed to the whole blood ADCC activity of these antibodies.

Material and Methods
Experiments reported here were approved by the Ethical Committee of the Christian Albrechts University, Kiel, Germany, in accordance with the Declaration of Helsinki. Blood donors were randomly selected from healthy volunteers, or from G-CSF-primed hematopoietic stem cell donors, who gave written informed consent before analyses.
MAb 2F8 was selected for its potency to block the interaction between EGF-R and its ligands, EGF and TGF-α. MAb 2F8-H was produced by the original 2F8 monoclonal hybridoma cells (derived from SP2/0). MAb 2F8-C was produced in a transfectoma cell line (derived from CHO-DG44cells). Culture supernatants of both cell lines were purified using protein A affinity chromatography, followed by size exclusion chromatography on an HR200 column (Pharmacia, Peapack, NJ), and were formulated in PBS containing Tween 80 and mannitol. 2F8 Fab fragments were made by papain digestion. Human IgG1κ, specific for keyhole limpet hemocyanin (KLH), developed using the same mouse strain, served as isotype control. Purity and monomerity were analysed by SDS-PAGE, isoelectric focusing (IEF) and high-performance size-exclusion chromatography (HP-SEC).
Degalactosylated and desialylated material of 2F8-H and 2F8-C was prepared by treatment with neuraminidase (acrobacter, Roche, Mannheim, Germany), 1,4 βgalactosidase (Streptococcus pneumoniae, Calbiochem, San Diego, CA), and αgalactosidase (Sigma-Aldrich) for 48 hours at 37º C. Unexpectedly, approximately 30% of the N-glycolylneuraminic acid present on 2F8-H appeared resistant for neuramidase-treatment. As a substitute we used material from mAb 2F8 produced in HEK-293 cells, which contains comparable levels of fucose as 2F8-H, but lacks sialic acids in the N-linked glycan structure. After enzymatictreatment the material was repurified and formulated as described above. Both preparations were analyzed by HPEAC-PAD to confirm removal of galactose and sialic acid. The degalactosylated and desialylated material of 2F8 produced in CHO cells was named 2F8-C-(F -/G -/S -), that of 2F8 produced in HEK-293 cells Plates were incubated at 37º C for 5 days, before AlamarBlue solution (Biosource, Camarillo, CA) was added. Plates were incubated for another 4 hours, transferred to room temperature (RT), and fluorescence of reduced AlamarBlue was measured by exciting at 528 nm and measuring emission at 590 nm on Synergy HT plate reader (Bio-Tek Instruments, Winooski, VT).

Inhibition of EGF-R phosphorylation.
Inhibition of EGF-induced EGF-R phosphorylation was measured using a two-step assay. Briefly, A431 cells were cultured overnight in serum-deprived medium. Cells were then incubated with serial dilutions of mAb 2F8 at 37º C. After 60 minutes, 50 ng/ml recombinant human EGF (Biosource) was added for an additional 30 minutes. Subsequently, cells were solubilized with lysis buffer (Cell Signaling Technology, Beverly, MA), lysates were transferred to ELISA plates coated with 1 μg/ml of mouse anti-EGF-R antibodies (mAb EGFR1, BD Pharmingen, San Diego, CA) and incubated for 2 hours at RT. Next, the plates were washed and binding of phosphorylated EGF-R was visualized using a europium-labelled mouse mAb, specific for Antibody dependent cell-mediated cytotoxicity (ADCC) assays. ADCC assays against 51 Cr labeled target cells were performed as described 37 . Whole blood (50 μL), plasma or isolated effector cells and sensitizing antibodies at varying concentrations were added to microtiter plates (Nunc, Neerijse, Belgium).
Assays were started by adding effector and target cells at a (E:T) ratio of 80:1, unless otherwise indicated. Isolated PMN were stimulated by GM-CSF (50 U/ml).
After three hours at 37º C, 51

Antibody production, characterization and glycosylation analyses
To analyze the impact of antibody Fc glycosylation on PMN-and MNC-mediated ADCC we compared glycosylation variants of a fully human EGF-R antibody of the IgG1 isotype. Two batches of this mAb were produced by hybridoma cells, However, their N-linked oligosaccharide structures -analyzed by HPAEC-PAD and MALDI-MS -showed some major differences (Table 1, supplementary Table   1, supplementary Figure S1A). The analysis of 2F8-C carbohydrate indicated that about 25 % of the complex type N-linked glycans were not core-fucosylated on the reducing N-acetylglucosamine (Peaks 2, 4, 6 and 7). In contrast, for 2F8 produced in hybridoma cells, non-core fucosylated glycans, i.e. peaks 2 and 4 were not detected -demonstrating that 2F8-H was fully fucosylated.
In addition, both batches showed differences in sialylation and galactosylation.
2F8-C mainly contained non-sialylated complex type glycans, with limited galactose. About 46 % of the glycans did not contain galactose, about 35 % contained only one galactose, and only 6 % were fully galactosylated. We did not observe the presence of α-galactosyl or N-glycolylneuraminic acid (NeuGc) on 2F8-C. Hybridoma-derived 2F8-H contained significantly more sialylated glycans (i. e. peaks eluting between 37 -43 and 55 -58 min) compared to CHO derived mAb 2F8-C. Characterization of these charged peaks revealed that they were mainly core-fucosylated complex type glycans with one (structures between 37 -43 min) or two N-glycolylneuraminic acids (structures between 55 -58 min).
Taken together, although the difference in the fucose content was most prominent, there were also differences in other monosaccharides, such as galactose and sialic acid.

Antigen binding and Fab-mediated direct effector functions
In a first set of functional experiments, the antigen binding characteristics of the two antibody preparations were investigated. 2F8-C was compared to it's highly fucosylated counterpart 2F8-H. Binding to purified EGF-R and to native EGF-R on the cell surface of A431 cells was analyzed by ELISA and indirect immunofluorescence staining, respectively. Both antibodies bound specifically and with similar EC50 to purified EGF-R (data not shown) and to cell-surface expressed EGF-R ( Figure 1A) -demonstrating that the differences in glycosylation did not influence antigen binding characteristics of the two antibody preparations.
To address whether also biological activity mediated by the antigen-binding Fab showed no significant differences in their ability to modulate EGF-R signaling, which demonstrated that differences in Fc fragment glycosylation did not impact the function of the antigen combining site.

Fc-mediated effector functions
It is well established that the Fc glycosylation profile of antibodies may affect Fcmediated effector functions such CDC 38 and ADCC [12][13][14] . However, in CDC assays with A431 tumor cells as targets and human plasma as source of complement, none of the tested antibody preparations triggered significant complement-mediated lysis (not shown). This is probably attributable to the high expression levels of membrane-bound complement regulators (e.g. CD55 and CD59) on most solid tumor cells.
In order to investigate whether the observed glycosylation differences resulted in altered Fc-mediated biological effector functions, the two glycosylation variants of 2F8 were compared for their ability to trigger ADCC (Figure 2). We first analyzed MNC, in which NK cells primarily serve as effector cells. Low-fucosylated 2F8-C induced superior ADCC compared to highly fucosylated 2F8-H (Figure 2A) Figure 2B).
Killing mediated by 2F8-C was inhibited to a lesser extent than lysis by 2F8-Hmost likely reflecting the higher affinity of 2F8-C for CD16. In contrast, no inhibition of MNC-mediated killing was observed with AT10 F(ab')2-fragmentsdemonstrating that FcγRII (CD32) was not involved in 2F8 mediated tumor cell lysis by MNC ( Figure 2B).
Next, we performed ADCC with PMN effector cells ( Figure 2C). Notably, both 2F8-C and 2F8-H antibodies mediated efficient and significant ADCC by PMN.
Compared to MNC, which triggered maximum lysis at low antibody concentrations, higher antibody concentrations were required for efficient ADCC by PMN. Interestingly, high-fucosylated 2F8-H antibody demonstrated enhanced maximum lysis compared to its low-fucosylated counterpart, but no significant differences in the EC50 values of 2F8-C and 2F8-H were observed. Thus, also PMN-mediated ADCC was affected by the glycosylation profiles of the antibody preparations ( Figure 2C). In contrast to MNC-mediated lysis, PMN-mediated killing was completely blocked by AT10 F(ab')2 fragments against FcγRII (CD32) -demonstrating that FcγRII engagement was necessary for efficient tumor cell lysis by PMN. Interestingly, PMN-mediated killing was not inhibited, but rather stimulated by 3G8 F(ab')2 fragments against FcγRIII (CD16) ( Figure 2D). These results with blocking antibodies and PMN effector cells were independent of the glycosylation status of the targeting antibodies.

Impact of sialic acid, galactose and fucose removal
Although the difference in fucose content between the two antibody batches was most prominent, we also observed differences in other monosaccharides, such as galactose and sialic acid, as discussed above (Table 1 Figure 3G), but not sialic acid ( Figure 3A) or galactose ( Figure 3C) was responsible for enhanced ADCC activity by MNC effector cells -as also published by others 12,14 . However, lack of fucose from the carbohydrate structure negatively affected PMN-mediated killing ( Figure 3H). To our knowledge, this is the first report that reduced levels of fucose in antibody Fc may adversely affect antibody-mediated effector functions.

Fc receptor binding of glycosylation variants
In Therefore, the major affinity difference of fully fucosylated 2F8-H for FcγRIIIa-V158 versus -F158 was minimized for low-fucosylated 2F8-C -consistent with previous findings from others 14,39 . In addition binding of low-fucose antibodies to FcγRIIIb was enhanced; more pronounced differences were observed for the FcγRIIIb-NA1 allotype. Interestingly, we observed an approximately twofold higher affinity of human IgG1 for FcγRIIa-H131 compared to -R131, which was not influenced by fucosylation levels. Binding to FcγRIIa, FcγRIa, and to FcγRIIb was not significantly affected by altering the glycosylation profile of the antibodies ( Figure 4). Binding of exo-glycosidase-treated batches to FcγRIa, FcγRIIIa (V/F158), FcγRIIa (R/H131) and FcγRIIb in ELISA confirmed that fucose content modulated the affinity to FcγRIIIa (V/F158), but did not affect binding affinity to FcγRIa, FcγRIIa (R/H131) and FcγRIIb. Removal of galactose and sialic acid did not impact on the binding of 2F8 to FcγRIA, FcγRIIa (R/H131) and FcγRIIb, but slightly decreased the affinity to FcγRIIIa (V/F158) (supplementary Figure S2). In conclusion, these data underline that MNC-mediated killing was primarily attributable to enhanced FcγRIIIa binding due to the lack of fucose. As no differences in binding to FcγRIIa were observed, the mechanism of superior PMN killing was not attributable to differences in binding affinity to this receptor. The superior PMN killing by high-fucosylated antibodies may therefore correlate with the lower affinity of high-fucose antibodies to FcγRIIIb. This is in accordance with CD16 blocking experiments demonstrating that blocking FcγRIIIb enhances PMN-mediated ADCC. Together these findings suggest that high affinity binding to FcγRIIIb partially inhibits PMN-mediated killing ( Figure 2D).

Effector cell recruitment in whole blood ADCC assays
In a further set of experiments the glycosylation variants 2F8-H and 2F8-C were tested in human whole blood to induce ADCC with a physiological mixture of effector cells. Interestingly, only small differences in lysis were observed between the two antibody variants ( Figure 5A). These differences were less pronounced than with isolated MNC or PMN (Figure 2A,C). Since the Fc receptor involvement in MNC-and PMN-mediated tumor cell killing was different (MNC via CD16, PMN via CD32), we aimed to investigate the contribution of both effector cell populations to whole blood ADCC. However, because in ADCC assays with isolated effector cells, F(ab')2-fragments against FcγRIII stimulated PMNmediated killing ( Figure 2D), we could not use this antibody in our whole blood ADCC assay. We therefore performed experiments with AT10 F(ab')2-fragments to selectively block PMN-mediated killing. In these experiments, blockade of CD32 resulted in partial inhibition of tumor cell lysis. Control F(ab')2-fragments did not affect whole blood ADCC ( Figure 5B). Together, these results clearly demonstrated that PMN contributed to tumor cell killing in whole blood. To further address the contribution of PMN in 2F8-mediated whole blood killing, we analyzed blood from G-CSF-primed donors. As shown in Figure 5C, ADCC activity in G-CSF-primed blood was significantly enhanced compared to healthy donor blood ( Figure 5A). Blocking experiments revealed that killing in G-CSFprimed blood was more efficiently blocked by AT10 F(ab')2-fragments than killing in healthy donor blood -indicating the PMN contribution to tumor cell lysis to be enhanced during G-CSF treatment. Notably, high-fucosylated 2F8-H was significantly more effective in blood from G-CSF-treated patients than lowfucosylated 2F8-C.

Discussion
In this manuscript, we investigated the impact of antibody Fc glycosylation on triggering Fc receptor-mediated tumor cell killing by different human effector cell types. Glyco-engineering of therapeutic antibodies currently receives great attention, because it is thought to represent a promising approach to improve antibody binding to activating, compared to inhibitory Fcγ  receptors 14,40 . As previously reported for antibodies against other target antigens 12-14,24,40 , we observed low-fucosylated batches of a human EGF-R-specific antibody to be more effective in binding to recombinant FcγRIIIa than its high-fucosylated variant, which led to enhanced tumor cell killing in ADCC assays with MNC effector cells.
Interestingly, low fucosylated 2F8-C showed smaller affinity differences between FcγRIIIa-V158 and -F158 than fully fucosylated 2F8-H. This observation suggests that the clinically relevant FcγRIIIa polymorphism may be less important for low compared to high fucosylated antibodies -as also proposed by others 14,39 . However, lower levels of fucose adversely affected PMN-mediated killingleading to similar killing levels by both antibody batches in human whole blood assays. Similarly, a fully fucosylated HLA class II antibody was more effective in recruiting PMN than its non-fucosylated variant (data not shown) -demonstrating that this observation is not dependent on one particular target antigen. Notably, the impact of antibody fucosylation on PMN-mediated killing has not been reported before and may have implications for therapeutic antibodies that recruit PMN for their in vivo effects.
In addition to fucosylation also antibody sialylation has been reported to impact on antibody efficacy 25 . However, these studies were performed in mice, which express Fc receptors with limited homology, different cellular expression patterns and different Fc-binding preferences compared to the human system 41 . With human effector cells, sialylation did not significantly affect antibody efficacy 40,42 [and our results], but also other observations have been reported 26 . These contradictionary results may be explained e.g. by differences in antibody preparations, selected target antigens or assay conditions. Antibody galactosylation has been observed to affect complement activation 27 , but did not impact on ADCC 40,42 [and our results]. Corresponding to our functional data, antibody fucose ( Figure 4) -but not galactose or sialic acid (supplementary Figure S2) -content affected binding to human FcR.
The contribution of NK cells to cell-mediated killing mechanisms of monoclonal antibodies is widely acknowledged 43 , and is strongly supported by clinical studies investigating the contribution of FcR polymorphisms 6,7,44         For personal use only. on August 21, 2017. by guest www.bloodjournal.org From