Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities.

Abstract

The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its "regular" fucosylated counterpart and a series of mixtures containing varying proportions of "regular" and afucosylated materials. Compared with the "regular" fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC 50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.

Full text

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©2011 Landes Bioscience.
afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa,
moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. the afucosylated
antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity.
Based on eC50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in
antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the
extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.
Do not distribute.
mAbs 4:3, 326-340; May 2012; © 2012 Landes Bioscience
Quantitative evaluation of fucose reducing
effects in a humanized antibody on Fcγ receptor
binding and antibody-dependent cell-mediated
cytotoxicity activities
RepoRt
326 mAbs Volume 4 Issue 3
*Correspondence to: Shan Chung; Email: chung.shan@gene.com
Submitted: 02/02/12; Revised: 03/05/12; Accepted: 03/08/12
http://dx.doi.org/10.4161/mabs.19941
Introduction
The glycans attached to the asparagine at the 297 position (N297)
of the Fc region of IgG play a critical role on the effector func-
tions of antibodies.1-3 These N-linked glycans are situated within
a cleft formed by the paired heavy chains in the CH2 domain of
IgGs such that they may undergo extensive non-covalent interac-
tions with the adjacent protein surface.4-6 There is evidence that
interactions between the IgG Fc region and the effector ligands
the presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward
FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature
of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-
CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively
evaluated using the afucosylated antibody, its “regular” fucosylated counterpart and a series of mixtures containing
varying proportions of “regular” and afucosylated materials. Compared with the “regular” fucosylated antibody, the
Shan Chung,1,* Valerie Quarmby,1 Xiaoying Gao,1 Yong Ying,1 Linda Lin,1 Chae Reed,1 Chris Fong,2 Wendy Lau,3 Zhihua J. Qiu,1
Amy Shen,4 Martin Vanderlaan2 and An Song1
1Department of BioAnalytical Sciences; Genentech, Inc.; San Francisco, CA USA; 2Department of Analytical operations; Genentech, Inc.; San Francisco, CA USA; 3Department of
protein Analytical Chemistry; Genentech, Inc.; San Francisco, CA USA; 4Department of early Stage Cell Culture; Genentech, Inc.; San Francisco, CA USA
Keywords: monoclonal antibody, antibody-dependent cellular cytotoxicity, FcγRIIIA, glycosylation, fucosylation, glycoform
variants, afucosylated antibody
Abbreviations: AbX, a humanized anti-CD20 IgG1 antibody; ADCC, antibody-dependent cell-mediated cytotoxicity; AICC,
antibody-independent cellular cytotoxicity; APTS, 8-aminopyrene-1,3,6-trisulfonic acid; BSA, bovine serum albumin; CDC,
complement-dependent cytotoxicity; CDR, complementarity determining region; CE-LIF, capillary electrophoresis-laser induced
fluorescence; CV, coefficient of variation; EC50, the concentration of test antibody at which 50% of its maximal binding activity
is observed; EDTA, ethylene-diamine-tetra-acetic acid; ELISA, enzyme linked immunosorbent assay; Fab, fragment of antigen-
binding; FBS, fetal bovine serum; Fc, fragment, crystalizable; GlcNAc, N-acetylglucosamine; G0 glycoform, no terminal galactose
on either arm of the oligosaccharide chain; G1 glycoform, terminal galactose on one arm of the oligosaccharide chain; G2 glycoform,
terminal galactose on both arms of the oligosaccharide chain; G0-F, afucosylated G0 glycoform; GST, glutathione S-transferase;
HPMC, hydroxypropylmethyl-cellulose; HRP, horseradish peroxidase; IgG, immunoglobulin G; MSD, meso scale discovery;
PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PNGase F, peptide, N-Glycosidase F; RFU, relative
fluorescence unit; RLU, relative luminescence unit; RT, room temperature; TMB, 3,3',5,5'-tetramethylbenzidine
(Fcγ receptors and C1q) are critically dependent on IgG Fc pro-
tein-glycan interactions.7,8 Both the conformation and function-
ality of antibodies can be modulated by manipulation of these
oligosaccharides.9,10 Antibodies depleted of N-linked glycans at
Asn-297 behave similarly to normal antibodies with respect to
antigen binding and Protein A binding capacity. However, they
are defective in binding to Fcγ receptors, activating complement
and inducing ADCC.4,11-13 Structural and thermodynamic data
have shown that the precise structure of the IgG-Fc N-linked

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ADCC activity by fucose removal has been demonstrated with
all four subclasses of IgGs, as well as with Fc fusion proteins in
both in vitro assays and in vivo animal models.23,25,28-32 Moreover,
the antigen density required to induce efficient ADCC activity is
lower when the IgG has low, rather than high, fucose content.24
Based on these data, afucosylated forms of therapeutic antibodies
are expected to have clinical advantages over the fucosylated forms
because of the increased efficiency of ADCC induction and its
ability to better compete with endogenous IgGs for binding with
FcγRIIIa on effector cells. To date, several antibodies with no or
low levels of fucose have been produced from genetically engi-
neered cell lines and are currently in clinical studies as candidate
therapeutics for various indications.
ADCC is a critical effector function that has been implicated
in the clinical efficacy of a number of therapeutic antibodies.33,34
In ADCC, target cells bound by the Fab regions of antibodies are
lysed by activated effector cells following interactions between Fc
regions of the antibody and activating Fcγ receptors on effector
cells. The potency of ADCC induction by therapeutic antibodies
is dependent on binding affinity to both the target ligand and
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RepoRt
RepoRt
to the activating Fcγ receptors. Therefore, both the efficacy and
safety of therapeutic antibodies may be dependent on the ADCC
activity, which in turn is closely influenced by the level of afu-
cosylated glycans in the antibody product. To ensure optimal
safety and efficacy, the level of afucosylated glycan in therapeutic
antibody products should be controlled during the manufactur-
ing process. However, at present, there is no known way to fully
regulate the glycosylation activities of CHO cells. Batch-to-batch
variations in glycan profiles, including the extent of afucosylated
glycans, are commonly observed in CHO cell-derived antibody
products. While the impact of afucosylated glycans on FcγRIIIa
binding and ADCC is established, the quantitative nature of this
structure-function relationship remains unclear. A better under-
standing of this relationship, specifically its quantitative nature, is
crucial for devising an assessment and control strategy to monitor
the impact of varying levels of afucosylated glycans (inherent to
the production processes) on effector functions of the antibody
product.
In this study, we sought to provide a comprehensive in vitro
characterization of biological activity of an afucosylated anti-
CD20 antibody and to generate quantitative data to measure the
affect of fucose reduction on Fc effector functions. We prepared
a sample set consisting of an afucosylated AbX produced from a
CHO cell line deficient in FUT8, the “regular” fucosylated AbX
produced from CHO cells, and a series of mixtures with vary-
ing degrees of “regular” and afucosylated AbX. This sample set,
hereafter referred to as AbX AF-blends, was quantitatively char-
acterized in a series of in vitro assays including antigen (CD20)
binding, Fcγ receptor binding, ADCC, C1q binding and CDC
assays. Results from these studies were used to delineate the rela-
tionship between the relative activity of FcγRIIIa binding/ADCC
and the level of afucosylated glycans in the antibody samples.
Results
Using capillary electrophoresis to determine fucosylation level.
Fluorescent derivatives of glycans were released by PNGase F
digestion and quantified by CE-LIF. Peaks were identified using
a combination of known standards purchased commercially
and enzymatic digests of the glycan structures. In this study,
β-galactosidase was used to convert all glycans to the G0 form and
the observed amount of G0-F reflected the aggregate of all non-
fucosylated species (except the high mannose structures). The
AbX AF-blends sample set consisted of 14 samples: the “regular”
fucosylated (0% afucosylated AbX); the afucosylated (100% afu-
cosylated AbX); and blended samples containing 1%, 2%, 3%,
4%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20% and 50% of the
afucosylated AbX, with the “regular” fucosylated AbX added to
make up the final content. Samples in the AbX AF-blends were
identified by the content of the afucosylated AbX, including the
starting material. Therefore “regular” fucosylated AbX is referred
as 0% AF, whereas the afucosylated AbX is referred as 100%
AF. An overlay of glycan analysis data from the various blends
is shown in Figure 1. The increase in the level of G0-F is appar-
ent, together with a corresponding decrease in G0F, as increasing
amounts of afucosylated material are added to the blend.
glycans helps to determine the binding affinity of the IgG to Fcγ
receptors and thus the effector functions of the antibodies.14,15
Specifically, the N-glycans stabilize particular conformations of
the CH2 domains and act as spacers, holding the CH2 domains
apart to provide an open state of the horseshoe-shaped IgG-Fc
fragment, allowing increased accessibility and tighter binding to
Fcγ receptors.7,16-18
The majority of human IgG-Fc N-linked glycans are based
on a common core structure of biantennary heptapolysaccharide
containing GlcNAc and mannose.19,20 Further modification of
the core carbohydrate structure through the addition of fucose,
as well as bisecting GlcNAc, galactose and sialic acid, substan-
tially increases structural heterogeneity, with more than 30 vari-
ant forms possible.21 For both serum-derived endogenous human
IgGs and IgG produced from engineered mammalian cell lines,
the majority of Fc N-linked glycans carry different degrees of
terminal galactosylation resulting in a G0 glycoform, a G1 glyco-
form and a G2 glycoform. Whereas these glycans are predomi-
nantly fucosylated, i.e., contain a fucose attached to the innermost
GlcNAc residue in the core structure, small amounts of naturally
occurring glycoforms that lack the core fucose have been observed
in both human serum-derived and CHO cell produced IgG. It is
well-documented that the absence of core fucose in IgG results in
higher affinity binding to the FcγRIIIa receptor (both the F158
and V158 allotypes of this receptor) and increased ADCC activ-
ity.22-27 In 2002, Shields et al. first reported that recombinant
human IgG1 produced from the CHO-Lec13 cell line showed
enhanced FcγRIIIa binding and ADCC activity compared with
IgGs produced by regular CHO cells. The CHO-Lec13 cell line is
deficient in its ability to add fucose to glycans, but produces IgGs
with oligosaccharides that are otherwise similar to those found
in normal CHO cell lines.22 Similar results were later reported
by other groups using afucosylated antibodies produced from
engineered CHO cell lines in which α-1,6-fucosyltransferase
(FUT8) was either downregulated by RNA interference tech-
nology or genetically knocked out.26,27 In fact, enhancement of

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©2011 Landes Bioscience.
total theoretical G0-F content in various AF-blends. As shown in
Table 1, the CE measurement of G0-F in the samples was consis-
tent with the expected values, but often under-recovered G0-F by
about 15% when measured within the 5–20% G0-F range (e.g.,
the observed level of 7.4% G0-F may reflect an underlying value
of 8.4%). The reason for this slight discrepancy between theo-
retical and actual CE measured values for G0-F is not clear, but
could be attributed to variations in the efficiency of PNGase F
digestion or the quantum efficiency of the fluorophore used for
CE, which was conjugated in close proximity to the fucose.
Antigen binding activity. Binding of selected AbX AF-blends
to target cells was measured with a cell-based assay using WIL2-S
cells, which express a high level of target antigen (CD20) on the
cell surface. Test antibodies bound to WIL2-S cells were detected
with an electrochemiluminescent method using the MSD tech-
nology. Since there is no glycosylation site in the CDR of AbX,
changes in the fucosylation level of this monoclonal antibody
should not affect its binding to cell surface target antigens.
Representative dose-response binding curves of AbX AF-blends to
WIL2-S cells are presented in Figure 2. Samples tested included
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328 mAbs Volume 4 Issue 3
AF-blends containing 0%, 5%, 10%, 20%, 50% and 100% afu-
cosylated AbX. As expected, there were no discernible differences
in CD20 binding among the AF-blends tested (Fig. 2).
C1q binding and CDC activities. The affect of fucose reduc-
tion on C1q binding was assessed by an ELISA using purified
human C1q. Test antibodies included the “regular” fucosylated
AbX (0% AF), the afucosylated AbX (100% AF) and 2 blended
samples containing 10% and 50% afucosylated AbX (10% AF
and 50% AF) respectively. As shown in Figure 3A, all four sam-
ples showed comparable C1q binding activity as demonstrated
by overlapping dose-response curves. Further, the CDC activity
of these samples was assessed in a cell-based assay using WIL2-S
as target cells and complement derived from normal human
serum. As shown in Figure 3B, comparable CDC activities were
observed for all 4 AbX samples with varying degrees of afucosyl-
ated glycans. This finding is consistent with published literature,
which reports that removal of core fucose in N-glycans of anti-
bodies showed no affect on CDC activity of the antibodies.25
Fcγ receptor binding activity. The Fcγ receptor binding
activity of AbX AF-blends was measured by ELISA-based meth-
ods using a panel of recombinant human Fcγ receptors (Ia,
IIa-R131, IIb, IIIa-F158 and IIIa-V158). Antibody samples in
Since the “regular” AbX material contained approximately
1.3% afucosylated glycans, this value was used to estimate the
Figure 1. overlay of electropherograms from Ce-LIF with blended AbX samples. Glycan analysis was performed following β-galactosidase treatment
of samples to convert all glycans to either G0F or G0-F. the top line corresponds to 100% afucosylated AbX, with a peak at G0-F but a flat line at G0F,
indicating that the AbX produced by that cell line is not fucosylated (100% G0-F, 0% G0F); the bottom line is the “regular” AbX which contains mostly
G0F. Intermediate lines show data from different blends of normal AbX and afucosylated AbX, showing the expected mixtures of G0-F and G0F peaks.
the amount of afucosylated material in AbX AF-Blends whose electropherograms were stacked from top to bottom are 100%, 50%, 20%, 17.5%, 15%,
12.5%, 10%, 7.5%, 5%, 4%, 3%, 2%, 1% and 0%. RFU = relative fluorescence unit.

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©2011 Landes Bioscience.
Representative binding curves are shown in Figure 4.
Overlapping dose-response curves were observed for the bind-
ing of AbX AF-blends to Fcγ receptor Ia (Fig. 4A). The binding
curves from samples containing increasing amounts of afucosyl-
ated glycans showed a small but apparent leftward shift for Fcγ
receptors IIa (Fig. 4B) as well as IIb (Fig. 4C) and a much more
profound leftward shift for both allotypes of FcγRIIIa (Fig. 4D
and E). Overall, the extent of leftward shifts in binding curves
among test samples appeared to be proportional to the respec-
tive content of afucosylated AbX. The leftward shift results in
reduced EC50 value and is indicative of increased binding affinity.
Overall, it appeared that removal of core fucose had a minimal
affect on the binding of AbX with FcγRIa, moderate impact on
the binding with Fcγ receptors IIa and IIb, and marked affect
on the binding with IIIa. The assays were performed in multiple
runs and the relative binding activity was calculated by inversely
normalizing the EC50 values of test samples with those of the
“regular” fucosylated AbX (0% afucosylated AbX). This exer-
cise provided better quantification of the effect of afucosylation
in different samples and allowed direct comparison of receptors
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with varying binding affinities. Of note, given that minimal dif-
ferences were observed at upper asymptote of binding curves, it
was justified to use EC50 values to calculate relative binding activ-
ity. The mean relative binding activity values are summarized
in Table 2. Compared with the “regular” fucosylated AbX that
had a relative binding activity value of 1, the afucosylated AbX
showed a respective relative binding activity of 35 and 9 for F158
and V158; 3.6 and 2.4 for FcγRIIa and IIb, and 1.1 for FcγRIa.
The relative binding activity was plotted against the level of
afucosylation and the data were fitted with a linear model to fur-
ther examine the nature of the relationship between FcγRIIIa
binding activity and afucosylation. Proportional increases in rela-
tive FcγRIIIa binding were observed for AF-blends with increas-
ing amount of afucosylated AbX (Fig. 5A); the R-squared values
for the linear fit with F158 and V158 allotypes are 0.994 and
0.985, respectively. Given that levels of afucosylated glycans in
CHO cell-derived IgG are usually less than 10%, further analy-
sis was performed using data generated from samples contain-
ing afucosylated glycan levels ranging from 0–10%. As shown
in Figure 5B, a positive linear correlation between relative bind-
ing activity and the amount of afucosylated AbX in AF-blends
(0%, 1%, 2%, 3%, 4%, 5%, 7.5% and 10%) was clearly dem-
onstrated; the R-squared values for the linear fit with the F158
and V158 allotypes are 0.996 and 0.985, respectively. It is worth
noting that this linear relationship was also observed with rela-
tive binding activity expressed as a function of the total content
of afucosylated glycans represented by the theoretical G0-F and
observed G0-F values (Table 1).
ADCC assay. The ADCC activity of selected AbX AF-blends
was assessed using a human B lymphoma cell line, WIL2-S, as
target cells and PBMC from healthy donors with pre-determined
FcγRIIIa genotype as effector cells. Due to constraints on the
volume of fresh blood readily available, we tested 8 samples in this
study: the “regular” fucosylated (0% AF-blend), the afucosyl-
ated (100% AF-blend), plus blended samples containing 2%,
5%, 7.5%, 10%, 20% and 50% of the afucosylated AbX. The
study involved 16 donors: 5 were homozygous for FcγRIIIa-F158
(FF), 5 were homozygous for FcγRIIIa-V158 (VV), and 6 were
heterozygous (VF). Dose-response curves from representative
experiments using effector cells carrying each of the 3 different
FcγRIIIa genotypes (VV, VF and FF) are presented in Figure
6A–C. Consistent with published results,23,35 samples with
higher levels of afucosylated material showed enhanced ADCC
activity with effector cells from donors of all three FcγRIIIa
genotypes as evidenced by leftward shifts, indicating reduced
EC50 and higher upper asymptote, indicating increased maximal
ADCC level. Of note, the differences in leftward shifts among
samples appeared to be more profound than differences at the
upper asymptote. Nevertheless, both changes appeared to cor-
relate with the amount of afucosylated glycans in the sample.
Figure 7A shows mean EC50 values calculated from dose-response
curves fitted with a 4 Parameter Logistic nonlinear regression
model. As evidenced by the large error bars, the variability for
most of the mean values were high even though the data were
calculated from donors carrying the same FcγRIIIa genotype.
This wide range of variability is typical of ADCC assays using
monomeric form were tested for binding with the high-affinity
receptor FcγRI (CD64) and in multimeric form (cross-linked
with a F(ab’)2 fragment of goat anti-human kappa chain) for
binding with the low-affinity receptors, i.e., FcγRII (CD32) and
FcγRIII (CD16). Twelve AbX AF-blends were tested for binding
with FcγRII and FcγRIII, including those containing 0%, 1%,
2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, 50% and 100% of
afucosylated AbX. Only six samples were tested for binding with
FcγRI; the amount of afucosylated AbX in these samples were
0%, 5%, 10%, 20%, 50% and 100%.
Table 1. Levels of afucosylated glycans in AbX AF-blends
Afucosylated
Material
Theoretical
G0-F (T)
Observed
G0-F (O)
Difference
(O-T)/T
100% 100%98%-2.2%
50%51% 46%-8.6%
20% 21% 18%-15%
17.5% 19%16%-16%
15% 16%14%-16%
12.5%14%11%-16%
10% 11%9.5% -15%
7.5%8.7%7.4%-15%
5% 6.2% 5.4%-13%
4%5.2%4.8%-8.2%
3%4.3%4.1%-4.0%
2%3.3%3.2%-3.8%
1% 2.3%2.3%0.1%
0% 0%1.3% 1.3%
theoretical G0-F was calculated assuming that the “regular” fucosylated
material contains 1.3% afucosylated glycans and the afucosylated mate-
rial contains 100% of afucosylated glycans; for example, the theoretical
G0-F value of AbX AF-blend containing 10% afucosylated material is
calculated as 0.1 x 100% + 0.9 x 1.3% = 11%. observed G0-F values are
obtained from the Ce-based glycan analysis.

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©2011 Landes Bioscience.
cells; an intermediate EC50 value was obtained from effector cells
from VF donors. This indicates that effector cells carrying VV
genotype exert the highest level of ADCC activity, followed by
those carrying VF, and the effector cells carrying the FF geno-
type have the lowest ADCC activity.
Relative ADCC activity was calculated by inversely normaliz-
ing the EC50 value of each sample to the EC50 value from the “reg-
ular” AbX (0% AF-blend) to better quantify the effect of fucose
reduction on ADCC activity. The resulting mean relative ADCC
activity values are summarized in Figure 7B. As evidenced by
the reduced size of error bars compared with those in Figure 7A,
normalization within a data set generated from a single donor sig-
nificantly reduces the variability of the mean value; this confirms
that the high variability seen in the raw data are primarily caused
by inter-donor variability. Whereas EC50 values appear to differ
according to the FcγRIIIa genotype of the effector cells (Fig. 7A),
the relative ADCC activity in relation to the extent of afucosyl-
ated glycans remains similar despite the difference in FcγRIIIa
genotypes (Fig. 7B). This result clearly demonstrates that the
effect of fucose reduction on ADCC activity is independent of
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330 mAbs Volume 4 Issue 3
the FcγRIIIa genotype of the effector cells. The relationship
between the amount of afucosylated glycan and ADCC activity
was further analyzed in a scatter graph (data not shown). A posi-
tive trend was observed where increasing amounts of afucosylated
material generally led to higher levels of ADCC activity. Fitting
the data to a linear model yielded R-squared values ranging from
0.94 to 0.97 depending on the FcγRIIIa genotype of the effector
cells (data not shown). However, due to the wide range of afu-
cosylated materials tested and the relatively small number of sam-
ples, specifically, those carrying low level (<10%) of afucosylated
AbX, the resulting R-squared values from the linear fit might be
skewed by samples carrying high levels of afucosylated material.
Further, given the high variability of the PBMC-based ADCC
assay, it is technically challenging to accurately detect small dif-
ferences in ADCC activity associated with samples that contain a
low level of afucosylated material. Engineered NK cell lines have
been shown to behave similarly to purified NK cells and to offer
improved performance in ADCC assays.36 To confirm the linear
relationship between levels of afucosylated glycan and relative
ADCC activity, additional ADCC studies were performed using
an engineered NK cell line that expresses a high level of stably
transfected human FcγRIIIa-F158 as effector cells. In this study,
AbX AF-blends containing 0%, 1%, 2%, 3%, 4%, 5%, 7.5%
and 10% of afucosylated material were tested in the NK cell line
based ADCC assays. As shown in Figure 8, a linear relationship
PBMC as effector cells and is largely due to donor-to-donor vari-
ability. Additionally, we observed a notable rank order of EC50
values from donors with different FcγRIIIa genotypes (Fig. 7A).
The lowest mean EC50 values came from effector cells carrying
the VV genotype; the highest EC50 value came from FF effector
Figure 2. Antigen binding activity of select AbX AF-blends measured in a cell-based binding assay. Human B lymphoma cell line WIL2-S were coated
on plates and incubated with various concentrations of AbX AF-blends. Antibodies bound to CD20 on WIL2-S were detected with goat anti-human IgG
polyclonal antibodies. Results for AbX AF-blends containing 0% (○), 5% (□), 10% (△), 20% (◇), 50% (●) and 100% (■) of the afucosylated material are
shown. RLU, relative luminescence unit.

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©2011 Landes Bioscience.
100% (◇) of the afucosylated material.
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Removal of core fucose from IgG-Fc N-glycans has been shown
to enhance FcγRIIIa binding and ADCC activity of antibodies.
Structural models indicate that the core fucose in Fc N-glycan
may present steric hindrance for the FcγRIII glycan at N162,
thereby preventing optimal interactions between the antibody
and the receptor.37 For therapeutic antibodies produced from
mammalian cell lines, some variation in the level of afucosyl-
ated glycans is routinely observed in different production
batches. Although these differences are usually small, given the
affect of afucosylated glycan on FcγRIIIa binding and ADCC,
it is important to monitor the content of afucosylated glycan
in the antibody product during production processes and assess
the affect on biological activities with appropriate assays. In this
study, we sought to quantitatively characterize effects of fucose
reduction on in vitro biological activities of a humanized anti-
body. The results of our study confirm that the content of afu-
cosylated glycans in antibodies does not affect target binding,
C1q binding and CDC activity. More importantly, our data
show a strong linear correlation between levels of afucosylated
glycans and both the FcγRIIIa binding and ADCC activity of
the antibody.
Fcγ receptors play a vital role in the in vivo mechanisms
by which antibodies mediate their activity. Activating Fcγ
receptors such as FcγRIIa and IIIa are involved in activation
of cytotoxic activity of effector cells such as NK cells, mono-
cytes, macrophages and neutrophils. On the other hand, the
inhibitory FcγRIIb has been shown to modulate B cell activ-
ity, humoral tolerance and plasma-cell survival.38 Since many
cell types co-express activating and inhibitory Fcγ receptors, the
effector functions of therapeutic antibodies are counterbalanced
by differential interactions with both the activating FcγRIa/IIa/
IIIa and the inhibitory FcγRIIb.38 The outcome of these interac-
tions may influence both the efficacy and safety of the therapeu-
tic antibodies. Using ELISA-based binding assays, we showed
that increasing the afucosylated glycan content of AbX led to
markedly increased binding activity toward FcγRIIIa, moderate
increases in IIa and IIb binding, but no affect on binding with
Ia. While it is difficult to ascertain the biological significance
of the moderate increase in binding with FcγRIIa and IIb by
afucosylated antibodies, the increase in binding activity toward
FcγRIIIa appeared to result in proportional increase in ADCC
activity. Additionally, the changes in afucosylated glycan con-
tent and relative ADCC potency derived from normalized EC50
values appeared to correlate in a linear fashion. Given that this
linear relationship was also observed among samples carrying
low (<10%) levels of afucosylated glycans, it is clear that small
differences in levels of afucosylated glycans routinely observed
in production batches of CHO cell produced antibody products
can result in considerable changes in biological activity. Due to
the lack of clinical data and the complexity of molecular mecha-
nisms underlying immune effector functions, the significance of
these differences relative to the in vivo functions of AbX remains
unclear. However, it does not minimize the importance of
monitoring levels of afucosylated glycans in antibody products.
Currently, there are several engineered antibodies with improved
effector functions under clinical development; the availability of
between relative ADCC activity and the amount of afucosylated
glycan in the AF-blends was clearly demonstrated; the R-squared
value for the linear fit was 0.967.
Effect of afucosylated glycans in AbX production batches
on FcγRIIIa binding and ADCC activities. Ten samples of
AbX from different production runs were evaluated to ver-
ify the effects of afucosylated glycans on effector functions of
AbX samples produced from wild type CHO cells. The relative
FcγRIIIa-F158 binding and relative ADCC activity were plotted
against the level of afucosylated glycans (% G0-F) measured by
glycan analysis. As shown in Figure 9A and B, linear relation-
ships between the amount of afucosylated glycan and either the
relative FcγRIIIa-F158 binding activity or the level of relative
ADCC activity based on EC50 values were clearly demonstrated.
The R-squared value for the FcγRIIIa binding data are 0.9688;
the R-squared value for the relative ADCC data are 0.9674.
Discussion
Figure 3. (A) C1q binding and (B) CDC activities of select AbX AF-blends.
C1q binding was assessed using purified human C1q by eLISA. CDC
against human B lymphoma cell line WIL2-S cells was induced by the hu-
man serum complement in the presence of the test antibodies. extent of
cell lysis was measured by Celltiter-Glo which detects Atp from live cells.
Samples tested included those containing 0% (○), 10% (□), 50% (△) and

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©2011 Landes Bioscience.
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332 mAbs Volume 4 Issue 3
dose-response curves, we were able to quantify the increase in
ADCC activity in relation to varying levels of afucosylated
glycans in the AbX AF-blends. Our data clearly indicate that
increasing levels of afucosylated glycans lead to increased ADCC
activity and that the extent of increase correlates linearly with
the amount of afucosylated glycans. Furthermore, the extent of
data from these clinical studies should further our understanding
on the correlation between in vitro effector functions and in vivo
efficacy of a therapeutic antibody.
The in vitro ADCC activities of the AbX AF-blends were
assessed using PBMC from donors with pre-determined
FcγRIIIa genotypes. Based on EC50 values derived from complete
Figure 4. Binding responses of AbX AF-blends to human Fcγ receptors. Dose-response binding curves of AbX AF-blends to various human Fcγ recep-
tors were generated from eLISA-based binding assays using soluble recombinant Fcγ receptors. the test antibodies were assayed in monomeric form
for (A) Fcγ Receptor Ia, and in multimeric forms for (B) IIa-R131, (C) IIb, (D) IIIa-F158, (e) IIIa-V158. All data points were collected in duplicate and the
mean absorbance values from duplicate wells at oD 450 nm were plotted against the antibody concentration. Binding curves for 0% (blue ○), 2%
(green □), 5% (orange △), 7.5% (magenta ◇), 10% (gray ●), 20% (brown ■), 50% (purple ▲), 100% (red ◆) of the afucosylated material are shown. the
results shown were obtained from one representative data set out of at least three in vitro binding experiments.

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©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com mAbs 333
increase in ADCC activity appeared to be minimally affected
by FcγRIIIa genotypes because AbX AF-blends showed compa-
rable relative ADCC activity regardless of the FcγRIIIa geno-
type of the effector cells. Overall, our data indicate an 8–10-fold
increase in ADCC activity with afucosylated AbX over the
regular fucosylated AbX. Some previous studies reported sub-
stantially higher (>100 fold) increases in ADCC activity due
to fucose depletion.23,39 However, the quantification in those
studies was largely based on estimated values extrapolated from
incomplete dose-response curves. In contrast, our data was based
on multiple samples with varying degrees of reduction in afu-
cosylated glycans and relative ADCC activity quantified with
EC50 values derived from complete dose-response curves. In
addition, the approximately 10-fold increase in ADCC activ-
ity found in afucosylated AbX was also observed in assays using
either fresh NK cells or engineered NK cell lines as target cells
(unpublished data). It is worth noting that whereas proportional
shifts were observed toward higher maximal levels of ADCC at
saturating concentrations, reflecting improvements in the rela-
tive efficacy of the variants, the differences were typically within
a two-fold range. This limited range of the upper asymptote,
which represents maximum ADCC activity, justifies the use of
EC50 values as an expression of relative ADCC activity. However,
this approach to quantification might under-estimate the effects
by failing to account for changes in the upper asymptote of the
dose-response curves. Nevertheless, given that movements in the
upper asymptote were also proportional to levels of afucosylated
glycans in the samples, the omission of this parameter should
not compromise the validity of the observed linear relationship
between the afucosylated glycan level and the ADCC activity.
Of note, both the ADCC and the FcγR binding assays used in
this study are non-validated characterization assays in which the
degree of similarity (parallelism) between dose response curves
was not assessed. The observation of significant shifts toward
higher maximal levels of ADCC activity, but not of FcgRIIIa
binding activity, at saturating concentrations reflects intrinsic
limitations of different in vitro effector function assays. For
Table 2. Summary of mean relative Fcγ receptor binding activity of AbX AF-blends
Percent
FcγRIIIa-F158
Afucosylated MaterialMean (n = 6)
FcγRIIIa-V158
Mean (n = 6)
FcγRIIa-R131
Mean (n = 4)
FcγRIIb FcγRIa
SDSDSD Mean (n = 4) SDMean (n = 3) SD
0%1.00 NA1.00 NA1.00NA 1.00NA1.00NA
1%1.34 0.111.10 0.26 1.160.091.13 0.01ND NA
2%1.49 0.161.160.171.37 0.181.310.03ND NA
3% 1.780.141.240.28 1.34 0.101.260.10NDNA
4%1.96 0.241.320.21 1.35 0.041.27 0.00 ND NA
5%2.310.221.55 0.441.30 0.081.20 0.050.88 0.06
7.5%2.810.311.810.50 1.620.021.600.09ND NA
10% 3.340.12 2.050.50 1.64 0.161.38 0.021.000.04
15%4.350.36 2.530.471.72 0.011.630.03 NDNA
20%6.000.462.980.761.660.111.500.220.940.03
50% 15.71.23 6.120.752.290.081.700.150.98 0.04
100%34.9 6.25 9.050.80 3.620.42 2.400.13 1.130.11
Relative Fcγ receptor binding activity = [eC50 0% afucosylated material/ eC50 sample]. SD = standard deviation; NA = not applicable; ND = not done.
Figure 5. Correlation between relative FcγRIIIa binding activity and
amount of afucosylated materials in AbX AF-blends. Relative bind-
ing activity was calculated by inverse normalization of eC50 values of
samples with eC50 of the sample containing 0% of the afucosylated
material (100% “regular” fucosylated material). Mean relative binding
activity values (Table 2) were plotted against levels of afucosylated ma-
terial in the samples and the data points were fitted to a linear model
using excel.

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©2011 Landes Bioscience.
ADCC activity than those bearing the F158 allele. In spite of
such differences in ADCC potency, effector cells carrying dif-
ferent FcγRIIIa genotypes showed similar degrees of increase in
relative ADCC activity by AbX AF-blends in our assays, indicat-
ing that the effect of N-glycan fucose reduction on ADCC activ-
ity of the antibody is independent of the FcγRIIIa genotype of
the effector cells.
Afucosylated glycan content, in contrast, had a more pro-
found affect on binding activity toward FcγRIIIa-F158 in the
ELISA based binding assays. Specifically, compared with the
“regular” fucosylated AbX, there were 9- and 35-fold increases
in the afucosylated AbX for binding with FcγRIIIa-V158 and
F158, respectively. This discrepancy could be due to differ-
ences in assay systems and the sample preparation involved
in the ELISA method. Due to the low binding affinity, bind-
ing between monomeric IgG and FcγRII and III could not be
detected in ELISA-based assays, which required multiple wash-
ing cycles between each incubation step. To overcome this obsta-
cle, the samples of antibodies were routinely tested in multimeric
forms via cross-linking with anti-kappa F(ab’)2. It is conceivable
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334 mAbs Volume 4 Issue 3
product release testing, the most relevant biological activity
(potency) of the product is typically measured by quantitative
comparison of dose-dependent response of reference and sample
preparations in a precise and accurate manner to ensure manu-
facture consistency. A more precise and accurate measurement of
ADCC activity of antibody product that takes into account the
differences in the upper asymptote may be achieved by employ-
ing a parallel-logistics assay where parallelism assessment is part
of the system suitability criteria and relative ADCC potency is
measured with a constrained 4- or 5-parameter parallel curve
model.
The ability of antibodies to induce ADCC depends on the
binding affinities to both the antigen on target cells and to the
Fcγ receptors on effector cells. One of the factors that influ-
ences binding affinity between IgG and FcγRIIIa is the genetic
polymorphism in FcγRIIIa that results in the expression of
valine (V) or phenylalanine (F) at amino acid 158. Structural
and functional studies have shown that FcγRIIIa residue 158 is
directly involved in hydrophobic contact with IgG-Fc and that
the FcγRIIIa-V158 allotype binds to IgG1 with higher affinity
than the FcγRIIIa-F158 allotype. Additionally, immune effector
cells bearing the FcγRIIIa-V158 allele have been shown to medi-
ate more potent ADCC activity than those bearing the F allele in
in vitro ADCC assays.40 Further, clinical studies have shown that
non-Hodgkin lymphoma patients carrying the FcγRIIIa-V158
allotype responded better to rituximab treatment than those car-
rying the FcγRIIIa-F158 allotype.41,42 Similar findings were also
reported for breast cancer patients treated with trastuzumab.43,44
Consistent with these published reports, we observed a clear and
notable rank order of EC50 values from donors with different
FcγRIIIA genotypes. The lowest EC50 values (highest ADCC
activity) came from effector cells carrying VV genotypes; the
highest EC50 value came from FF effector cells; an intermedi-
ate EC50 value was obtained with effector cells from the VF
donors. This result confirms that immune effector cells bearing
the FcγRIIIa-V158 allele are generally more potent in inducing
Figure 6. Activity of AbX samples consisting of varying mixtures of
fucosylated and afucosylated materials in ADCC assays with effec-
tor cells carrying different FcγRIIIa genotypes. All experiments were
performed using human B lymphoma WIL2-S cells as target cells and
human peripheral blood mononuclear cells (pBMC) as effector cells
at an effector cell/target cell ratio of 25:1. the extent of cell lysis was
measured by LDH activity assay. (A) Representative ADCC curves from
assays using pBMC expressing homozygous FcγRIIIa-V158 (VV); (B)
Representative ADCC curves from assays using pBMC expressing both
FcγRIIIa-V158 and -F158 (VF); (C) Representative ADCC curves from
assays using pBMC expressing homozygous FcγRIIIa-F158 (FF). Data
are expressed as percent cell lysis by ADCC and concentration of the
antibodies. Samples tested included those containing 0% (blue ○), 2%
(green □), 5% (orange △), 7.5% (magenta ◇), 10% (gray ●), 20% (brown
■), 50% (purple ▲), 100% (red ◆) of afucosylated material. the results
shown were obtained from one representative data set out of at least
five in vitro ADCC assays.

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©2011 Landes Bioscience.
genotypes (VV: homozygous V158; VF: heterozygous with both V158
and F158; FF: homozygous F158). Relative ADCC activity was calculated
by inverse normalization of eC50 values of samples with eC50 value of the
sample containing 0% afucosylated material. Data presented are mean
values from at least five in vitro ADCC experiments using different donors;
error bars indicate corresponding standard deviations.
Do not distribute.
www.landesbioscience.com mAbs 335
effector function assays is typically based on the effector func-
tion potential of the product and depends on its IgG subclass,
glycoform and mechanism of action, as well as the biological
property of the target molecule.3 For antibody products where
Fc effector function is a part of the mechanism of action, effec-
tor function testing should be tightly coordinated with other
analytical assessments of product quality attributes. The char-
acterization of IgG1 Fc glycosylation is of particular importance
because of the established correlation between fucose reduction
and effector functions.
In both serum-derived IgG and IgG produced from CHO
cells with conventional processes, there are typically less than
10% afucosylated glycans. An antibody could carry an afu-
cosylated N-glycan in each of the two heavy chains. However,
given the small amount of the afucosylated glycans present
and the fact that glycan biosynthesis is intrinsically random
as well as heterogeneous, it is more likely that most naturally
occurring afucosylated glycans exist in antibodies that are
half-afucosylated, that is, where one heavy chain carries an
afucosylated glycan and the other heavy chain in the same
molecule carries a fucosylated glycan. Crystal structures show
that the Fcγ receptor binding site on IgG-Fc is localized to
the hinge proximal region of the CH2 domain and includes
the lower hinge region.4-7 More importantly, the binding site
is asymmetrical with both heavy chains making contact with
a single FcγRIII molecule, which dictates that one receptor
can only bind to one IgG-Fc at a time.4-7 This 1:1 stoichi-
ometry of the IgG-Fc/FcγRIII complex is critical for normal
immune functions because it prevents constant stimulation
of the immune system by monomeric immunoglobulins that
are present at high concentrations in the serum. The struc-
tural model of IgG-Fc/FcγRIII complex also predicts that
only one of the two Fc-fucose residues needs to be absent for
increased binding affinity toward FcγRIII.37 It implies that a
homogenously afucosylated antibody should behave similarly
to a half-afucosylated antibody in terms of FcγRIII binding
and ADCC activities. Most of the data in this study were gen-
erated from antibody samples carrying mixtures of homog-
enously fucosylated or homogenously afucosylated glycans
and not from half-afucosylated antibodies. To ensure that
our findings are generally applicable to “regular” antibody
samples, a panel of 10 AbX samples produced from wild-
type CHO cells were evaluated for the FcγRIIIa binding and
ADCC activities. Linear relationships between the amount of
afucosylated glycans and either the FcγRIIIa binding activity
or the ADCC activity were clearly demonstrated, suggesting
that the quantitative data generated by blended samples can
be extended to batch samples that presumably contain mostly
half-afucosylated antibodies. The observation of over three-
fold increase in ADCC activity with product batches contain-
ing varying amount of afucosylated glycans demonstrates that
glycoform can profoundly influence effector functions of the
product and underscores the importance to better control the
production process to provide adequate manufacturing consis-
tency. However, controlling glycoform fidelity remains a huge
challenge to pharmaceutical industry to date. Manipulation of
that cross-linked samples with high afucosylated glycan content
might exert avidity approaching the limit of the dynamic range of
the assay, resulting in under-quantification of the binding activity
with the V158 allotype. Further, additional binding studies using
a surface plasmon resonance-based immunoassay showed com-
parable changes of 20-fold in binding affinity between “regular”
and afucosylated AbX for both FcγRIIIA allotypes (unpublished
data). Together, the present observation of the differential affect
of afucosylated glycans on the binding with FcγRIIIa allotypes is
likely due to inherent limitation of the ELISA-based assay system.
During clinical development and manufacturing, different
assay strategies appropriate to different types of antibody prod-
ucts should be devised to assess effector functions. Selection of
Figure 7. (A) Mean eC50 value and (B) Mean Relative ADCC activity of
select AbX AF-blend samples with effector cells carrying different FcγRIIIa

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©2011 Landes Bioscience.
over the time of a production run.45 Other control strategies
include cell engineering, glycoengineering and development of
alternative production vehicles.
Our observation of linear relationships between the afu-
cosylated glycan content of IgGs and both FcγRIIIa binding
and ADCC activity provides direct evidence that helps to define
the quantitative nature of this structure-activity relationship. A
well-defined structure-activity relationship is invaluable for the
manufacture and control of therapeutic antibody products. It
allows cross-validation of analytical and activity assays for the
control system and provides meaningful biological information
to the product development process where function assays are
not routinely performed. Nevertheless, given the complexity of
molecular interactions between Fcγ receptors and IgG glyco-
forms, and the fact that the present study involves a humanized
IgG1 with a native Fc sequence, the observed linear relationship
between afucosylated glycan content and Fc effector function
should not be overly generalized. It is possible that alternative
relationships exist for other antibody molecules with different
isotypes/subclasses or alternative Fc sequences.
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336 mAbs Volume 4 Issue 3
Materials and Methods
Preparation of AbX AF-blends. AbX is a humanized anti-CD20
antibody based on IgG1 heavy chains and kappa light chains. For
this study, greater than 98% fucosylated AbX was produced from
the CHO DP12 cell line. Completely afucosylated AbX was pro-
duced from a CHO cell line deficient in FUT8. Both materials
showed comparable physicochemical properties including purity,
solubility and low level of high mannose glycoform or aggrega-
tion (data not shown). Blends of afucosylated variants were gen-
erated by mixing the “regular” fucosylated antibodies with the
afucosylated materials. The resulting AbX AF-blends consisted
of 14 samples: the “regular” fucosylated, the afucosylated, plus
blended samples containing 1%, 2%, 3%, 4%, 5%, 7.5%, 10%,
12.5%, 15%, 17.5%, 20% and 50% of the afucosylated AbX.
“Regular” fucosylated AbX was added to make up the final con-
tent. Of note, samples in the AbX AF-blends were identified by
the content of the afucosylated AbX, including the starting mate-
rial. Thus, the “regular” fucosylated sample is referred as 0% AF,
whereas the afucosylated AbX is referred as 100% AF.
Using CE-LIF to determine the fucose content of antibody
glycans. The fucose content of Fc glycans was determined by CE
using a LIF method as the detection mode.46 Briefly, the samples
culture medium and culture conditions for mammalian cells
can have substantial influence on the glycoform profiles of prod-
ucts and may allow for manipulation of the glycoform profiles
Figure 8. Activity of AbX samples consisting of varying mixtures of fucosylated and afucosylated materials in ADCC assays using an engineered NK
cell line as effector cells. the engineered NK cell line expresses stably transfected human FcγRIIIa-F158 and was used in conjunction with the human
B lymphoma WIL2-S as target cells at an effector cell/target cell ratio of 5:1. the extent of cell lysis was measured by LDH activity assay. Samples tested
included those containing 0%, 1%, 2%, 3%, 4%, 5%, 7.5% and 10% of afucosylated material. Data presented are mean values from three in vitro ADCC
experiments; error bars indicate corresponding standard deviations. Relative ADCC activity was calculated by inverse normalization of eC50 values of
samples with eC50 value of the sample containing 0% afucosylated material. the data was fitted to a linear model using excel.

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©2011 Landes Bioscience.
(300 μg) were transferred to pH 7.5 buffer containing 20 mM
sodium phosphate, 50 mM EDTA and 0.02% NaN3; then
digested with 10 milliunits of PNGase F (New England Biolabs,
Ipswich, MA) for 15 h at 37°C. The antibody was precipitated
by heating the solution at 95°C for 5 min and the precipitate
was removed by centrifugation. The supernatant solutions were
further treated with 10 milliunits of β-galactosidase (Prozyme)
for 2 h at 37°C. The solutions were then dried in a centrifu-
gal vacuum evaporator and the released glycans were used to
generate derivatives with APTS (Beckman Coulter) in a 15%
acetic acid solution containing sodium cyanoborohydride. The
derivative formation reaction was conducted for 2 h at 55°C. A
Beckman PA-800 system was used for the CE analysis in a run-
ning buffer of 40 mM ε-amino caproic acid/acetic acid (pH 4.5)
and 0.2% HPMC. This system was equipped with a N-CHO
coated capillary (50 μm i.d. × 60.2 cm, Beckman Coulter) and
a LIF detection module using an argon-ion laser (488 nM exci-
tation, 520 nM emission). A water plug was injected into the
capillary at 0.2 psi for 5 sec prior to sample loading. Samples
were then injected into the capillary at 0.5 psi for 10 sec. The
Do not distribute.
www.landesbioscience.com mAbs 337
separation of the oligosaccharide derivatives was performed at
500 V/cm (30 kV) with the capillary temperature maintained
at 20°C.
CD20 binding assay. Binding of AbX to CD20 antigen was
measured using a cell-based electrochemiluminescent assay as
described by Lu et al. Briefly, WIL2-S cells, a human B lym-
phoma cell line expressing a high level of CD20, were washed
with phosphate buffered saline (PBS) and seeded at 25,000
cells/well in 25 μL PBS on 96-well MULTI-ARRAY High
Bind plates (MSD). WIL2 cells were incubated for one hour
at room temperature (RT) to allow cell attachment to the car-
bon surface. The plates were then blocked with 15% FBS in
PBS for 30 min at RT with mild agitation and incubated with
serial dilutions of antibodies (2.74–2,000 ng/mL) in assay buf-
fer (PBS containing 10% FBS). After 1 h incubation at RT with
mild agitation, the plates were washed and bound antibodies
were detected by adding ruthenium-labeled F(ab’)2 goat anti-
human IgG Fc in assay buffer in the presence of Tris-based
Read Buffer T (MSD). Upon electrochemical stimulation,
luminescent signals were quantified with a Sector Imager 6000
reader (MSD). Dose-response binding curves were generated
by plotting the mean luminescent signals from duplicates of
sample dilutions against the sample concentrations and fitting
the data points to a four-parameter model using SoftMax Pro
(Molecular Devices).
Fcγ receptor binding by ELISA. The binding activities
of test antibodies toward various human Fcγ receptors were
assessed by ELISA-based ligand binding assays.48 The human
Fcγ receptors were expressed as fusion proteins containing the
extracellular domain of the Fcγ receptor linked to a Gly/6x
His/glutathione S-transferase (GST) polypeptide tag at the
C-terminus. For the low-affinity receptors [Fcγ RIIa (CD32A),
Fcγ RIIb (CD32B) and Fcγ RIIIa (CD16)], antibodies were
tested as multimers by cross-linking with F(ab’)2 fragments of
goat anti-human kappa chain antibodies (MP Biomedicals) at
an approximate molar ratio of 1:3. For the high-affinity recep-
tor (FcγRIa), the antibodies were assayed as monomers without
cross-linking. Sample and reagent dilutions were prepared in
an assay buffer containing PBS, 0.5% BSA and 0.05% Tween-
20. Plates were washed with PBS containing 0.05% Tween-20
using an ELx405TM plate washer (Biotek Instruments) after
each incubation step.
Plates were coated with an anti-GST antibody (Genentech)
in a 0.05 M sodium carbonate buffer (pH 9.6) overnight
at 4°C. After blocking with the capture antibody, the plates
were incubated with Fcγ receptors for 1 h at RT. Serially
diluted test antibodies were added either as monomers (for
binding with FcγRIA) or as multimeric complexes (for bind-
ing with FcγRIIa, IIb and IIIa) and the plates were incu-
bated for 2 h at RT. Antibodies bound to the Fcγ receptor
were linked to HRP conjugated F(ab’)2 fragment of goat
anti-human F(ab’)2 (Jackson ImmunoResearch Laboratories,
West Grove, PA) and substrate TMB (Kirkegaard and Perry
Laboratories, Gaithersburg, MD) was added. The plates were
incubated for 5–20 min at RT, depending on the Fcγ recep-
tors tested, to allow color development. The reaction was
Figure 9. Relationship between amount of afucosylated glycans and
(A) relative FcγRIIIa-F158 binding activity (B) relative ADCC activ-
ity of selected AbX samples. Samples were tested in an eLISA-based
FcγRIIIa-F158 binding assay and engineered NK cells-based ADCC
assays. Relative activity (RA) of FcγRIIIa-F158 binding and ADCC activity
based on eC50 values were calculated by inverse normalization with the
corresponding eC50 value of the “regular” fucosylated AbX, which is one
of the samples tested. %G0-F is the level of afucosylated glycans in the
sample measured by the glycan analysis.

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©2011 Landes Bioscience.
out the antibody.
The extent of specific ADCC was calculated as follows:
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338 mAbs Volume 4 Issue 3
the incubation time was reduced to 3 h. The rest of the proce-
dures were the same as described above.
C1q binding assay. Human C1q binding to AbX-AF blends
was measured by ELISA.49 Antibodies were serially diluted and
coated on high binding microtiter plate wells at 2–8°C with
overnight incubation. The plates were sequentially incubated
with 2 mg/mL human C1q (Quidel Corporation) for 2 h at
RT, goat anti-human C1q (Accurate Chemical, Westbury, NY)
for 1 h at RT, and donkey anti-goat IgG-HRP (Millipore) for
1 h at RT after blocking with 3% BSA/PBS (1 h at RT). Plates
were washed with PBS containing 0.05% Tween-20 using an
ELx405TM plate washer (Biotek Instruments) after each incuba-
tion step. Peroxidase activity was detected with TMB substrate
(Kirkegaard and Perry Laboratories) and the plates incubated
at RT for 15 min to allow color development. The reaction
was terminated with 1 M H3PO4 and absorbance measured at
450 nm (the background measured at 650 nm was subtracted
for each well) using a microplate reader SpectraMax®190
(Molecular Devices). Dose-response binding curves were gen-
erated by plotting the mean absorbance values from duplicates
of sample dilutions against the sample concentrations and then
fitting the data points to a four parameter logistic model using
SoftMax Pro. In a separate experiment, an anti-human Fab-
HRP conjugate (Jackson ImmunoResearch Laboratories) was
used to determine equivalent coating of antibodies to the wells
of the microtiter plate.
CDC assay. CDC activities of test antibodies were assessed
by a cell-based assay using WIL2-S cells as target cells. AbX-AF
blends were serially diluted in assay medium (RPMI 1640
medium supplemented with 1% FBS) and distributed into a
96-well opaque-walled microtiter plate (Costar Corning Inc.).
The plate was incubated with 5% CO2 for 2 h at 37°C after
WIL2-S cells (5 × 104 cells/well) and normal human serum
complement (Quidel Corporation) were added. After the incu-
bation, the CellTiter-Glo reagent (Promega Corp.), which
assays for ATP in metabolically active cells, was added and the
plate was incubated for 10 min at RT with constant shaking.
The extent of cell lysis was quantified by measuring intensity of
luminescence with a plate reader SpectraMax M5 (Molecular
Devices). Dose-response binding curves were generated by plot-
ting the mean luminescence signal from duplicates of sample
dilutions against the sample concentrations and then fitting
the data points to a four-parameter model using SoftMax Pro
(Molecular Devices).
Disclosure of Potential Conflicts of Interest
All the authors are employees of Genentech, Inc., which sup-
ported the study financially.
Acknowledgements
The authors would like to thank Patty Siguenza for support on
the project, Kyra Cowan and Bob Kelley for helpful comments
on the manuscript, and colleagues at the research blood program,
the sample transportation program, and the CritRS group for
providing critical reagents.
terminated with 1 M H3PO4 and absorbance determined at
450 nm. Dose-response binding curves were generated by plot-
ting the mean absorbance values from duplicates of sample
dilutions against the sample concentrations and fitting the data
points to a four-parameter model using SoftMax Pro (Molecular
Devices) to obtain the EC50 values. For comparison, relative
activity was calculated for each sample by inversely normalizing
its EC50 value with that of a reference molecule using the follow-
ing formula:
Relative Activity = EC50 Reference/EC50 Sample
ADCC assay. ADCC assays were performed using PBMC
from healthy donors as effector cells and WIL2-S, a human B
lymphoma cell line, as target cells. Briefly, PBMC were isolated
from fresh blood of healthy human donors by Ficoll-Paque (GE
Healthcare, Milwaukee, WI) density gradient centrifugation.
Target cells (4 × 104) prepared in assay medium (RPMI 1640
with 1% BSA and 100 units/mL penicillin and streptomycin)
were added to each well in round-bottom, 96-well tissue culture
plates. Serial dilutions of antibodies (10,000 to 0.0038 ng/mL
following 4-fold dilutions in series) were added to the plates
containing the target cells (50 μL/well) and incubated for 30
min at 37°C with 5% CO2 to allow opsonization. After incu-
bation, PBMC (1.0 × 106) were added to each well, in assay
medium, for a 25:1 effector:target cell ratio and the plates were
incubated further for 4 h. The plates were then centrifuged
and the supernatants were assayed for lactate dehydrogenase
activity using a Cytotoxicity Detection Kit (Roche Diagnostics
Corporation). Cell lysis was quantified by measuring absor-
bance at 490 nm using a microplate reader SpectraMax®190
(Molecular Devices). Absorbance of wells containing only the
target cells served as the control for background (Low Control),
whereas wells containing target cells lysed with Triton-X100
provided the maximum available signal (High Control). AICC
was measured in wells containing target and effector cells with-
%ADCC = [A490 nm (sample) - A490 nm (AICC)]/[A490 nm (High
Control) - A490 nm(Low Control)]
Dose-response curves were generated by plotting the mean
ADCC values from duplicates of antibody sample dilutions
against the antibody concentration. The EC50 values were cal-
culated by fitting the data points to a four-parameter equation
using SoftMax Pro. For comparison, relative activity was cal-
culated for each sample by inversely normalizing its EC50 value
with that of a reference molecule using the following formula:
Relative Activity = EC50 Reference/EC50 Sample
In select experiments, an engineered NK cell line expressing
stably transfected human Fcγ RIIIa-F158 was used as effector
cells,36 but the effector:target cell ratio was changed to 5:1 and

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©2011 Landes Bioscience.
org/10.1074/jbc.M107478200.
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Do not distribute.
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