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Departments of
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Analytical Chemistry,
Immunology,
QC Clinical Development,
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BioAnalytical Technology, and
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Protein Engineering, Genentech, South San Francisco, CA 94080
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Complement activation occurs by the binding of C1q to the Fc domain of
Igs, IgG or IgM, complexed with Ags (3). C1q is a large
structurally complex glycoprotein of 
410 kDa present in human serum
at a concentration of 70 µg/ml (4). Together with two
serine proteases, C1r and C1s, C1q forms the complex C1, the first
component of complement. At least two of the N-terminal globular heads
of C1q must be bound to the Fc of Igs for C1 activation, hence for
initiation of the complement cascade (4).
Various investigations with a focus on the C1q binding region of IgG have led to some understanding of the recruitment of complement by Abs (5). By assessment of solvent-accessible amino acid residues, sequence comparison, chemical modification studies, domain swapping, and site-directed mutagenesis experiments, the CH2 domain has been shown to be required for C1q binding (6, 7, 8, 9, 10). More specifically, by focusing on conserved residues capable of polar interactions, Duncan and Winter revealed that N297, E318, K320, and K322 in murine IgG2b are important for C1q binding and proposed E318, K320, and K322 as the core of the C1q binding site (7). The presence of an oligosaccharide at position N297 has been shown to be important for most Ab effector functions (11, 12, 13, 14). In their study, Duncan and Winter demonstrated that alanine substitution at E318, K320, and K322 in murine IgG2b resulted in mutants that were nonlytic and at least 30-fold lower in binding to guinea pig C1q than the wild-type Ab. Because these three residues, E318, K320, and K322, are conserved in human IgG and IgGs of several other species, they have been designated as the C1q binding motif (7). However, several studies implicate that the contact sites for C1q in murine IgG2b may be different from that of human IgG1 (9, 10, 13, 15, 16). In one of these studies, a K320A mutation in a human IgG1 Ab was shown to have little or no effect on C1q binding or CDC activity (10). Also, a few residues, L235, D265 and P331, which are important for C1q binding and CDC activity in a human IgG background (9, 10, 13, 15, 16), are of minimal importance for complement activation in a murine IgG2b background (7). Substitution of P331 with serine in human IgG1 resulted in a 60% decrease in binding to C1q and complete loss of complement activity (15). Vice versa, substitution of S331 to proline in IgG4 resulted in a molecule that could bind C1q and activate complement (15, 16). Substitution of D265, an oligosaccharide interaction site, with alanine in human IgG3 was shown to have impaired CDC and ADCC activity (13). Likewise, a leucine to glutamic acid substitution at position 235 in human IgG1 resulted in a mutant with impaired CDC and ADCC activity (10). Together, these data suggest that the contact site for C1q on murine IgG2b is perhaps different from that on human IgG1.
In this study, our aim was to identify mutations in the CH2 domain of Rituximab that ablate binding to C1q without altering ADCC or binding to the CD20 and FcRn receptors, thereby designing a molecule that is deficient in complement activation exclusively. It is important to retain optimal binding to the neonatal Fc receptor, FcRn, because the ability of an IgG to bind FcRn determines its in vivo half-life (17, 18). By alanine substitutions, we demonstrate that two mutants of human IgG1, D270A and P329A, are deficient in complement activation and C1q binding. We confirm that the amino acid residues, D265 and P331, are important for C1q binding and complement activation in a human IgG1 background. In accordance with Morgan et al. (10), we show alanine substitution at position 320 in Rituximab had no effect on complement activation. Also, substitution of E318 to alanine in Rituximab had no effect on complement-mediated lysis. In addition, some of these mutants with low affinity for C1q recruit ADCC in a manner similar to the wild-type Ab and bind normally to the CD20 Ag and the FcRn receptor. Our results demonstrate that there are species differences in complement activation and reiterate the core C1q binding site in a human IgG1 is different from that of murine IgG2b.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The chimeric L and H chains of Rituximab (IDEC Pharmaceuticals, San Diego, CA) (1) subcloned separately into previously described pRK vectors (19) were used. By site-directed mutagenesis (20), alanine variants of the CH2 domain of the H chain were constructed. The H and L chain plasmids were cotransfected into the adenovirus-transformed human embryonic kidney 293 cell line (American Type Culture Collection, Manassas, VA) as previously described (21). The media was changed to serum-free 24 h after transfection, and the secreted Ab was harvested after 5 days. The Abs were purified using protein A-Sepharose CL-4B (Pharmacia, Piscataway, NJ), buffer exchanged with PBS and concentrated to 0.5 ml using a Centricon-30 (Amicon, Beverly, MA), and stored at 4°C. The concentration of the Ab was determined using total Ig-binding ELISA. The results reported here were consistent in two separate transfections and preparations of Ab. Also, Rituximab and mutants had the same the binding efficiency to the 96-well plates used in each assay.
C1q binding assay
The binding of human C1q (Quidel, San Diego, CA) to Rituximab and mutants was assessed by an ELISA binding assay. High binding Costar 96-well plates (Corning, NY) were coated overnight at 4°C with varying concentrations of Rituximab in coating buffer (0.05 M sodium carbonate buffer, pH 9). The plates were washed after each incubation step with PBS/0.05% Tween 20, pH 7.4, and incubations after coating were performed at room temperature. After coating, the plates were blocked with 200 µl of ELISA diluent (0.1 M NaPO4/0.1 M NaCl/0.1% gelatin/0.05% Tween 20/0.05% ProClin300) for 1 h, and incubated for 2 h with 100 µl of 2 µg/ml human C1q in ELISA diluent. Then, 100 µl of a 1:1000 dilution of sheep anti-human C1q peroxidase-conjugated Ab (Biodesign, Kenne- bunkport, ME) in ELISA diluent was added and incubated for 1 h. The plates were developed with 100 µl of substrate buffer (PBS/0.012% H2O2) containing o-phenylenediamine dihydrochloride (Sigma, St. Louis, MO). The reaction was stopped by the addition of 100 µl of 4.5 N H2SO4, and the OD was measured at 492 nm using a microplate reader Spectra MAX 250 (Molecular Devices, Sunnyvale, CA). To correct for background, the OD at 405 nm was subtracted from the OD at 492 nm. The binding efficiency of each mutant to the plate was examined using an anti-human IgG Fc peroxidase-conjugated Ab as the probe (Jackson ImmunoResearch, West Grove, PA).
CDC assay
The ability of Rituximab and mutants to promote cell killing of a CD20-expressing B lymphoblastoid cell line, WIL2-S (American Type Culture Collection), was assessed by a method previously described (22). Serum complement from human (Quidel, San Diego, CA), rabbit (ICN, Costa Mesa, CA), or guinea pig (Life Technologies, Grand Island, NY) was used. Rituximab (0.08–100 µg/ml) was diluted with RHB buffer (RPMI 1640 (Life Technologies), 20 mM HEPES, pH 7.2, 2 mM glutamine, 0.1% BSA, 100 µg/ml gentamicin). WIL2-S cells were washed in RHB buffer and resuspended at a density of 106 cells/ml. In a typical assay, 50 µl of Rituximab, 50 µl of diluted complement, and 50 µl of a cell suspension (50,000 cells/well) were added to a flat-bottom tissue culture 96-well plate. The mixture was incubated for 2 h at 37°C in a 5% CO2 incubator to facilitate complement-mediated cell lysis. Then, 50 µl of Alamar Blue (Accumed International, Westlake, OH) was added to each well and incubated overnight at 37°C. Fluorescence was read using a 96-well fluorometer with excitation at 530 nm and emission at 590 nm. As previously described, the results are expressed in relative fluoresence units (RFU) that are proportional to the number of viable cells. The activity of the various mutants was examined by plotting the percent CDC activity against the log of Ab concentration (final concentration before the addition of Alamar Blue) using a four-parameter curve fitting program (Kaleidagraph).
The percent CDC activity was calculated as follows: % CDC activity = (RFU test - RFU background) x 100 (RFU at total cell lysis - RFU background).
ADCC assay
Blood from normal volunteers was drawn into heparinized syringes, mixed with an equal volume of HBSS without Ca2+/Mg2+ (Life Technologies), layered onto a Lymphoprep gradient (Life Technologies), and centrifuged at 800 x g for 20 min. PBMC at the interface were harvested and washed in HEPES-buffered saline and resuspended in assay medium (RPMI 1640 media (Life Technologies) containing 1% heat-inactivated FBS (HyClone Laboratories, Logan, UT), 2 nM L-glutamine, 10 mM HEPES, and 50 µg/ml gentamicin). WIL2-S cells (104 cells/well) in 50 µl of assay buffer, and varying concentrations of Ab samples in 50 µl of assay buffer were added into round-bottomed 96-well plates. The mixture was preincubated for 30 min at 37°C. Then, 50 µl of the effector cells (2.5 x 105) were dispensed into the wells and incubation was continued for 4 h at 37°C. An E:T ratio of 25:1 was used. The plates were centrifuged at 250 x g for 10 min, and the supernatants were harvested. The activity of lactate dehydrogenase in the supernatants were determined by using a Cytotoxic Detection kit (Boehringer Mannheim, Indianapolis, IN) and the manufacturer’s protocol. The average absorbance of triplicates was used to calculate the percentage of cytotoxicity.
The percentage of cytotoxicity was calculated as follows: % cytotoxicity = (experimental - effector spontaneous - target spontaneous) x 100 (target maximum - target spontaneous)
CD20 binding potency of the Rituximab mutants
The binding of Rituximab and mutants to the CD20 Ag was assessed by a method previously described (1, 22). This assay is a cell-based FACS assay in which WIL2-S cells are used as the CD20-expressing cell line. The binding of Rituximab and mutants to WIL2-S cells was detected by a goat anti-human IgG-FITC Ab (American Qualex, San Clemente, CA). The results expressed as RFU are proportional to the amount of FITC-labeled dectection Ab bound to the cell.
FcRn binding ELISA
The 
and ß2-microglobulin clones of
the human FcRn were obtained (Research Genetics, I.M.A.G.E. Consortium,
Huntsville, AL). Each polypeptide was subcloned separately into
previously described pRK vectors (19) and cotransfected
into human embryonic kidney 293 cells. The human FcRn was expressed as
a His6-tagged extracellular domain, purified by
Ni-NTA column (Qiagen, Valencia, CA) chromatography, and buffer
exchanged into PBS. The concentration was measured by absorbance at 280
nm using an extinction coefficient (0.1%, 1 cm, 280 nm) of 1.9.
Maxisorb Nunc 96-well immunoplates (Nalge Nunc International,
Naperville, IL) were coated with 100 µl of 2 µg/ml of streptavidin
(Zymed Laboratories, South San Francisco, CA) in coating buffer (0.05 M
carbonate/bicarbonate, pH 9.6) at 4°C overnight. The plates were
washed after each incubation step with PBS/0.05% Tween 20, pH 7.2, and
incubations were performed at room temperature. After coating, the
plates were blocked with PBS/0.5% BSA, pH 7.2, for 1 h, incubated
with 100 µl of 1–2 µg/ml biotinylated FcRn for 1 h, and then
100 µl containing various concentrations of Rituximab in dilution
buffer (PBS/0.5%BSA/0.05% Tween 20, pH 6.0) was added to each well.
After 2 h incubation, 100 µl of goat
F(ab')2 anti-human IgG
F(ab')2 HRP conjugate (Jackson ImmunoResearch) in
dilution buffer was added and incubated for 1 h. The plates were
developed using a 3,3',5,5'-tetramethylbenzidine substrate detection
method (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and OD
values were measured at 450 nm using a Vmax microplate
reader (Molecular Devices, Sunnyvale, CA). To correct for background,
the OD at 650 nm was substracted from the OD at 450 nm.
Determination of C3 and C4 levels in complement
C3 and C4 levels in complement from the three different species used in this study, human, rabbit, and guinea pig, were measured by radial immunodiffusion (National Jewish Center Complement Laboratory, Denver, CO.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptors and FcRn binding. The alanine mutants,
E318A, K320A, and K322A, having been found to ablate binding to murine
IgG2b (7), were initially constructed and assessed for
their ability to bind C1q and activate complement. When compared with
Rituximab, there appeared to be little difference in the binding of
E318A and K320A to C1q (Fig. 1
A). The IgG2 negative control
appears to have a much lower affinity for C1q than the E318A and K320A
mutants. Also, the ability of E318A and K320A to activate complement
was essentially identical with the wild-type Ab (Fig. 1B).
These results demonstrate that the effect of the E318A and the K320A
substitution in a human Fc on complement activation and C1q binding was
minimal. Conversely, we found that the K322A substitution had a
significant effect on both complement activity and C1q binding. The
K322A mutant displayed no CDC activity when a 1/12 dilution of human
complement was used and was significantly lower in binding to C1q
when compared with wild-type (Fig. 1, A and B).
Under these conditions, the IgG2 construct did not promote cell killing
(Fig. 1B).
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A) in our standard assay and
no CDC activity (Fig. 1B) when a 1/12 dilution of human
complement was used. Although considerably impaired in its ability to
bind C1q (Fig. 1A), only a slight decrease in CDC activity
(Fig. 1B) was observed for the P331A mutant when a 1/12
dilution of human complement was used. An additional mutant, D265A, was
also constructed to determine whether D265 was important for complement
activation in a human IgG1 background. As shown in Fig. 1, the D265A
substitution in Rituximab severely impaired C1q binding and decreased
CDC activity.
We observed that the activity of these low-affinity mutants, D270A,
K322A, and P329A, could be rescued by increasing the complement
concentration from a 1/12 to a 1/3 dilution, which approaches
physiological levels of complement (Fig. 2, A–C). At a 1/6 dilution of
human complement, some activity was observed for D270A, K332A, and
P329A, and at a 1/3 dilution of human complement, although decreased in
bioactivity when compared with Rituximab, these mutants were able to
confer >60% lysis (Fig. 2, B and C). To ensure
that the activity of the low-affinity C1q binding mutants at high
concentrations of complement was not due to activation of the
alternative pathway, the CDC assays were also performed in the presence
of Mg2+ and EGTA. The addition of
Mg2+-EGTA at either a 1/12, 1/6, or 1/3 dilution
of human complement reduced the CDC activity of Rituximab and mutants
to background (data not shown). In the presence of
Mg2+-EGTA, the CDC activity observed for the
mutants as well as wild type ranged from 2 to 6% when probed with a
1/3 dilution of human complement. This experiment was performed at a
fixed concentration of Ab (0.6 µg/ml). Under the same experimental
conditions but without Mg2+-EGTA, the wild type
showed about 65% activity and the activity of the mutants was between
30 and 40%. In addition, in the presence of
Mg2+-EGTA the CDC activity of the mutants as well
as the wild type did not change with an increase in complement
concentration from a 1/12 to a 1/3 dilution. This indicates the
observed CDC activity of the low-affinity binding mutants at high
concentration of complement is not due to activation of the alternative
pathway. Nonetheless, our results demonstrate that the human C1q
binding epicenter of human IgG1 is centered around these four spatially
close residues, D270, K322, P329, and P331 (Fig. 3). For the most part, single point
mutations at these sites, D270, K322, P329, and P331, do not alter any
other Ab effector functions. All four mutants, D270A, K322A, P329A, and
P331A, bound normally to the CD20 and FcRn receptors (data not shown).
However, unlike the D270A, K322A, and P331A mutants, which were
able to recruit ADCC to an appreciable extent, the P329A mutant was
markedly impaired in its ability to recruit ADCC (Fig. 4).
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A). The binding of the K322R
mutant to C1q was comparable to that of the wild-type Ab, indicating
that the K322R mutation did not alter C1q binding. The lack of binding
of the K322M mutant suggests that a positive charge is required at this
position. Substitution of D270 with alanine, valine, or lysine resulted
in mutants that were severely deficient in C1q binding (Fig. 5A). At P329 and P331, any amino acid substitution resulted
in mutants that were deficient in C1q binding and CDC activity (data
not shown). Due to the unique restriction on backbone conformation
imposed by proline, it is possible that point mutations at these sites,
329 and 331, cause local perturbations in structure. Among the P331
mutants constructed were P331S and P331G. Although the binding of the
P331S mutant was substantially decreased when compared with the
wild-type Ab and the P331A mutant, the P331S mutant was able to bind
C1q and promote cell killing in our system (Fig. 5, A and
B). In contrast, the P331G mutant showed no binding to C1q
and no CDC activity when a 1/12 dilution of human complement was used
(Fig. 5, A and B).
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shows the E318A and K320A mutants were
as efficient as Rituximab in complement-mediated lysis when probed with
either human (Fig. 6A), rabbit (Fig. 6B), or
guinea pig (Fig. 6C) complement. Likewise, D270A, K322A,
P329A, and P331A showed a deficiency in CDC when tested with the
different sources of complement (Fig. 6, A–C). However,
there were a few differences in CDC activity with the different sources
of complement. For example, Rituximab and the mutants appeared to be
less effective in promoting cell lysis when probed with guinea pig
complement. At the same dilution (1/9) of human and rabbit complement,
at least 100-fold higher concentration of Rituximab was required for
50% cell lysis when guinea pig complement was used as the probe (Fig. 6, A–C). The mutant D270A appeared to be much more active
when challenged with rabbit (Fig. 6B) than with human
complement (Fig. 6A) and showed no activity with guinea pig
complement (Fig. 6C). At a concentration of 1 µg/ml of
D270A, 80% cell lysis was observed with rabbit complement, and 20%
cell lysis was observed with human complement. In contrast, the CDC
activity of the wild-type Ab (100% cell lysis at 1 µg/ml of Ab)
was essentially the same with either rabbit or human complement. Also,
the P331A mutant showed only a slight deficiency in the ability to
recruit complement-mediated cell lysis when probed with a 1/9 dilution
of rabbit and human complement, but appeared to be completely inactive
when probed with the same dilution of guinea pig complement. To
determine whether the disparity between the three complement sources
was to some extent a reflection of concentration differences, the
levels of complement components, C3 and C4, in the complement from the
three different species was determined. Table I shows the levels of C3 and C4 in the
human, rabbit, and guinea pig sera were comparable and within the
reference range for human sera (P. C. Giclas, unpublished
observations). Also, C1q is present in similar concentrations (70
µg/ml) in human (4) and guinea pig (23)
sera. These data indicate the concentration of complement components in
sera of human, rabbit, and guinea pig complement is similar and thus
the discrepancy in CDC activity between the three sources of complement
is probably not due to concentration differences.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). This is
consistent with the previous finding of Morgan et al. that a K320A
mutation in human IgG1 had no effect on complement lysis
(10). Also, P331 in human IgG1 has been shown by other
investigators to be important for C1q binding (15, 16). We
attribute the incongruity between our results and those obtained by
Duncan and Winter to the Ab background used. The results reported here,
on the C1q binding sites of human IgG1, were consistent with three
different sources of complement (Fig. 6, A–C). With either
guinea pig, rabbit, or human complement as the probe, the D270A, K322A,
P329A, and P331A mutants were deficient in CDC activity and the ability
of the E318A and K320A mutants to activate complement was comparable to
Ritiuximab (Fig. 6). The Duncan and Winter study was performed using
murine IgG2b with guinea pig complement as the probe (7).
Comparing the guinea pig data, the major difference between our study
and that of Duncan and Winter is the Ab background used, human IgG1 vs
murine IgG2b. Our data and that of other investigators reveal several
differences in the C1q binding site between murine IgG2b and human
IgG1. For example, E318 and K320 play only a minor role in the C1q
binding site on human IgG1 but play a major role in murine IgG2b (Refs.
7 and 10 and this work). The residues, D265,
D270, and P331, which are important for CDC in human IgG1, do not
effect a change in CDC activity when altered in a murine IgG2b
background (Refs. 7, 9, 10, 13, 15 , and 16
and this work). In addition, a L235E substitution had no effect on CDC
when constructed in murine IgG2b (7), but abolished the
ability of human IgG1 to activate complement (10). These
findings strongly suggest the C1q binding sites on murine IgG2b are
different from human IgG1. Notably, however, extremely dilute guinea
pig complement (1/40 dilution) was used to assess the activity of the
murine IgG2b mutants (7), and this may have affected the
results to some extent.
The core C1q binding site in human IgG1 was, for the most part,
independent of the source of complement (Fig. 6). However, there are
species differences in complement activation. One important difference
was at P331. Tao et al. (15) and Xu et al.
(16) demonstrated that a P331S mutation in human IgG1
resulted in a molecule that was unable to promote cell killing; their
studies were performed using guinea pig complement as the probe.
Consistent with their results, we show in this study that a mutation at
P331 (P331A) in human IgG1 resulted in a molecule that was unable to
confer complement lysis when guinea pig complement was used as the
probe (Fig. 6C). However, with either rabbit or human
complement, a substantial amount of CDC activity was observed for the
P331A mutant (Fig. 6, A and B). Also, a P331S
mutation resulted in a molecule, although impaired, was still able to
promote cell killing when challenged with human complement (Fig. 5B). The mutant D270A was much more active with rabbit
complement than with human or guinea pig complement. In addition,
Rituximab appeared to be less effective in conferring lysis when probed
with guinea pig complement than with human or rabbit complement. These
results demonstrate that there are species-specific differences in
complement, although complement concentration may also play a role in
some of the observed discrepancies.
As demonstrated here, and by other investigators (24, 25),
complement concentration is an important factor in complement-mediated
cytotoxicity. By increasing the human complement concentration, we were
able to recruit the activity of the D270A, K322A, and P329A mutants,
which appeared to be completely inactive at lower concentrations (Fig. 2, A–C). The results at higher concentrations of complement
indicate that impaired C1q binding by single point mutations at D270,
K322, and P329 will not lead to complete ablation of CDC activity.
In terms of the interaction between C1q and the core C1q binding site
in Rituiximab, our data indicates that lysine at position 322 may be
involved in an electrostatic interaction with C1q (only the K322R
mutant retained binding; loss of a positive charge, e.g., methionine,
reduced binding). At position D270, a hydrophobic or positively charged
residue does not promote C1q binding (Fig. 5). Besides possible
hydrophobic interactions with C1q, the two surface-exposed prolines
(P329 and P331) may play an important structural role; the fact that
the P329A mutant was also severely deficient in ADCC (Fig. 4) would
support this hypothesis.
In conclusion, our results demonstrate the concentration and source of
complement are important considerations in these types of studies. We
have also shown that for human IgG1, four spatially close sites on the
surface of the Ab (Fig. 3), D270, K322, P329, and P331, constitute the
C1q binding epicenter (Fig. 3). Because most of these sites are
conserved in human IgG isotypes that are deficient in C1q binding and
complement activation (5), it is evident that there are
other factors that influence complement activation. Also, other amino
acid residues in human IgG1 have been shown to be important for C1q
binding and complement activation (9, 10, 15). Perhaps,
the differences in function between these IgG isotypes is due to the
conformation of the Ab modulated by a few residues, the hinge
(26), and/or carbohydrate (27). With respect
to our goal, which is to completely ablate the ability of
Rituximab to mediate complement lysis, continued efforts are being made
to construct a mutant molecule with no detectable CDC activity.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: CDC, complement-dependent cytotoxicity; ADCC, Ab-dependent cell-mediated cytotoxicity; RFU, relative fluorescence units.
Received for publication September 2, 1999. Accepted for publication February 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI and FcRIII binding. Immunology 86:319.[Medline]
receptor I and influence the synthesis of its oligosaccharide chains. J. Immunol. 157:4963.[Abstract]
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 R. Bang, L. Marnell, C. Mold, M.-P. Stein, K. T. D. Clos, C. Chivington-Buck, and T. W. D. Clos Analysis of Binding Sites in Human C-reactive Protein for Fc{gamma}RI, Fc{gamma}RIIA, and C1q by Site-directed Mutagenesis J. Biol. Chem., July 1, 2005; 280(26): 25095 - 25102. [Abstract] [Full Text] [PDF]
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 P. Carter and C. F. McDonagh Designer Antibody-Based Therapeutics for Oncology Am. Assoc. Cancer Res. Educ. Book, April 1, 2005; 2005(1): 147 - 154. [Full Text] [PDF]
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G. Cartron, H. Watier, J. Golay, and P. Solal-Celigny From the bench to the bedside: ways to improve rituximab efficacy Blood, November 1, 2004; 104(9): 2635 - 2642. [Abstract] [Full Text] [PDF]
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S. Dall'Ozzo, S. Tartas, G. Paintaud, G. Cartron, P. Colombat, P. Bardos, H. Watier, and G. Thibault Rituximab-Dependent Cytotoxicity by Natural Killer Cells: Influence of FCGR3A Polymorphism on the Concentration-Effect Relationship Cancer Res., July 1, 2004; 64(13): 4664 - 4669. [Abstract] [Full Text] [PDF]
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M. S. Kojouharova, M. G. Gadjeva, I. G. Tsacheva, A. Zlatarova, L. T. Roumenina, M. I. Tchorbadjieva, B. P. Atanasov, P. Waters, B. C. Urban, R. B. Sim, et al. Mutational Analyses of the Recombinant Globular Regions of Human C1q A, B, and C Chains Suggest an Essential Role for Arginine and Histidine Residues in the C1q-IgG Interaction J. Immunol., April 1, 2004; 172(7): 4351 - 4358. [Abstract] [Full Text] [PDF]
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F. Hong, R. D. Hansen, J. Yan, D. J. Allendorf, J. T. Baran, G. R. Ostroff, and G. D. Ross {beta}-Glucan Functions as an Adjuvant for Monoclonal Antibody Immunotherapy by Recruiting Tumoricidal Granulocytes as Killer Cells Cancer Res., December 15, 2003; 63(24): 9023 - 9031. [Abstract] [Full Text] [PDF]
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C. Gaboriaud, J. Juanhuix, A. Gruez, M. Lacroix, C. Darnault, D. Pignol, D. Verger, J. C. Fontecilla-Camps, and G. J. Arlaud The Crystal Structure of the Globular Head of Complement Protein C1q Provides a Basis for Its Versatile Recognition Properties J. Biol. Chem., November 21, 2003; 278(47): 46974 - 46982. [Abstract] [Full Text] [PDF]
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 R. Bannerji, S. Kitada, I. W. Flinn, M. Pearson, D. Young, J. C. Reed, and J. C. Byrd Apoptotic-Regulatory and Complement-Protecting Protein Expression in Chronic Lymphocytic Leukemia: Relationship to In Vivo Rituximab Resistance J. Clin. Oncol., April 15, 2003; 21(8): 1466 - 1471. [Abstract] [Full Text] [PDF]
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A. D. Kennedy, M. D. Solga, T. A. Schuman, A. W. Chi, M. A. Lindorfer, W. M. Sutherland, P. L. Foley, and R. P. Taylor An anti-C3b(i) mAb enhances complement activation, C3b(i) deposition, and killing of CD20+ cells by rituximab Blood, February 1, 2003; 101(3): 1071 - 1079. [Abstract] [Full Text] [PDF]
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R. L. Shields, J. Lai, R. Keck, L. Y. O'Connell, K. Hong, Y. G. Meng, S. H. A. Weikert, and L. G. Presta Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human Fcgamma RIII and Antibody-dependent Cellular Toxicity J. Biol. Chem., July 19, 2002; 277(30): 26733 - 26740. [Abstract] [Full Text] [PDF]
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G. Cartron, L. Dacheux, G. Salles, P. Solal-Celigny, P. Bardos, P. Colombat, and H. Watier Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor Fcgamma RIIIa gene Blood, February 1, 2002; 99(3): 754 - 758. [Abstract] [Full Text] [PDF]
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R. F. Montano and S. L. Morrison Influence of the Isotype of the Light Chain on the Properties of IgG J. Immunol., January 1, 2002; 168(1): 224 - 231. [Abstract] [Full Text] [PDF]
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 M. Hezareh, A. J. Hessell, R. C. Jensen, J. G. J. van de Winkel, and P. W. H. I. Parren Effector Function Activities of a Panel of Mutants of a Broadly Neutralizing Antibody against Human Immunodeficiency Virus Type 1 J. Virol., December 15, 2001; 75(24): 12161 - 12168. [Abstract] [Full Text] [PDF]
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W.-K. Weng and R. Levy Expression of complement inhibitors CD46, CD55, and CD59 on tumor cells does not predict clinical outcome after rituximab treatment in follicular non-Hodgkin lymphoma Blood, September 1, 2001; 98(5): 1352 - 1357. [Abstract] [Full Text] [PDF]
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E. E. Idusogie, P. Y. Wong, L. G. Presta, H. Gazzano-Santoro, K. Totpal, M. Ultsch, and M. G. Mulkerrin Engineered Antibodies with Increased Activity to Recruit Complement J. Immunol., February 15, 2001; 166(4): 2571 - 2575. [Abstract] [Full Text] [PDF]
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R. L. Shields, A. K. Namenuk, K. Hong, Y. G. Meng, J. Rae, J. Briggs, D. Xie, J. Lai, A. Stadlen, B. Li, et al. High Resolution Mapping of the Binding Site on Human IgG1 for Fcgamma RI, Fcgamma RII, Fcgamma RIII, and FcRn and Design of IgG1 Variants with Improved Binding to the Fcgamma R J. Biol. Chem., February 23, 2001; 276(9): 6591 - 6604. [Abstract] [Full Text] [PDF]
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L. Vidarte, C. Pastor, S. Mas, A. B. Blazquez, V. de los Rios, R. Guerrero, and F. Vivanco Serine 132 Is the C3 Covalent Attachment Point on the CH1 Domain of Human IgG1 J. Biol. Chem., October 5, 2001; 276(41): 38217 - 38223. [Abstract] [Full Text] [PDF]
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), E318A (
), K320A (•), K322A (
), P329A (
),
and P331A (
). An IgG2 (
) construct of Rituximab was used as a
negative control. C1q binding (A) and CDC activity
(B) using a 1/12 dilution of human complement as a
probe.
), K322R (
), K322N
(
), P331S (•), D270A (+), D270K (–),
and D270V (
). B, CDC activity of wild type (x) and
the mutants, P331A (