From the Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan
Received for publication, October 18, 2002, and in revised form, November 8, 2002
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ABSTRACT |
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An anti-human interleukin 5 receptor (hIL-5R)
humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1
produced by rat hybridoma YB2/0 cell lines showed more than 50-fold
higher antibody-dependent cellular cytotoxicity (ADCC)
using purified human peripheral blood mononuclear cells as effector
than those produced by Chinese hamster ovary (CHO) cell lines.
Monosaccharide composition and oligosaccharide profiling analysis
showed that low fucose (Fuc) content of complex-type oligosaccharides
was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was
subjected to Lens culinaris aggulutin
affinity column and fractionated based on the contents of Fuc. The
lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In
contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC
much less than that of Fuc. In addition, the correlation between
Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells
had lower expression level of FUT8 mRNA, which codes
Antibody-dependent cellular cytotoxicity
(ADCC),1 a lytic attack
on antibody-targeted cells, is triggered upon binding of lymphocyte receptors (Fc One IgG molecule contains two N-linked oligosaccharide sites
in its Fc region (3). The general structure of N-linked
oligosaccharide on IgG is complex-type, characterized by a
mannosyl-chitobiose core (Man3GlcNAc2-Asn) with or without bisecting
GlcNAc/L-fucose (Fuc) and other chain variants including
the presence or absence of Gal and sialic acid. In addition,
oligosaccharides may contain zero (G0), one (G1), or two (G2) Gal.
Recent studies have shown that engineering the oligosaccharides of IgGs
may yield optimized ADCC. ADCC requires the presence of
oligosaccharides covalently attached at the conserved
Asn297 in the Fc region and is sensitive to change in the
oligosaccharide structure. In the oligosaccharide, sialic acid of IgG
has no effect on ADCC (4). The relationship between the Gal residue and
ADCC is controversial. Boyd et al. (4) have shown that
obvious change was not found in ADCC after removal of the majority of
the Gal residues. However, several reports have shown that Gal residues enhance ADCC (5, 6).
Several groups have focused on bisecting GlcNAc, which is a
Recently, Shields et al. have revealed the effect of
fucosylated oligosaccharide on antibody effector functions, including binding to human Fc Here, we describe the correlation between glycosylation of human IgG1
and ADCC and demonstrate that Fuc showed the critical role for
enhancing ADCC out of several sugar residues reported previously. We
unexpectedly found that human IgG1 produced by rat hybridoma YB2/0
cells showed extremely high ADCC at more than 50-fold lower
concentration of those produced by CHO cells. YB2/0-produced IgG1 had
lower Fuc content than CHO-produced IgG1. IgG1 containing lower
fucosylated oligosaccharides, which was fractionated by Lens
culinaris aggulutin (LCA) lectin affinity chromatography, showed higher ADCC before separation. In contrast, the addition of
bisecting GlcNAc to IgG1 enhanced ADCC much less effectively than
defucosylation. The effect of bisecting GlcNAc was only observed in
highly fucosylated IgG1. YB2/0 cells expressed a lower level of FUT8
( Cell Lines--
Rat hybridoma YB2/0 cells were purchased from
the American Type Culture Collection (ATCC; CRL-1662). CHO cell line
DG44 (12), for wild type IgG1 production, was kindly provided by Dr.
Lawrence Chasin (Columbia University). LEC10, a variant CHO cell line
overexpressing GnTIII (13), was kindly provided by Dr. Pamela Stanley
(Albert Einstein College of Medicine).
Expression of IgG1s--
For the generation of human IgG1 of
humanized anti-human interleukin-5 receptor (hIL-5R) Production of IgGs--
The anti-hIL-5R IgG1-producing YB2/0
cell line was suspended in GIT medium (Wako, Osaka, Japan) containing
0.5 mg/ml G418 and 200 nM methotrexate to give a density of
3 × 105 cells/ml and dispensed in suspension culture
flasks (Greiner, Frickenhausen, Germany). The anti-hIL-5R
IgG1-producing CHO cell line was suspended in the EX-CELL302 medium
(JRH, Kansas City, MO) containing 3 mM L-Gln,
0.5% chemically defined lipid concentrate (Invitrogen), and 0.3%
PLURONIC F-68 (Invitrogen) to give a density of 3 × 105 cells/ml and cultured using spinner flasks (Asahi
Techno Glass, Tokyo, Japan) under agitating at a rate of 100 rpm. The
anti-CD20 IgG1-producing YB2/0 cell line was suspended in the
hybridoma-SFM medium (Invitrogen) containing 5% Daigo's GF21 (Wako)
and 200 nM methotrexate to give a density of 1 × 105 cells/ml and dispensed in suspension culture flasks.
The flasks were incubated under conditions of 37 °C in humid air
containing 5% CO2. After 8 or 10 days of incubation, the
culture supernatants were recovered.
Purification of IgG1s--
The culture supernatants containing
anti-hIL-5R IgG1 from YB2/0 cells and CHO cells and anti-CD20 IgG1 from
YB2/0 cells were clarified by centrifugation and passed through a
0.2-µm filter. The IgG1 bound to a PROSEP-A (Millipore) column was
eluted with 0.1 M citrate buffer (pH 3.5). Then the
antibody was subjected to a Sephacryl S-300 (Amersham Biosciences)
column. The buffer composition of the YB2/0-produced anti-CD20 IgG1 was
changed to that for RituxanTM (9.0 mg/ml sodium chloride,
7.35 mg/ml sodium citrate dihydrate, 0.7 mg/ml polysorbate 80). The
purity of IgG1 was confirmed by SDS-PAGE. YB2/0- and CHO-produced
humanized anti-hIL-5R IgG1 were designated as KM8399 and KM8404,
respectively. YB2/0-produced chimeric anti-CD20 IgG1 was designated as
KM3065. RituxanTM (chimeric mouse/human anti-CD20
monoclonal antibody derived from the CHO cell line) was purchased from
Genentech (South San Francisco, CA)/IDEC Pharmaceutical (San Diego, CA).
ADCC Assay for the Anti-hIL-5R IgG1--
An ADCC assay was
performed by 51Cr release assay as reported previously
(15). Briefly, target cells (1 × 106), a murine T
cell line CTLL-2 (h5R) expressing hIL-5R ADCC Assay for the Anti-CD20 IgG1--
An ADCC assay was
performed by a lactate dehydrogenase release assay. Aliquots of target
cells, a human B lymphoma cell line Raji (number 9012, purchased from
JCRB, Tokyo, Japan), were distributed into 96-well U-bottomed plates
(1 × 104/50 µl) and incubated with serial dilutions
of antibodies (50 µl) in the presence of human effector cells (100 µl) at an E/T ratio of 25:1 or 20:1. Human
effector cells were PBMC purified from healthy donors using Lymphoprep
(Axis Shield, Dundee, UK). After a 4-h incubation at 37 °C, the
plate was centrifuged, and the lactate dehydrogenase activity in the
supernatants was measured using a nonradioactive cytotoxicity assay kit
(Promega, Madison, WI). The percentage of specific cytolysis was
calculated from the activities of samples according to the formula,
Profiling Analysis of N-Linked
Oligosaccharides--
Oligosaccharides were prepared from 100 µg of
IgG1 by hydrazinolysis and N-acetylation by the methods
reported previously (17). Oligosaccharides were pyridylaminated (17)
and analyzed by reverse-phase HPLC using a Shim-pack CLC-ODS column
(60 × 150 mm; Shimadzu, Kyoto, Japan) (18) with slight
modifications. Elution was performed at a flow rate of 1.0 ml/min at
55 °C using two solvents, A and B. Solvent A was 10 mM
sodium phosphate buffer (pH 3.8), and solvent B was 10 mM
sodium phosphate buffer (pH 3.8) containing 0.5% 1-butanol (Sigma).
The column was equilibrated with solvent A. After injection of sample,
the ratio of solvent B to A was increased with a linear gradient to
60:40 in 80 min. The elution profile was monitored by fluorescence
detection with excitation at 320 nm and emission at 400 nm. The
oligosaccharide peak assignments were made according to retention time
comparison with PA-labeled oligosaccharide standards (TaKaRa Bio, Otsu,
Japan). The peak area was used to calculate the percentage of each
oligosaccharide, since the relative fluorescence was the same on a
molar basis for each component (17). To identify the structure, each
oligosaccharide fraction that separated as a peak was collected and
evaporated to dryness under vacuum prior to matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry.
Monosaccharide Composition of IgG1s--
Monosaccharides were
released from an aliquoted quantity of IgG1 by heating with 4 M trifluoroacetic acid at 100 °C for 2 h.
Monosaccharides were dried under vacuum and reconstituted in water prior to high performance anion exchange chromatography analysis.
Monosaccharides were analyzed using a waveform and DX500 system
(DIONEX, Sunnyvale, CA) described previously (19). A CarboPac PA-1
column (DIONEX) was used to resolve monosaccharides with a flow rate of
0.8 ml/min at 35 °C. After injection of samples, the monosaccharides
were resolved with 18 mM NaOH for 30 min, and the column
was regenerated by elution with 500 mM NaOH for 10 min. The
column was held 18 mM NaOH for 30 min prior to the next injection.
Lectin Affinity HPLC--
LCA or Phaseolus
vulgaris E4 (PHA-E4) were used as
ligands of lectin affinity HPLC for separation of IgG1 based on the
content of Fuc or bisecting GlcNAc, respectively. An LA-LCA (4.6 × 150 mm; HONEN, Tokyo, Japan) column was installed in the LC-6A HPLC system (Shimadzu). Purified antibodies dissolved in 10 mM
KH2PO4 were applied to the column previously
equilibrated with 50 mM Tris-H2SO4
(pH 7.3). The column was eluted with a linear gradient of the buffer
containing 0.2 M
Establishment of Competitive RT-PCR Analysis of FUT8--
Total RNA was isolated
from 1.0 × 107 YB2/0 cells, FUT8-overexpressing YB2/0
cells, or CHO/DG44 cells using the RNeasy minikit (Qiagen, Tokyo,
Japan) and incubated for 1 h at 37 °C with 20 units of
RNase-free DNase (RQ1; Promega) to degrade genomic DNA. After DNA
digestion, the total RNA was purified again using the RNeasy minikit.
The single-strand cDNA was synthesized from 3 µg of each total
RNA using the Superscript first strand synthesis system for RT-PCR
(Invitrogen). The 50-fold diluted reaction mixture was used as a
template for the following competitive RT-PCR. Quantification of FUT8
transcripts was carried out using competitive RT-PCR in which a 979-bp
partial fragment of rat FUT8 cDNA was used as a standard DNA and a
772-bp fragment deleting a 207-bp ScaI
(blunt)-HindIII (blunt) fragment from standard DNA was used
as a competitor. Standard DNA was amplified from single-stranded
cDNAs of YB2/0 cells by PCR using primers
5'-ACTCATCTTGGAATCTCAGAATTGG-3' and
5'-CTTGACCGTTTCTATCTTCTCTCG-3'. PCRs for detection of FUT8 were
carried out by heating at 94 °C for 3 min and subsequent 32 cycles
of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min in 20 µl of reaction mixture containing 5 µl of the 50-fold diluted
single-stranded cDNA, 10 fg of linearized competitors, 10 pmol of
FUT8-specific primers, 4 nmol of dNTP mixture, 5% dimethyl sulfoxide,
and ExTaq polymerase (Takara Bio). The sense primer
5'-GTCCATGGTGATCCTGCAGTGTGG-3' and antisense primer
5'-CACCAATGATATCTCCAGGTTCC-3' were employed to amplify an FUT8
fragment. Aliquots of PCR products (7 µl) were subjected to
electrophoresis in 1.75% agarose gel and stained with SYBR Green
I nucleic gel stain (Molecular Probes, Inc., Eugene, OR). The amount of products was quantified by measuring luminescence intensity using a FluoroImager SI (Amersham Biosciences), calculated from standard curves, and converted into molar numbers. To normalize the synthesis efficiency of first-strand cDNAs, the amount of ADCC of YB2/0- and CHO-produced IgG1--
The purified
humanized anti-hIL-5R IgG1 antibodies, KM8399 (YB2/0-produced) and
KM8404 (CHO-produced), were compared for their ability to induce ADCC
against a murine T cell line CTLL-2 (h5R) expressing hIL-5R
To confirm the reproducibility of this result, we assessed the ADCC of
another antibody, chimeric anti-CD20 IgG1. RituxanTM was
CHO-produced chimeric anti-CD20 IgG1 approved as a therapeutic agent in
non-Hodgkin's lymphoma. We originally established YB2/0-produced chimeric anti-CD20 IgG1, KM3065, that had the same V region amino acid
sequences as RituxanTM. Both chimeric RituxanTM
and KM3065 exhibited the same antigen binding activity in
flow cytometric analysis using CD20-positive cell lines (data not
shown). On the other hand, the ADCC of KM3065 was at least 100-fold
higher than that of RituxanTM at the concentration of
antibody of 20% cytotoxicity that was the maximum activity of
RituxanTM (Fig. 1B). Moreover, the maximum
cytotoxicity of YB2/0-produced KM8399 and KM3065 was 2-3-fold higher
than that of CHO-produced KM8404 and RituxanTM,
respectively (Fig. 1, A and B).
Oligosaccharide Analysis--
To elucidate the molecular basis of
the difference of ADCC between YB2/0- and CHO-produced IgG1, we
analyzed the protein portion and oligosaccharide portion of KM8399 and
KM8404. There was no significant difference in SDS-PAGE, peptide
mapping, and CD (data not shown), suggesting the importance of
oligosaccharides for controlling ADCC.
Hydrazinolysis-derived oligosaccharides were labeled with
2-aminopyrizine and separated by HPLC (Fig.
2, A and B). As
shown in Fig. 2, A and B, peak patterns and
content of oligosaccharide between YB2/0-produced KM8399 and
CHO-produced KM8404 were quite different. KM8399 contained nine major
oligosaccharides (peaks a, b, c, e, f, g, h, k, and l); in contrast,
KM8404 contains five major oligosaccharides (peaks a, e, f, g, and h).
The oligosaccharide structure of each peak is shown in Fig.
2C, and all structures have been found in human IgG
N-linked oligosaccharides as natural structures (22,
23).
To clear the difference of oligosaccharides found in Fig. 2,
quantitative monosaccharide compositions of IgG1s were determined (Table I). The contents of Fuc, Gal, and
GlcNAc were different between YB2/0-produced IgG1s and CHO-produced
IgG1s. KM8399 and KM3065 (YB2/0-produced) contained 0.8- and 0.09-fold
lower content of Fuc than KM8404 and RituxanTM
(CHO-produced), respectively. In contrast, the two
YB2/0-produced IgG1s showed a higher content of GlcNAc than the two
CHO-produced IgG1s. The difference in the content of Gal between
YB2/0-produced IgG1s and CHO-produced IgG1s was not consistent. These
results suggest the difference of ADCC between YB2/0-produced IgG1s and CHO-produced IgG1s is caused by that of oligosaccharide structure, especially Fuc- and/or GlcNAc-containing oligosaccharides.
Oligosaccharide profiling analysis also showed that content of
nonfucosylated oligosaccharides of KM8399 (34%, Table
II) and KM3065 (91%, Table IV) were
higher than those of KM8404 (9%, data not shown) and
RituxanTM (6%, data not shown). Fuc composition of four
IgG1s (KM8399, KM8404, KM3065, and RituxanTM), which was
calculated from oligosaccharide profiling analysis (0.71, 0.91, 0.09, and 0.94, respectively) coincided well with the result of
monosaccharide analysis (0.76, 0.91, 0.08, and 0.94, respectively)
(Table I). Based on these results, we selected the oligosaccharide
profiling analysis for further study.
Lectin Affinity Chromatography--
To analyze the effect of
Fuc-containing and bisecting GlcNAc-containing oligosaccharides on
ADCC, YB2/0-produced anti-hIL-5R IgG1 KM8399 was fractionated based on
the content of Fuc or bisecting GlcNAc by two lectin affinity
chromatographies, LCA (Fig. 3) or PHA-E4 (Fig. 4). KM8399 was
fractionated by LCA lectin affinity chromatography to the fraction I
with lower Fuc content and the fraction II with higher Fuc content
before separation (Fig. 3A and Table II). The contents of
nonfucosylated IgG1 of the fraction I, II, and unseparated fraction
were 100, 15, and 34%, respectively. As shown in Fig. 3B,
ADCC of the fraction I was enhanced 10-fold before separation;
nevertheless, that of the fraction II was decreased 10-fold.
These results indicated that Fuc content is inversely correlated to
ADCC of anti-hIL-5R IgG1.
Several groups have focused on the relationship between ADCC and
bisecting GlcNAc, although KM8399 had few contents of bisecting GlcNAc-binding oligosaccharides (4%, Table II). To examine the relationship, KM8399 was separated to fractions III and IV based on the
content of bisecting GlcNAc by PHA-E4 column (Fig.
4A) followed by sequential separation of fraction III' and
IV' by LCA chromatography (Fig. 4, B and C) to
reduce the effect of nonfucosylated oligosaccharides. As shown in Table
III, fraction III' contained no bisecting
GlcNAc-binding oligosaccharide (0%), and fraction IV' had more content
of bisecting GlcNAc-binding oligosaccharides (30%) than before
separation (4%). On the other hand, the content of Fuc of each
fraction was consistent (fraction III', 88%; fraction IV', 90%). As
shown in Fig. 4D, fractions III' and IV' showed no
significant difference in ADCC, suggesting bisecting GlcNAc at least
under 30% content in the oligosaccharides has little effect in ADCC of
anti-hIL-5R IgG1.
YB2/0-produced KM3065 contained bisecting GlcNAc-binding nonfucosylated
oligosaccharides with a relatively high percentage of 16%. Although
oligosaccharides of such structure are quite small in quantity (less
than 1%), those oligosaccharides are detectable in serum IgG derived
from normal human (22, 24). To analyze the combined effect of Fuc and
bisecting GlcNAc, KM3065 was separated to four fractions, V, VI, VII,
and VIII, based on the content of bisecting GlcNAc by
PHA-E4 lectin affinity chromatography (Fig. 5A). As shown in Table
IV, each fraction had a different content of bisecting GlcNAc-binding oligosaccharides (from 0 to 45%); however,
the content of nonfucosylated oligosaccharides was not significantly
different. All four fractions showed almost the same ADCC to
unseparated KM3065 (Fig. 5B), indicating that the content of
bisecting GlcNAc at least under 45% did not affect the ADCC of
anti-CD20 chimeric IgG1, which contained around 90% content of
the nonfucosylated oligosaccharides.
To verify the combined effect of bisecting GlcNAc with Fuc, LEC10
cells, a variant CHO cell line overexpressing GnTIII (12), were used to
produce chimeric anti-CD20 monoclonal IgG1. In oligosaccharide analysis, bisecting GlcNAc-binding fucosylated oligosaccharides were
the majority on LEC10-produced anti-CD20 IgG1 (74% of bisecting GlcNAc
and 100% of Fuc; data not shown), whereas bisecting GlcNAc-nonbinding fucosylated oligosaccharides were the majority of
RituxanTM (0% of bisecting GlcNAc and 94% of Fuc; data
not shown). In the ADCC assay, LEC10-produced IgG1 showed only
severalfold enhancement of ADCC compared with RituxanTM
(Fig. 5B). In addition, YB2/0-produced KM3065 with low Fuc
content (19% of bisecting GlcNAc and 9% of Fuc; Table IV) exhibited
~100-fold higher ADCC than LEC10-produced IgG1. These results suggest
that a relatively high content of bisecting GlcNAc (74%) improves ADCC when IgG1 has a high content of fucosylated oligosaccharides, although
the impact of the content of bisecting GlcNAc in ADCC is much less than
that of Fuc.
Expression Levels of
Quantification of FUT8 transcripts was performed using competitive
RT-PCR. To normalize the synthesis efficiency of first-strand cDNAs, the amounts of Overexpression of In this study, we analyzed the molecular basis of extremely high
ADCC of recombinant IgG1 produced by rat hybridoma YB2/0 cells, which
produced IgG1 with at least 50-fold higher ADCC than that produced by
CHO cells, one of the most widely used host cell lines for production
of recombinant antibodies (Fig. 1). Our conclusion of the present study
is that nonfucosylated oligosaccharide of YB2/0-produced IgG1 has a
more critical role in enhancing ADCC than Gal-binding or
bisecting GlcNAc-binding oligosaccharides according to the following
evidence. First, monosaccharide composition and oligosaccharide
profiling analysis showed that high content of nonfucosylated
complex-type oligosaccharides were characteristic in YB2/0-produced
anti-hIL-5R IgG1 (34%, Table II) and anti-CD20 IgG1 (91%, Table IV)
compared with low content of those in CHO-produced anti-hIL-5R IgG1
(9%, data not shown) and anti-CD20 IgG1 (6%, data not shown). Second,
ADCC assay of the anti-hIL-5R IgG1 separated by LCA affinity
chromatography demonstrated that Fuc content of IgG1 was inversely
correlated with ADCC (Fig. 3, A and B). Third, quantitative PCR analysis indicated that YB2/0 cells had a 10-fold lower expression level of FUT8 mRNA than CHO cells (Fig.
6A). Fourth, overexpression of FUT8 in YB2/0 cells increased
the content of fucosylated oligosaccharides and also decreased ADCC of
anti-CD20 IgG1 (Fig. 6B).
L-Fuc residues in an The importance of nonfucosylated oligosaccharide on ADCC has been
reported very recently by Shields et al. (11). They have shown that nonfucosylated anti-Her2 humanized IgG1 and anti-IgE humanized IgG1 produced by a variant of CHO cells, Lec13, had enhanced
ADCC relative to fucosylated IgG1s produced by normal CHO cells.
However, they have only focused on Fuc, because no appreciable
differences in the content of the other sugar residues have been found
in Lec13-produced IgG1 and normal CHO-produced IgG1. Until now, the
effects of Fuc, Gal, or bisecting GlcNAc on ADCC have been analyzed
independently (4-7, 9-11); therefore, comparison of the effect
of each sugar residue or the combined effect of each sugar residue has
not yet been reported.
In this report, we could not find any correlation between the content
of Gal and ADCC. A difference in the content of Gal between
YB2/0-produced IgG1s and CHO-produced IgG1s was not correlated to ADCC
(Table I). As a result of the separation of KM8399 using LCA lectin
affinity chromatography, the compositions of G0, G1, and G2 of fraction
II were very similar to that of KM8399 (Table II); nevertheless, ADCC
of fraction II and KM8399 was quite different (Fig. 3B). Our
results show a good coincidence with the report of Boyd et
al. (4), in which obvious change was not found in ADCC after
removal of the majority of the Gal residues of anti-CDw52 IgG1 produced
by CHO cells. In contrast, Kumpel et al. (5, 6) reported
that highly galactosylated anti-D-antigen IgG1s have higher ADCC,
although that effect was only 2-3-fold.
Two groups independently reported that increasing the level of
bisecting GlcNAc of anti-neuroblastoma IgG1 and anti-CD20 IgG1 could
enhance ADCC (9, 10). GnTIII-transfected CHO cells produced anti-CD20
IgG1 with a high content of bisecting GlcNAc (48-71%), which showed a
10-20-fold enhancement of ADCC compared with that with no content of
bisecting GlcNAc (0%). In the present study, we carefully examined the
effect of bisecting GlcNAc in ADCC in comparison with that of Fuc. We
prepared anti-hIL-5R IgG1 with different content of bisecting GlcNAc
(0-30%). To avoid the effect of fucosylation, nonfucosylated IgG1s
were depleted by LCA lectin affinity chromatography. To our surprise,
we could not detect any correlation between ADCC and content of
bisecting GlcNAc. One possible explanation of the discrepancy with the
results of Umana et al. (9) and Davies
et al. (10) might be that 30% content of bisecting GlcNAc
is not enough to enhance ADCC. We next produced anti-CD20 IgG1 by LEC10
cells, a variant CHO cell that overexpressed GnTIII. The resultant IgG1
(74% bisecting GlcNAc and 100% Fuc) had shown only severalfold higher
ADCC than normal CHO-produced IgG1 (0% bisecting GlcNAc and 94% Fuc);
in contrast, YB2/0-produced IgG1 (19% bisecting GlcNAc and 91%
non-Fuc) had 100-fold higher ADCC than LEC10-produced IgG1 (Fig.
5B). These results suggest that an extremely high content of
bisecting GlcNAc (74%) has a relatively weak effect for enhancing
ADCC. More importantly, nonfucosylated oligosaccharide was shown to
have a prominent effect in enhancement of ADCC of IgG1 compared with
bisecting GlcNAc-containing oligosaccharide. We further evaluated the
combined effect of bisecting GlcNAc with nonfucosylated
oligosaccharides. YB2/0-produced anti-CD20 IgG1 was separated based on
the content of bisecting GlcNAc-binding oligosaccharides (0, 8, 33, and
45%), which contained the same content of nonfucosylated
oligosaccharides (around 90%). These four fractions did not show any
significant difference in ADCC, indicating that the presence of
bisecting GlcNAc-binding oligosaccharides, at least under 45%, does
not have any additional effect in ADCC of highly nonfucosylated IgG1
(90%). To our knowledge, this is the first report that shows the
effect of Fuc, Gal, and bisecting GlcNAc simultaneously and also shows
the combined effect of Fuc and bisecting GlcNAc.
The ADCC have been believed to be a result of specific killing of
antigen-positive cells by natural killer cells through binding of the
IgG Fc domain to Fc Several recombinant monoclonal antibodies are being used as human
therapeutics. Some of these are blocking monoclonal antibodies to
receptors or soluble ligands and therefore may function without utilizing antibody effector functions. However, ADCC is still considered to be one of the most important anti-tumor mechanisms of
clinically effective anti-Her2 humanized IgG1 and anti-CD20 chimeric
IgG1 at least in animal models, since they are supposed to have
multiple anti-tumor mechanisms (2). More importantly, Cartron et
al. (32) have reported recently that therapeutic activity of
anti-CD20 chimeric IgG1 in patients with non-Hodgkin's lymphoma has
been correlated with polymorphism in the Fc1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8
mRNA in YB2/0 cells led to an increase of fucosylated
oligosaccharides and decrease of ADCC of the IgG1. These results
indicate that the lack of fucosylation of IgG1 has the most critical
role in enhancement of ADCC, although several reports have suggested
the importance of Gal or bisecting GlcNAc and provide important
information to produce the effective therapeutic antibody.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Rs) to the constant region (Fc) of the antibodies. ADCC
is considered to be a major function of some of the therapeutic antibodies, although antibodies have multiple therapeutic functions (e.g. antigen binding, induction of apoptosis, and
complement-dependent cellular cytotoxicity) (1, 2).
1,4-GlcNAc residue transferred to a core
-mannose (Man) residue, and it has been implicated in biological activity of therapeutic antibodies (7). N-Acetylglucosaminyltransferase III
(GnTIII), which catalyzes the addition of the bisecting GlcNAc residue
to the N-linked oligosaccharide (8), has been expressed in a
Chinese hamster ovary (CHO) cell line with an anti-neuroblastoma IgG1 and resulted in greater ADCC (9). Moreover, expression of GnTIII in a
recombinant CHO cell line has led to the increase in ADCC of the
anti-CD20 antibody (10).
R, human C1q, human FcRn, and ADCC (11). The
Fuc-deficient IgG1s have shown 50-fold increased binding to Fc
RIIIa
and enhanced ADCC. Nevertheless, there are no data on comparison
of the effect of Fuc, Gal, and GlcNAc or the combined effect of Fuc and
bisecting GlcNAc.
1,6-fucosyltransferase gene) mRNA than CHO cells, and overexpression of FUT8 in YB2/0 led the increase of fucosylation of
IgG1 and the decrease of ADCC.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain and
chimeric anti-CD20, the appropriate humanized or murine VL and VH
cDNAs were subcloned into the previously described pKANTEX93 vector
(14). The cDNA coding for the VL and VH region of each antibody was
constructed by the PCR-based method (14). In the case of chimeric
anti-CD20 antibody, the cDNA sequence of each V region was designed
as the same with that of RituxanTM (VL:
GenBankTM accession number AR015962; VH:
GenBankTM accession number AR000013). Establishment of
anti-hIL-5R humanized IgG1 will be described
elsewhere.2 Antibody
expression vectors were introduced into YB2/0 cells or DG44 cells via
electroporation and selected for gene amplification in
methotrexate-containing medium (14).
-chain and
-chain (16),
were labeled with 3.7 MBq of
Na251CrO4 at 37 °C for
1.5 h. Human effector cells were peripheral blood mononuclear
cells (PBMC) purified from healthy donors using Polymorphprep (Nycomed
Pharma AS, Roskilde, Norway). Aliquots of the 51Cr-labeled
target cells were dispensed into 96-well U-bottomed plates (1 × 104/50 µl) and incubated with serial dilutions of
antibodies (50 µl) in the presence of human effector cells (100 µl)
at an E/T ratio of 90/1. After 4 h of incubation at 37 °C, the
plates were centrifuged, and the radioactivity in the supernatants was
measured using a
counter. The percentage of specific cytolysis was
calculated from the counts of samples according to the formula,
where E represents the experimental release (cpm in
the supernatant from target cells incubated with antibody and effector cells), S is the spontaneous release (cpm in the supernatant
from target cells incubated with medium alone), and M is the
maximum release (cpm released from target cells lysed with 1 mol/liter HCl).
(Eq. 1)
where E represents the experimental release (activity
in the supernatant from target cells incubated with antibody and
effector cells), SE is the spontaneous release
in the presence of effector cells (activity in the supernatant from
effector cells), ST is the spontaneous release
of target cells (activity in the supernatant from target cells
incubated with medium alone), and M is the maximum release
of target cells (activity released from target cells lysed with 9%
Triton X-100).
(Eq. 2)
-methyl-D-mannoside (Nacalai Tesque, Kyoto,
Japan) in 60 min. An LA-PHA-E4 (4.6 × 150 mm; HONEN)
column was installed in the LC-6A HPLC system (Shimadzu). Purified
antibodies were applied to the column, previously equilibrated with 50 mM Tris-H2SO4 (pH 8.0) (A buffer).
The column was eluted with the buffer containing 0.1 M
K2B4O7 (B buffer). Elution was
followed by linear gradients from 0 to 58% B buffer in 35 min and then 100% B buffer for 5 min. The column was equilibrated with 100% A
buffer for 20 min before the next injection. Each chromatography was
performed at room temperature and a flow rate of 0.5 ml/min.
1,6-Fucosyltransferase-overexpressing
YB2/0 Cells--
A mammalian expression vector,
pAGE249, which was derived by excision of a 2.7-kb
SphI-SphI fragment containing the dihydrofolate reductase gene expression cassette from pAGE249 (20), was employed. This plasmid contained a hygromycin-resistant gene driven by the herpes simplex virus thymidine kinase gene promoter. The murine FUT8
cDNA (1728 bp) (21) was inserted into pAGE248 under the control of
the Moloney murine leukemia virus 3'-LTR promoter to form a plasmid
designated as pAGEmfFUT8. Two FspI restriction sites located
within the plasmid backbone enabled linearization of the expression
vector construct, prior to transfection of cells. FUT8 expression
vector, pAGEmfFUT8, was introduced into anti-CD20-IgG1-producing YB2/0
cells via electroporation, and FUT8-overexpressing YB2/0 cells,
3065ft8-72, were selected in 0.5 mg/ml hygromycin-B (Sigma)-containing medium.
-actin transcripts was also quantified by competitive RT-PCR in
which a 1128-bp partial fragment of rat
-actin cDNA was used as
a standard DNA and a 948-bp fragment deleting a 180-bp
DraIII (blunt)-DraIII (blunt) fragment from
standard DNA as a competitor were employed. Standard DNA was amplified
from single-stranded cDNAs of YB2/0 cells by PCR using primers
5'-ATTTAAGGTACCGAAGCATTTGCGGTGCACGATGGAGGGG-3' and
5'-AAGTATAAGCTTACATGGATGACGATATCGCTGCGCTCGT-3'. PCRs for
detection of
-actin were carried out by heating at 94 °C for 3 min and subsequent 17 cycles of 94 °C for 30 s, 65 °C for 1 min, and 72 °C for 2 min in 20 µl of reaction mixture containing 5 µl of the 50-fold single-stranded cDNAs, 1 pg of linearized
competitors, 10 pmol of
-actin-specific primers, 4 nmol of dNTP
mixture, 5% dimethyl sulfoxide, and ExTaq polymerase (TakaRa Bio). The
sense primer 5'-GATATCTGCTGCGCTCGTCGTCGAC-3' and antisense
primer 5'-CAGGAAGGAAGGCTGGAAGAGAGC-3' were designed to amplify an
-actin fragment. Aliquots of PCR products (7 µl) were subjected to
electrophoresis in 1.75% agarose gel for analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain
and
-chain. Human peripheral blood mononuclear cells were used as
effector cells for ADCC. Both humanized KM8399 and KM8404 showed high
affinity to the soluble hIL-5R
-chain antigen and had no differences
in antigen binding in enzyme-linked immunosorbent assay (data not
shown). In contrast, the ADCC of KM8399 was ~50-fold higher than that
of KM8404 at the concentration of antibody of 25% cytotoxicity that
was the maximum activity of KM8404 (Fig.
1A), indicating that
YB2/0-produced IgG1 promoted killing of IL-5R-positive cells at an
~50-fold lower concentration than the CHO-produced IgG1.
View larger version (22K):
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Fig. 1.
Evaluation of ADCC of anti-hIL-5R IgG1
(A) and anti-CD20 IgG1 (B).
Lysis (%) of target cells by human PBMC as effector cells
(E/T ratio of 90:1 (A) or 25:1
(B)) in the presence of antibody at the indicated
concentrations was measured via release of 51Cr
(A) or lactate dehydrogenase (B). The percentage
of cytotoxicity was calculated as described under "Experimental
Procedures." A, filled circles,
KM8399; open squares, KM8404. B,
filled circles, KM3065; open
squares, RituxanTM.
View larger version (34K):
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Fig. 2.
Oligosaccharide profiles of anti-hIL-5R
IgG1. Oligosaccharides derived from IgG1s produced by YB2/0
(A) and CHO (B) cell cultures were
pyridylaminated and applied to reverse-phase HPLC as described under
"Experimental Procedures." The alphabetical peak codes correspond
to those of oligosaccharide structure in C. A conserved
oligosaccharide core, linked to the Asn, is composed of three Man and
two GlcNAc monosaccharide residues. Additional GlcNAcs are normally
1,2-linked to the
6 Man and
3 Man (
6 and
3 arms,
respectively), whereas the monosaccharide residues in
boldface type, Gal, Fuc, and the bisecting GlcNAc
(boxed), can be present or absent. Peaks indicated with
asterisks are artifacts of pyridylamino derivatives (33).
The percentage of nonfucosylated oligosaccharides was calculated by the
following equation: total peak area of a, b, c, d, i, and j)/(total
peak area) × 100.
Monosaccharide composition of IgGls
Oligosaccharide composition of IgGls
), total percentage of nonfucosylated
oligosaccharides. Bis(+), total percentage of bisecting GlcNAc-binding
oligosaccharides. G0, G1, and G2, total percentage of
nongalactosylated, monogalactosylated, and digalactosylated
oligosaccharides, respectively.
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Fig. 3.
LCA lectin affinity chromatography and ADCC
assay of KM8399. A, purified KM8399 was applied on an
LA-LCA column. The column was eluted with a linear gradient of 0.5 M -methyl-D-mannoside as
described under "Experimental Procedures." Fractions I and II were
analyzed in Table II. B, lysis (%) of target cells by human
PBMC as effector cells (E/T ratio of 90:1) in the
presence of antibody at the indicated concentrations was measured via
release of 51Cr. Filled circles,
KM8399 before separation; filled diamonds,
fraction I; filled squares, fraction II.
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Fig. 4.
Serial lectin affinity chromatography and
ADCC assay of KM8399. A, purified KM8399 was applied on
an LA-PHA-E4 column. The column was eluted with a linear
gradient of 0.1 M
K2B4O7 as described under
"Experimental Procedures." Fractions III (B) and IV
(C) were then applied to the LA-LCA column. The column was
eluted with linear gradient of 0.2 M
-methyl-D-mannoside as described under "Experimental
Procedures." Fraction III' and IV' were analyzed in Table III.
D, lysis (%) of target cells by human PBMC as effector
cells (E/T ratio of 90:1) in the presence of
antibody at the indicated concentrations was measured via release of
51Cr. Open squares, fraction III';
filled squares, fraction IV'.
Oligosaccharide composition of PHA-LCA-separated fractions
), total percentage of nonfucosylated
oligosaccharides. Bis(+), total percentage of bisecting GlcNAc-binding
oligosaccharides. G0, G1, and G2, total percentage of
nongalactosylated, monogalactosylated, and digalactosylated
oligosaccharides, respectively.
View larger version (56K):
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Fig. 5.
PHA-E4 lectin affinity
chromatography and ADCC assay of KM3065. A, purified
KM3065 was applied on an LA-PHA-E4 column. The column was
eluted with a linear gradient of 0.1 M
K2B4O7. Fractions were analyzed in
Table IV. B, lysis (%) of target cells by human PBMC as
effector cells (E/T ratio of 20:1) in the
presence of antibody at the indicated concentrations was measured via
release of lactate dehydrogenase. Filled circles,
KM3065 before separation; open circles, fraction
V; open triangles, fraction VI; open
diamonds, faction VII; filled
diamonds, fraction VIII; filled
triangles, anti-CD20 IgG1 derived from LEC10;
open squares, RituxanTM.
Oligosaccharide composition of PHA-E4-separated fractions
), total percentage of nonfucosylated
oligosaccharides. Bis(+), total percentage of bisecting GlcNAc-binding
oligosaccharides. G0, G1, and G2, total percentage of
nongalactosylated, monogalactosylated, and digalactosylated
oligosaccharides, respectively.
1,6-Fucosyltransferase in YB2/0
Cells and CHO Cells--
FUT8 is considered to be the only gene coding
1,6-fucosyltransferase, which catalyzes the transfer of Fuc from
GDP-Fuc to GlcNAc in
1,6-linkage of complex-type oligosaccharides,
because no homologous gene has been found (25). To elucidate the
mechanism of lower Fuc content of YB2/0-produced IgG1 than that of
CHO-produced IgG1, we examined expression levels of FUT8 in each cell line.
-actin transcripts were also quantified by
competitive RT-PCR. These PCR analyses, which were performed independently three times, revealed that an expected FUT8 fragment from
YB2/0 cells has poor intensity compared with CHO cells (Fig. 6A). The expression level of
FUT8 transcripts in YB2/0 cells was shown to be 0.1% relative to
-actin transcripts, whereas CHO cells have 2.0% FUT8 transcripts to
-actin transcripts. There was no significant difference observed
when we performed PCR analysis using primers and competitor DNAs
specific for Chinese hamster FUT8 and
-actin. These results suggest
that lower expression of FUT8 mRNA is the cause for lower content
of Fuc of IgG1 produced by YB2/0 cells.
View larger version (45K):
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Fig. 6.
Competitive PCR analysis and ADCC assay of
FUT8 expression in YB2/0 cells and CHO/DG44 cells. A,
single-stranded cDNAs prepared from each cell line or standard FUT8
DNAs (0.1, 1, 10, 100, and 1,000 fg) were subjected to PCR with 10 fg
of competitor DNA as described under "Experimental Procedures."
Arrows, the amplified products with the expected size.
B, lysis of Raji human B by human PBMC at a target/effector
ratio of 1:20 in presence of different concentrations of antibodies was
quantified by detecting lactate dehydrogenase activity. The percentage
of cytotoxicity is calculated relative to a total lysis control, after
subtraction of the signal in the absence of IgG1s. Filled
circles, KM3065; open squares,
RituxanTM; filled squares, anti-CD20
IgG1 derived from FUT8-overexpressed YB2/0 cells.
1,6-Fucosyltransferase in YB2/0
Cells--
To confirm the effects of FUT8 expression on the ADCC of
IgG1 produced by YB2/0 cells, FUT8-overexpressing YB2/0 cells were established by transfection of murine FUT8 cDNA to anti-CD20
IgG1-producing YB2/0 cells. The most highly FUT8-expressing clone that
showed 145.5% FUT8 transcripts relative to
-actin transcripts was
selected and designated as 3065ft8-72 (Fig. 6A). The ADCC of
anti-CD20 IgG1 produced by 3065ft8-72 cells was 100-fold lower than
that produced from the original YB2/0 cells and equivalent to the
commercially available anti-CD20 IgG1 RituxanTM produced by
normal CHO cells (Fig. 6B). Monosaccharide analysis of each
IgG1 showed that there was no significant difference between the IgG1
from FUT8-overexpressing cells and the original YB2/0 cells except that
a higher amount of Fuc (81%; data not shown) was detected in the IgG1
from FUT8-overexpressing cells. These findings strongly suggest that
FUT8 acts as a key gene in YB2/0 cells to affect the Fuc content as
well as ADCC of IgG1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,6-linkage to the GlcNAc of the
reducing end ("core Fuc") are relatively common in mammalian
N-linked oligosaccharide. FUT8, considered to be the only
gene that codes
1,6-fucosyltransferase, catalyzes the transfer of
Fuc from GDP-Fuc to GlcNAc of the reducing end. Therefore, we focused
on FUT8 as a key gene controlling the low Fuc content of IgG1 produced
by YB2/0 and indicate that the cells produce low Fuc content IgG1 simply due to the low expression level of FUT8. Since biosynthesis of
N-linked oligosaccharides is controlled by a number of
glycosyltransferases, their acceptors and substrates, etc., it remains
to be determined whether there is possible involvement of the
other factor in biosynthesis of nonfucosylated IgG1 in YB2/0 cells.
RIIIa. Recently, Shields et al. (11) have revealed that binding of the Fuc-deficient IgG1 (produced by Lec13
cells) to Fc
RIIIa was enhanced up to 50-fold. They have shown that
improved binding to Fc
RIIIa has translated into improved ADCC
in vitro, using PBMC or natural killer cells. These results suggest that Fuc-deficient IgG1 may require a lower concentration of
antibody on the surface of the target cell to activate an
effector cell. There are a few possible explanations of why the
antibodies with nonfucosylated oligosaccharides give rise to stronger
binding to Fc
RIIIa than those in which the glycoforms are absent. A
core Fuc has been shown to influence the conformational flexibility of
biantennary oligosaccharides (26, 27). The oligosaccharides of IgG
appear to be largely sequestered between the CH2 domains and may help
to stabilize the CH2 domain (28). In the co-crystal structure of IgG1
Fc:Fc
RIIIb, Fuc is orientated away from the interface and making no
specific contacts with the receptor (29); nevertheless, Harris et
al. (30, 31) have supposed that Fuc could have influence on the
binding by the receptor. We speculated that the absence of Fuc provided
a more suitable conformation for the binding of IgG1 to Fc
RIII than
the presence of bisecting GlcNAc or Gal, although structural analyses
of a series of IgG1 with or without Fuc, bisecting GlcNAc, or Gal are
needed for further discussion.
RIIIa gene. They have
shown that FCGR3A-158V patients showed a better response to anti-CD20
chimeric IgG1 because they have higher ADCC against lymphoma cells.
These reports have suggested that therapeutic antibodies with enhanced
ADCC including the nonfucosylated IgG1 would result in the improvement
of clinical response. Moreover, these findings may allow for use of the
nonfucosylated IgG1 at lower doses with no reduction in efficacy.
Antibody therapeutics effective in lower doses might reduce the cost of
antibody therapy.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 81-42-725-2555; Fax: 81-42-725-2689; E-mail: kshitara@kyowa.co.jp.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M210665200
2 M. Koike, E. Shoji-Hosaka, K. Nakamura, A. Furuya, K. Shitara, and N. Hanai, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: ADCC, antibody-dependent cellular cytotoxicity; hIL-5R, human interleukin 5 receptor; Fuc, fucose; Man, mannose; CHO, Chinese hamster ovary; Fc, the constant region of the antibody; PBMC, peripheral blood mononuclear cell; LCA, L. culinaris aggulutin; PHA-E4, P. vulgaris E4; HPLC, high performance liquid chromatography; LTR, long terminal repeat; GnTIII, N-acetylglucosaminyltransferase III.
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REFERENCES |
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