IMMUNOBIOLOGY
Complement-mediated lysis by anti-CD20 mAb correlates with
segregation into lipid rafts
Mark S. Cragg, Suzanne M. Morgan, H. T. Claude Chan, B. Paul Morgan, A. V. Filatov, Peter W. M. Johnson, Ruth R. French,
and Martin J. Glennie
Despite the clinical success of anti-CD20
monoclonal antibody (mAb) in the treatment
of lymphoma, there remains considerable
uncertainty about its mechanism of action.
Here we show that the ability of mAbs to
translocate CD20 into low-density, detergentinsoluble membrane rafts appears to control how effectively they mediate complement lysis of lymphoma cells. In vitro studies
using a panel of anti–B-cell mAbs revealed
that the anti-CD20 mAbs, with one exception (B1), are unusually effective at recruit-
ing human complement. Differences in
complement recruitment could not be explained by the level of mAb binding or
isotype but did correlate with the redistribution of CD20 in the cell membrane following
mAb ligation. Membrane fractionation confirmed that B1, unlike 1F5 and rituximab,
was unable to translocate CD20 into lipid
rafts. In addition, we were able to drive B1
and a range of other anti–B-cell mAbs into a
detergent-insoluble fraction of the cell by
hyper–cross-linking with an F(abⴕ)2 anti-Ig
Ab, a treatment that also conferred the ability to activate lytic complement. Thus, we
have shown that an important mAb effector
function appears to be controlled by movement of the target molecule into membrane
rafts, either because a raft location favors
complement activation by mAbs or because
rafts are more sensitive to complement penetration. (Blood. 2003;101:1045-1052)
© 2003 by The American Society of Hematology
Introduction
The success of the chimeric anti-CD20 monoclonal antibody
(mAb), rituximab (Ritux), in treating B-cell malignancies and,
more recently, autoimmune diseases has continued to focus attention on antibody effector mechanisms in order to explain how this
mAb operates in vivo.1-4 What makes anti-CD20 mAbs so clinically effective is not clear, however, at least in vitro, they mediate
both complement-dependent cytotoxicity (CDC) and antibodydependent cell-mediated cytotoxicity (ADCC) and, unlike many
other anti–B-cell mAbs, are also able to mediate growth arrest and
apoptosis in certain cell lines.5-9 At present, it is not known which
of these mechanisms predominates in vivo or in fact whether
anti-CD20 mAbs are able to use different effector mechanisms,
according to where the target cells are located or their stage of
differentiation. The most persuasive evidence supporting natural
effector (ADCC and CDC) mechanisms in the therapeutic response
comes from work in primates showing that an IgG4 variant of
Ritux, unlike the clinically approved IgG1, was unable to deplete
normal B cells.10 Since this IgG4 variant would be unable to recruit
natural effectors effectively but should retain the binding and
cross-linking functions needed for any direct cytotoxic activity,
including the signaling of apoptosis, these data point toward CDC
and ADCC as the major effectors in vivo. However, in vitro
experiments show that signaling activity is often enhanced by
hyper–cross-linking either using anti-Ig antisera or Fc␥R-bearing
cells, such as ADCC effector cells, or with anti-CD20 mAb
multimers.8,11 Thus, it is conceivable that switching the isotype of
Ritux may have reduced transmembrane signaling by interfering
with the hyper–cross-linking following Fc␥R-Fc interactions. The
importance of Fc␥R in vivo also is supported by elegant work from
Clynes et al12 using Fc␥R-deficient mice, which indicated that
Fc␥R on macrophages are critical to the ability of mAbs to control
subcutaneous B-cell lymphoma. Similarly, interesting clinical data
now has revealed that non-Hodgkin lymphoma (NHL) patients
expressing the high-affinity variant of Fc␥RIIIa (158V allotype)
respond better to Ritux than those carrying the low-affinity 158F
allotype.13 Thus, there is data to support a role for Fc␥R-bearing
effectors in mediating the activity of anti-CD20 mAbs.
In contrast, understanding the role of complement in anti-CD20
therapy is more problematic, and while complement is consumed during
Ritux treatment and appears to be responsible for much of the associated
toxicity, its importance as an effector mechanism is still controversial.14-19 One of the main arguments against complement being a major
effector is that the therapeutic activity of Ritux does not appear to
correlate with the expression levels of the complement defense molecules CD55 or CD59.15 Interestingly, however, a brief report of chronic
lymphocytic leukemia (CLL) patients receiving Ritux showed that cells
circulating 8 weeks after treatment did have increased CD59 expression,
consistent with the idea that they had been subject to complement
selection.20 Furthermore, most studies on anti-CD20 mAb effector
mechanisms have tended to consider ADCC and CDC as separate
processes and thus exclude any possible cooperative activity, for
example, where complement opsonization might be important for
From the Tenovus Research Laboratory and Medical Oncology, Cancer
Sciences Division, School of Medicine, General Hospital, Southampton, United
Kingdom; University of Wales, Department of Medical Biochemistry, College of
Medicine, Heath Park, Cardiff, United Kingdom; Institute of Immunology
Kashirskoye Shosse, Moscow, Russia.
(BBSRC), Tenovus of Cardiff, Cancer Research United Kingdom, and the
Leukaemia Research Fund.
Submitted June 19, 2002; accepted September 12, 2002. Prepublished online
as Blood First Edition Paper, September 19, 2002; DOI 10.1182/blood-200206-1761.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by the Biotechnology and Biological Sciences Research Council
© 2003 by The American Society of Hematology
BLOOD, 1 FEBRUARY 2003 䡠 VOLUME 101, NUMBER 3
Reprints: Martin J. Glennie, Tenovus Laboratory, Cancer Sciences Division,
General Hospital, Southampton, SO16 6YD, United Kingdom; e-mail:
[email protected].
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BLOOD, 1 FEBRUARY 2003 䡠 VOLUME 101, NUMBER 3
CRAGG et al
phagocytosis and regulation of immune responses. It is also unknown
whether products arising from complement activation, such as the
membrane attack complex (MAC), could have important signaling
functions via glycosylphosphatidylinositol (GPI)–linked receptors that
might modulate signaling activity provided by anti-CD20 mAbs.21,22
The CD20 molecule is without known function or ligand, and
knock-out mice show no obvious phenotype.23 It is a 33 to 37-kDa
nonglycosylated surface phosphoprotein that is predicted to cross
the plasma membrane 4 times, leaving a single detectable extracellular loop and the N- and C-termini inside the cell.24,25 CD20
probably exists in an oligomeric form in the plasma membrane,
where it may either assist in, or directly contribute to, the formation
of cellular ion channels.26,27 Until recently, blocking studies
showed very limited epitope diversity in the extracellular loop of
CD20, with only 2 overlapping epitopes, one recognized by the
vast majority of anti-CD20 mAbs, and a second recognized by a
unique reagent, 1F5.28 However, Deans and colleagues26 have just
revealed a more complex situation, placing 16 anti-CD20 mAbs
into 4 different subgroups according to their ability to bind to a
crucial alanine-X-proline motif in the CD20 extracellular loop.
These mAbs also could be grouped by their ability to translocate
CD20 into the detergent-insoluble lipid microdomains, generally
known as “rafts” (most mAbs), trigger extensive homotypic
cellular adhesion (3 mAbs), or perform both of these activities (1
mAb). In our previous work we found that a similar panel of
anti-CD20 mAbs could be subdivided into mAbs that were
effective or not at inducing CDC and speculated that these
functions might be related to the ability of the mAbs to translocate
into membrane rafts.29 In the current study we show a close
relationship between the ability of a mAb to translocate CD20 into
lipid rafts and to activate lytic complement. Thus, it appears that
a key effector function of a mAb may be related to its ability to
redistribute target antigen into specific compartments of the
plasma membrane.
Materials and methods
Cell lines
Human cell lines were obtained from the European Collection of Cell
Cultures (ECACC, Salisbury, United Kingdom) and maintained in antibioticfree RPMI 1640 medium with fetal calf serum (Myoclone, 10%), glutamine
(2 mM), and pyruvate (1 mM) (Gibco, Paisley, United Kingdom) at 37°C,
5% CO2. BCL1-3B3 is a mouse cell line derivative of the BCL1 tumor
maintained in tissue culture under the conditions described above.
Table 1. Sources, specificities, and isotypes of antibodies used in study
mAb
Specificity
Isotype
Source
2H7
CD20
IgG2b
Serotec, Oxford, United Kingdom
IF5
CD20
IgG2a
ECACC Hybridoma
B1
CD20
IgG2a
Coulter, Miami, FL
HI47
CD20
IgG3
ECACC Hybridoma
Rituximab
CD20
Chimeric Hu Fc
IDEC, San Francisco, CA
AT80
CD20
IgG1
In-house
LT20
CD20
IgG1
In-house
MB1/7
CD37
IgG1
Richard Miller30
WR17
CD37
IgG2a
Keith Moore31
AT13/5
CD38
IgG1
In-house
IB4
CD38
IgG2a
Fabio Malavasi32
F3.3
MHC II
IgG1
In-house
A9-1
MHC II
IgG2a
In-house
CAMPATH-1H
CD52
Chimeric Hu Fc
Geoff Hale33
Chalfont, United Kingdom) as described in the manufacturer’s instructions. The labeled mAb was isolated using a PD10-Sephadex G25
column. Molar ratios of coupling were determined spectrophotometrically from ⑀552 ⫽ 150/mM/cm for Cy3, ⑀650 ⫽ 250/mM/cm for Cy5, and
⑀280 ⫽ 170/mM/cm for protein, and ranged from 5- to 8-fold excess
dye/protein. Aggregates were removed by centrifugation.
Fluorescence resonance energy transfer (FRET) analysis was based on
the method described previously.37 Cells were resuspended at 3 ⫻ 106
cells/mL in phosphate-buffered saline (PBS)/0.1% bovine serum albumin
(BSA)/1% heat-inactivated mouse and human serum, and equimolar donor
(Cy3)–conjugated and acceptor (Cy5)–conjugated mAbs were combined
and added to the cell suspension (10 ␮g/mL, final). Cells were incubated for
50 minutes in the dark, on ice, or at 37°C. Each experiment included cells
labeled with donor- and acceptor-conjugated mAbs after preincubation with
a 20-fold molar excess of unconjugated mAbs, and cells labeled with donoror acceptor-conjugated mAbs in the presence of equimolar unlabeled
mAbs. FRET was assessed using flow cytometry on a FACSCalibur (BD
Biosciences, San Jose, CA). The fluorescence intensities at 585 nm (FL2)
and 650 nm (FL3), both excited at 488 nm, and the fluorescence intensity at
661 nm (FL4), excited at 635 nm, were detected and used to calculate FRET
according to the equation below, where A is acceptor (Cy5) and D is donor
(Cy3). All values obtained were corrected for autofluorescence:
FRET ⫽ FL3(D, A) ⫺ FL2(D, A)/a ⫺ FL4(DA)/b, where: a ⫽ FL2(D)/
FL3(D), and b ⫽ FL4(A)/FL3(A).
Correction parameters were obtained using data collected from singlelabeled cells, and side scatter was used to gate out dead cells. FRET
between donor and acceptor mAbs on the cells is expressed as acceptor
sensitized emission at 488 nm.37 Larger FRET efficiencies suggest closer
association of the donor and acceptor mAbs or a high density of acceptor
mAbs in the vicinity of donor mAbs.38,39
Preparation of lipid raft fractions and Western blotting
Antibody production and labeling
The mAbs used are shown in Table 1. mAb-producing hybridomas were
expanded in tissue culture and purified on Protein A column. F(ab⬘)2
fragments of IgG were produced by standard pepsin digestions.34 Flow
cytometry and 125I-labeled mAb binding were as described previously.35
CDC assay
Serum for complement lysis was prepared as follows: blood from healthy
volunteers was allowed to clot at room temperature for 30 to 60 minutes and
then centrifuged at 900g for 20 minutes. Serum was harvested and then
either used fresh or stored at ⫺80°C. To determine the CDC activity of the
various mAbs, elevated membrane permeability was assessed using a rapid
and simple propidium iodide (PI) exclusion assay, as previously described.36
Monoclonal Ab (1 ␮g/106 cells) was added to cells at 37°C. Following 20
minutes’ incubation, cells were pelleted and lysed in ice-cold 1.0% Triton
X-100 in 2-[N-morpholino] ethanesulfonic acid (MES)–buffered saline (25
mM MES, pH 6.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5
␮g/mL aprotinin, 5 ␮g/mL leupeptin, 10 mM EDTA [ethylenediaminetetraacetic acid]). Lipid raft fractions were then prepared by sucrose density
gradient centrifugation as described by Deans et al.40 Fractions were
resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis,
transferred onto polyvinylidenefluoride membranes, and incubated with
primary antibody (mouse anti-CD20, clone 7D1, or anti-Lyn rabbit
polysera; Serotec, United Kingdom), followed by horseradish-peroxidase–
conjugated secondary antibody (Amersham Biosciences). Blots were
visualized using ECL⫹plus (Amersham Biosciences).
Fluorescence resonance energy transfer analysis
Assessment of raft-associated antigen by Triton
X-100 insolubility
mAbs were directly conjugated to bisfunctional N-hydrosuccinimidyl
(NHS)–ester derivatives of Cy3 and Cy5 (Amersham Biosciences, Little
As a rapid assessment of antigen presence in raft microdomains, we used a flow
cytometry method based on Triton X-100 insolubility at low temperatures, as
BLOOD, 1 FEBRUARY 2003 䡠 VOLUME 101, NUMBER 3
ANTI-CD20 mAb EFFECTIVE IN RECRUITING COMPLEMENT
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chem-Novabiochem, San Diego, CA) with 0.1% Citifluor (Agar Scientific,
Stanstead, United Kingdom). YFP-HuCD20 clustering was visualized
using a Leica SP2 confocal microscope (Bannockburn, IL).
Results
CD20 mAb mediates potent recruitment of complement
Figure 1. CDC activity and binding levels of various antibodies directed to the
B-cell surface. (A) CDC induced by a panel of mouse IgG1 and IgG2a isotype mAbs
directed at CD20, CD37, CD38, and MHCII. Daudi (f) or EHRB (o) cells (1 ⫻ 106/
mL) were incubated with mAbs (10 ␮g/mL) for 15 minutes at room temperature.
Normal human serum (NHS, 20% vol/vol) was then added as a source of complement
and the cells incubated at 37°C for 30 minutes. Cell lysis was assessed by flow
cytometry using a PI exclusion assay, and the level of CDC expressed as PI-positive
cells as a percentage of total cells. The results show the means and SDs of 3
separate experiments. (B) Surface expression of CD20, CD37, CD38, and MHCII
antigens on Daudi or EHRB cells. Daudi or EHRB cells (5 ⫻ 105/mL) were incubated
with mAbs (10 ␮g/mL) for 30 minutes at room temperature, then washed and
incubated with goat anti–mouse IgG F(ab⬘)2 FITC for 30 minutes on ice, prior to
washing and analysis by flow cytometry.
described previously.41,42 In brief, cells were washed in RPMI/1% BSA and
resuspended at 2.5 ⫻ 106/mL. Cells were then incubated with 10 ␮g/mL of
fluorescein isothiocyanate (FITC)–conjugated mAbs for 15 minutes at 37°C,
washed in cold PBS/1% BSA/20 mM sodium azide, and then the sample divided
in half. One half was maintained on ice to allow calculation of 100% surface
antigen levels, while the other was treated with 0.5% Triton X-100 for 15 minutes
on ice to determine the proportion of antigens remaining in the insoluble raft
fraction. Cells were then maintained at 4°C throughout the remainder of the
assay, washed once in PBS/BSA/azide, resuspended, and assessed by flow
cytometry as detailed above. Similar results were obtained using indirect methods
of detection. To determine the constitutive level of raft association of target
antigens, cells were first treated with 0.5% Triton X-100 for 15 minutes on ice
and washed in PBS/BSA/azide prior to binding of FITC-labeled mAbs. To assess
whether more antigen could be moved into the Triton X-100–insoluble fraction
by additional cross-linking, cells were incubated with FITC-mAb as before,
washed, and then divided into 4 samples. Two of these samples were incubated
with goat anti–mouse Ig F(ab⬘)2 fragments for 15 minutes on ice. After washing,
one of the cross-linked and one of the non–cross-linked samples were lysed in
Triton X-100 and washed as detailed above, prior to flow cytometry.
Recently, we have been struck by the unusual potency with which
CD20 mAbs recruit human complement for the lysis of B-cell
lymphoma cells. Figure 1 shows CDC results for a panel of IgG1
and IgG2a isotype mAbs directed at CD20, CD37, CD38, and
MHCII. Ritux (chimeric Ab, with human IgG1␬ constant domains)
is included as a positive control. Against both Daudi and EHRB
cells, the rodent anti-CD20 mAbs were consistently the most active
of these reagents in CDC, usually mediating lysis of approximately
90% of the target cells. Such results cannot be explained by the
relative levels of binding, because flow cytometric analysis showed
that the anti-MHCII mAb bound at the highest levels (Figures 1B
and 5 show the mean fluorescence intensity [MFI] of these mAbs)
and yet were not the most active. The differences in potency were
most striking when comparing IgG1 isotype mAbs, because under
these conditions only the CD20 reagents initiated detectable CDC
(18% and 90% lysis of Daudi and EHRB cells, respectively).
Potency of different anti-CD20 mAbs
in complement-mediated lysis
To investigate whether this high CDC activity was a property of all
anti-CD20 mAbs, we compared a large panel of anti-CD20 mAbs that
included the clinically relevant Ritux and B1 reagents. Figure 2 shows
that the mAbs could be divided into 2 distinct groups by this assay: the
first, which included Ritux, 1F5, and HI47, contained reagents that were
potent in CDC; the second, which included AT80, 2H7, LT20, and B1,
contained those with relatively poor cytolytic activity. In most cases
such activity is explicable on the basis of mAb isotype with human
IgG1, mouse IgG2a, and IgG3 performing well, and mouse IgG1 and
IgG2b performing poorly. It is important to bear in mind that even this
poorly performing group was surprisingly active given that mouse IgG1
mAbs are generally considered unable to recruit human complement.43,44 Indeed, these mAbs were more active than those carrying the
mouse IgG2a isotype, directed to the CD37 and CD38 antigens. The one
exception in this distribution was B1, which, despite being a mouse
IgG2a, performs poorly in CDC. It also should be noted that kinetics of
CDC were examined in preliminary experiments, which revealed that
optimal lysis with 1F5 was observed as early as 5-10 minutes after the
addition of serum. However, our CDC assays were routinely performed
for 30-45 minutes to allow suboptimal mAbs to induce their maximal
Transfection of YFP-Hu CD20 and confocal microscopy
A chimera of yellow fluorescent protein (YFP) fused to the C-terminal
region of human CD20 (YFP-Hu20) was constructed by fusing full-length
human CD20 into the pEYFP-C1 plasmid (Clontech, Basingstoke, United
Kingdom) via XhoI and BamHI sites. YFP-Hu20 transfections of BCL13B3 cells were carried out using the GenePorter reagent (Gene Therapy
System, San Diego, CA) and transfectants selected with geneticin (Invitrogen, Paisley, United Kingdom) and identified by flow cytometry.
For visual redistribution studies, cells were stimulated with mAbs (10
␮g/mL) for 20 minutes at room temperature. Cells were then cytospun,
fixed (3.7% paraformaldehyde), washed, and mounted in Mowiol (Calbio-
Figure 2. Dose dependence of CDC activity with different anti-CD20 mAbs. To
determine the relative efficacy with which various anti-CD20 mAbs evoked CDC,
mAbs were titrated from 10 to 0.08 ␮g/mL and incubated with EHRB cells for 15
minutes; NHS was added at 20% and assessed for CDC activity as detailed in the
legend to Figure 1. As controls, anti-CD37 (WR17) and anti-CD38 (1B4) mAbs were
included and assessed in the same manner.
1048
CRAGG et al
CDC activity. Longer durations (ie, ⬎ 45 minutes) were not assessed to
minimize other forms of cell death, for example, apoptosis.
Binding properties of CD20 mAbs on B-cell lines
We next investigated the relative binding activity of these mAbs using
radiolabeled F(ab⬘)2 fragments to see if this might explain the differences in complement activity. Figure 3A shows saturation curves that
confirmed the binding constants for Ritux (5-8 ⫻ 10-9 M), B1 (3-6 ⫻ 109 M), and 1F5 (1-3 ⫻ 10-8 M) on Daudi and EHRB cells. Each mAb
gave a range of affinities, indicating interexperimental variation and
binding activity on different cell lines. Radiolabeled IgG gave similar
curves (results not shown, except for 1F5 IgG binding to EHRB cells),
showing that the Fc region was not involved in binding to the cells. The
surprising finding was that B1 and Ritux, despite binding to the same
target CD20 loop with high affinity, saturated at different levels, with
Ritux binding almost twice as many molecules on each cell. 1F5 has a
weaker binding affinity but, despite generating flatter binding curves,
eventually reaches similar levels of binding to those achieved by Ritux.
These differences are the subject of ongoing investigation. Despite these
distinct binding patterns, we do not believe that they explain the
observed differences in CDC activity, mainly because 1F5 and Ritux
gave clear cytotoxic activity at concentrations well below their binding
maxima. For example, Figure 2 shows that even at 0.08 ␮g/mL, a
concentration that would have achieved less than 20% Ritux or 10%
1F5 saturation (Figure 3A), we recorded at least 50% lysis for Ritux and
1F5. In contrast, B1 could achieve this level of lytic activity only when
added at 10 ␮g/mL, representing 125 times less potency. We conclude
that at saturation, B1 is capable of reaching levels on the cell that should
mediate CDC as effectively as 1F5 or Ritux.
During this investigation we performed microscopic examination of anti-CD20 mAb binding to cells using FITC-mAb. These
experiments revealed clear differences in the distribution of CD20
bound by the different mAbs, in that B1 bound over the cell surface
homogeneously, while other anti-CD20 mAbs appeared to bind in
clusters, revealed by punctate staining (data not shown). In order to
investigate this phenomenon more closely, we used a mouse cell
line (BCL1-3B3) transfected to express YFP-tagged human CD20,
which bypasses any variation in mAb-FITC conjugation and the
need of secondary amplifying reagents. These data reveal (Figure
3B) that, as in the Burkitt lines, B1 caused minimal redistribution
of CD20 upon binding, while other anti-CD20 mAbs such as Ritux
and 1F5 caused marked clustering of YFP-CD20.
BLOOD, 1 FEBRUARY 2003 䡠 VOLUME 101, NUMBER 3
Redistribution of CD20 and anti-CD20 mAbs on target cells
The reorganization of CD20 in the plasma membrane following
binding by mAbs is likely to be important in providing the
appropriate distribution of Fc regions for the recruitment of C1q
and the triggering of the classical pathway of complement. To
examine this reorganization further, we used FRET analysis to
measure the molecular proximity of anti-CD20 mAbs on intact
Daudi and EHRB cells. In these experiments, separate batches of
each anti-CD20 mAb were labeled with Cy3 or Cy5 and energy
transfer values obtained for 50:50 mixtures of Cy3- and Cy5labeled mAbs (eg, B1 Cy3 ⫹ B1 Cy5). For comparison, 3 other
non-CD20 mAb specificities were similarly assessed (MHCII,
CD37, and CD38). A high FRET value indicates close proximity of
neighboring Cy3- and Cy5-labeled mAbs38,39 and can be caused
either by mAb binding to a surface molecule that is expressed at
high density or by mAb ligation causing redistribution and closer
association of the molecular target. Figure 4A shows histograms
from a typical experiment, illustrating FRET data for Ritux and B1
(open histograms) compared with controls (shaded histograms).
The clear shift in the open histogram with Ritux represents
demonstrable FRET due to the close proximity of the acceptor and
donor-labeled mAbs. In comparison, B1 generated insignificant
levels of FRET. These experiments were performed 3 times with
the panel, and the results are presented as a bar chart in Figure 4B.
The results show that while Ritux and 1F5 were very effective in
this assay, with Ritux recording between 50 and 70 FRET units on
EHRB and Daudi cells, respectively, B1, which was poor at
activating complement, was completely inactive in generating a
FRET signal. In this regard, it performed like anti-CD37 and
anti-CD38 mAbs, other targets that are relatively poor in CDC. For
both Ritux and 1F5, the levels of FRET were higher at 37°C than at
4°C. Because the plasma membrane is more mobile at higher
temperatures, these results are consistent with FRET values being
dependent on molecular reorganization in the plasma membrane
rather than on antigen density. Conversely, mAb binding to MHCII
gave only a modest level of FRET, which was not greatly enhanced
at 37°C, indicating that these FRET values were predominantly due
to the high surface expression of MHCII. Thus, these results
indicate a good correlation between the ability of a mAb to perform
CDC and to mediate FRET.
Figure 3. Binding and redistribution properties of
different anti-CD20 antibodies. (A) Binding of 125Ilabeled F(ab⬘)2 fragments of 1F5, Ritux, and B1 antiCD20 mAbs to EHRB and Daudi cells. 125I-labeled F(ab⬘)2
fragments of anti-CD20 or control (anti-CD3) mAbs were
incubated with Daudi or EHRB cells for 2 hours at 37°C.
The exception to this is the curve for IF5 binding to Daudi
cells, for which 125I-labeled whole IgG was used. The
cell-bound and free 125I-labeled mAbs were then separated by centrifugation through phthalate oils and the cell
pellets together with bound antibody counted for radioactivity. (B) 1F5 and Ritux, but not B1, induce clustering of
YFP-HuCD20 in BCL1-3B3–transfected cells upon binding to CD20. BCL1-3B3 cells transfected with YFPHuCD20 were incubated with various anti-CD20 mAbs
for 20 minutes at room temperature. Cells were then
cytospun onto slides and fixed with 3.7% paraformaldehyde for 10 minutes at room temperature. YFP-HuCD20
clustering was visualized using confocal microscopy.
Representative images from 3 separate experiments are
shown (original magnification, ⫻ 100). During this investigation we performed microscopic examination of antiCD20 mAbs binding to cells using FITC-mAbs. These
experiments revealed clear differences in the distribution
of CD20.
BLOOD, 1 FEBRUARY 2003 䡠 VOLUME 101, NUMBER 3
Figure 4. Comparison of the abilities of antibodies to redistribute antigens on
the cell surface assessed by FRET and Triton X-100 insolubility. Analysis of
CD20 FRET (A and B) and the association of CD20 into membrane rafts (C) in the
presence of various anti-CD20 mAbs. (A) Homoassociation analysis of CD20 with B1
and Ritux mAb. Daudi cells were labeled with equimolar mixtures of Cy5 (acceptor)–
conjugated mAbs and either unconjugated mAbs (filled histograms) or Cy3 (donor)–
conjugated mAbs (open histograms) for 50 minutes at 37°C. Associations were
estimated by flow cytometric analysis as described in “Materials and methods.” (B)
Comparison of FRET with anti-CD20, anti-MHCII (A9-1), anti-CD37 (WR17), and
anti-CD38 (1B4). EHRB and Daudi cells were labeled with equimolar mixtures of Cy3
(donor)– and Cy5 (acceptor)–conjugated mAb pairs for 50 minutes at 4°C (s) or 37°C
(f) as indicated. Associations were estimated by flow cytometric analysis as
described in “Materials and methods.” Data illustrate FRET values ⫾ SEMs,
expressed in terms of Cy5 emission at 488 nm, for 3 independent experiments for
CD20, MHCII, CD37, and CD38 homoassociation as indicated. (C) Daudi cells were
treated with anti-CD20 mAbs (B1, Ritux, 1F5, as indicated) or left untreated (Cont,
Ct/Lyn), lysed after 20 minutes in 1% Triton X-100 lysis buffer, and fractionated on a
discontinuous sucrose gradient as described in “Materials and methods.” Gradient
fractions were resolved on 15% sodium dodecyl sulfate gels, and Western blots
developed using 7D1 primary antibody to identify CD20 or rabbit anti-Lyn polyclonal
antisera. The pelleted fraction from each sucrose density gradient did not contain
CD20 (data not shown).
On the basis of this information, we decided to investigate the
association of CD20 with lipid rafts. It is known that, without crosslinking, CD20 is not associated with rafts and is readily soluble in
nonionic detergents such as TX-100, at low temperatures.40,45 We
investigated the distribution of CD20 between the raft and nonraft
membrane fractions using the sucrose gradient fractionation method
described by Deans and colleagues.40 Figure 4C shows that following
sucrose gradient fractionation of Triton X-100–solubilized Daudi cells,
CD20 molecules are confined to high-density fractions 6-8. Cells treated
with B1 for 30 minutes showed a similar CD20 distribution to untreated
cells, in fractions 6-8. In contrast, cells treated with Ritux or 1F5 showed
a distinct shift in CD20 distribution with a significant proportion now
found in the lower density fractions 4, or 3 and 4, respectively,
coincident with where membrane rafts are expected to occur. To verify
the location of rafts in our preparations, we reprobed the membranes for
Lyn (Figure 4C), a known raft marker that, as expected, was found in the
low-density fractions.3-5 Thus, it appears that the ability of anti-CD20
mAbs to demonstrate FRET and activate complement shows a tight
correlation with the ability to redistribute CD20 into membrane rafts.
The distinct redistribution of CD20 to fraction 4 following cross-linking
with Ritux, compared with fractions 3 and 4 with 1F5 treatment, was a
consistent but unexplained finding (Figure 4B).
CD20 becomes Triton X-100–insoluble when cross-linked
by mAbs
To continue these investigations and study a range of other antigen
specificities without having to perform sucrose gradient sedimentation and Western blotting, we moved to a flow cytometric method
ANTI-CD20 mAb EFFECTIVE IN RECRUITING COMPLEMENT
1049
in which cells are treated with dilute Triton X-100 before or after
staining with fluorescent mAbs.41,42 Under the conditions used, it is
possible to remove nonraft lipid and most of the cytoplasm from
the cells, leaving only the insoluble parts of the cell together with
the lipid rafts. Figure 5 shows staining of Daudi and EHRB cells,
first using mAbs on intact cells (solid bars), then on cells that have
been treated with Triton X-100 before staining with mAbs (open
bars, not shown for EHRB), and finally for cells stained with mAbs
and then treated with Triton X-100 (hatched bars). The results
clearly show that in all cases except one, treating with Triton X-100
removes all cell-associated molecules, making them unavailable
for staining. The one exception is Campath (CD52), which is
GPI-linked and therefore constitutively associated with the outer
leaflet of rafts. The GPI-linked molecules CD55 and CD59 are
similarly retained following such treatment (data not shown).
These results are consistent with raft material being retained with
the Triton X-100–treated cells in this technique. EHRB cells do not
express GPI-linked proteins and therefore do not express CD52. In
contrast to this situation, when the cells were stained with these
mAbs before being treated with Triton X-100 (hatched bars), we
found that most anti-CD20 mAbs remained with the cells. Furthermore, B1, the one anti-CD20 mAb that did not move CD20 into
rafts, also failed to remain with the Triton X-100–treated cells. All
other mAb specificities, with the exception of CD52, failed to
remain with the Triton X-100 insoluble fraction.
Given that the ability of anti-CD20 mAbs to move CD20 into
rafts appeared to correlate with the ability to cross-link neighboring
CD20 molecules, we next asked if more surface molecules became
associated with the Triton X-100 insoluble fraction as they were
cross-linked. Figure 6A shows very clearly that as the primary
mAbs became cross-linked with F(ab⬘)2 fragments of polyclonal
anti-Ig Abs, so more molecules were able to remain with the cells
following treatment with Triton X-100. Under these conditions, all
mAbs, including B1, showed a proportion of binding remaining
following detergent treatment. In the most striking case, 80% of
MHCII molecules remained with the Triton X-100–treated cells
following hyper–cross-linking of the mAbs, whereas only 4%
remained when additional cross-linking was not added. In contrast,
B1 appeared the most reluctant to move into the insoluble fraction,
Figure 5. Triton X-100 insolubility of different surface antigens before and after
binding of mAbs. Daudi or EHRB cells were incubated with 10 ␮g/mL FITC-mAbs
for 15 minutes at 37°C, before washing and dividing the sample in half. One half was
maintained on ice in PBS (f), allowing calculation of 100% binding levels, while the
other was treated with 0.5% Triton X-100 (labeled Tx-100) for 15 minutes on ice (s;
stained before Triton X-100 lysis). Cells then were washed once, resuspended, and
assessed by flow cytometry. To determine levels of constitutively Triton X-100–
insoluble antigens, cells were treated with 0.5% Triton X-100 for 15 minutes on ice
and washed prior to staining with FITC-mAbs (䡺; Triton X-100 lysis before staining).
Shown are the mean fluorescence intensity (MFI) values for each sample. Error bars
show ⫾ SD of triplicate samples.
1050
CRAGG et al
and in this case about 25% of the mAbs remained after the
detergent treatment.
Monoclonal antibodies mediate complement lysis more
efficiently when transferred into the detergent-insoluble
fraction of the cell membrane
Finally, we asked if cross-linking a primary mAb in this way not
only shifted the target to the more insoluble part of the plasma
membrane but also changed the ability of the mAb to activate
complement. Figure 6B shows that for all mAbs investigated,
additional cross-linking with F(ab⬘)2 anti-Ig promoted CDC. This
was particularly striking in the case of the anti-MHCII IgG1 mAb,
which, without cross-linking, was unable to lyse cells but became
as potent as Ritux when cross-linked with 5 ␮g/mL F(ab⬘)2 anti-Ig.
Similar potentiation in CDC was obtained when the IgG2a mAb
against CD37 (WR17) and CD38 (1B4) were treated with just 1
␮g/mL F(ab⬘)2 anti-Ig. Interestingly, the B1 mAb (IgG2a) against
CD20, which was least inclined to move into the Triton X-100
insoluble fraction following cross-linking (Figure 6A), also showed
an appreciable improvement in CDC activity following the addition of the polyclonal cross-linker. Thus, in each case, a treatment
that is known to drive Ab:Ag complexes into a detergent-insoluble
fraction, which includes membrane rafts, improves the ability of
mAbs to recruit lytic complement.
Discussion
The ability of mAbs to recruit and activate complement at the cell
surface is dependent not only on isotype and target density but also the
lateral mobility of Ab:Ag complexes in the plasma membrane and the
distance between the target epitope and the lipid bilayer.46 Thus,
the specificity of mAbs can have profound and subtle influences on the
efficiency with which complement is recruited. Here we have shown
that anti-CD20 mAbs are unusually effective at recruiting complement
for the lysis of neoplastic B cells and that this activity is closely linked to
the ability of the mAbs to translocate CD20 into membrane rafts
Figure 6. Ability of cross-linking to enhance Triton X-100 insolubility and CDC
activity of different antigens. (A) To assess whether more antigen could be moved
into the Triton X-100–insoluble fraction by additional cross-linking, EHRB cells were
incubated with FITC-mAbs as before, washed, and then divided into 4. Two of these
samples were incubated with goat anti–mouse IgG F(ab⬘)2 fragments for 15 minutes
on ice to facilitate cross-linking. After washing, one of the cross-linked and one of the
non–cross-linked samples were lysed in Triton X-100 and washed as detailed above,
and the remaining 2 samples retained to determine total binding with and without
cross-linking. Results were then expressed as the percent of antigen retained in the
Triton X-100–insoluble fraction using the equations: (MFI Triton X-100 insoluble/MFI
total) ⫻ 100 and (MFI cross-linked Triton X-100/MFI cross-linked total) ⫻ 100 for
non–cross-linked and cross-linked, respectively. Error bars represent SD of results
for at least 3 experiments. (B) To assess whether cross-linking could enhance CDC
activity, mAb (10 ␮g/mL) was bound to cells for 15 minutes at room temperature as
before and then washed once, prior to addition of cross-linking agent at a range of
concentrations (0, 5, 1, 0.2 ␮g/mL) for 10 minutes at room temperature. NHS (5%
vol/vol) was then added and CDC measured as described in the legend to Figure 1.
Similar data were obtained in 3 experiments.
BLOOD, 1 FEBRUARY 2003 䡠 VOLUME 101, NUMBER 3
following ligation.26,40,45 All but one of the anti-CD20 mAbs were
effective in mediating CD20 raft translocation and in recruiting human
complement in CDC assays. The exception, B1, was unable to deliver
CD20 into rafts and, despite being of a complement-activating isotype
(IgG2a) and causing C1s and C3 deposition equivalent to 1F5 in
solid-phase enzyme-linked immunosorbent assay experiments (data not
shown), failed to lyse cells in CDC.
It is not clear why translocation of Ab:Ag complexes into lipid rafts
should influence the ability of mAbs to activate complement so
markedly, even to the point where mouse IgG1 anti-CD20 isotypes,
which normally activate human complement comparatively poorly,
were able to lyse malignant B cells.43,44 Because of the requirement for
at least 2-headed binding to engage and activate C1q, the most
straightforward explanation is that concentrating mAbs into a comparatively small area of the plasma membrane provides an ideal density of
juxtaposed Fc regions for engaging the C1q heads, thereby triggering
the classical complement pathway.47,48 Our FRET data give good
support for this suggestion, showing that ligation of CD20 by complement-lytic mAbs is associated with a sharp rise in FRET, consistent with
the Ab:Ag complexes becoming redistributed into rafts. A second
possibility is that the cholesterol-rich environment provided by rafts is
advantageous for complement activation through C3 deposition or by
providing a region of the membrane that is more susceptible to
penetration by MAC, perhaps because the lipid is more rigid or the
MAC are less likely to be removed by internalization. Previous work
supports this, showing that red blood cells became more susceptible to
lysis as their cholesterol content was increased.49,50 We are currently
investigating these 2 possibilities by asking whether the relative potency
of anti-CD20 mAbs lies in their ability to activate C1, that is, provide an
optimal Fc binding platform, or in the efficiency with which they use
MAC, that is, membrane penetration. It is interesting that most
complement-defense molecules, being GPI-linked, are constitutively
associated with rafts and consequently ideally placed to protect cells
from any damage focused on the rafts.
Cross-linking of cell-surface antigen by fluid-phase antibody,
with subsequent aggregation and lateral movement of Ab:Ag
complexes, has long been associated with the phenomenon of
antigenic modulation, whereby the cell acquires resistance to
complement lysis induced by that antibody.51 This process can be
inhibited with azide or by using univalent mAbs that are unable to
provide cross-linking activity.52 With many target antigens, such as
the thymus leukemia antigen TL and the B-cell receptor BCR, the
redistribution of Ab:Ag complexes is followed by their internalization and degradation, leaving the cell free of target antigen. Thus,
antigenic modulation and internalization often has been cited as
one of the reasons certain mAb specificities fail to perform in the
clinic and may partly explain why anti-CD20 mAbs, which do not
modulate or internalize even in vivo,53 are effective in CDC and
clinically successful. Interestingly, we have shown previously that
when the target antigen concerned is the BCR on leukemic B
lymphoblasts, anti–complement modulation was established in 2 to
5 minutes following exposure to anti-Ig at 37°C and appeared to
require only limited surface reorganization of Ab:Ag complexes on
the cell surface.54,55 This modulated state occurred sufficiently
rapidly to protect the cell when lytic syngeneic complement was
present and persisted in the presence of “patched” Ab:Ag complexes on the surface. It is not clear at this stage what relationship
this patched material has to lipid rafts, but recent cell signaling
studies show that the BCR can become raft-associated following
cross-linking in some B-cell types. The situation in the current
work appears quite different, since here we find that redistribution
of anti-CD20:CD20 complexes on the cell surface was required for
full CDC activity. Furthermore, the complement-activating activity
BLOOD, 1 FEBRUARY 2003 䡠 VOLUME 101, NUMBER 3
ANTI-CD20 mAb EFFECTIVE IN RECRUITING COMPLEMENT
of other anti–B-cell mAbs (anti-CD37, -CD38, and -MHCII) also
was promoted following cross-linking with F(ab⬘)2 anti-Ig and
movement into the detergent insoluble fraction of the plasma
membrane. Thus, our earlier observations, taken in conjunction
with the present report, suggest that a critical decision may be made
when Ab:Ag complexes, presumably with their attached C1, are
reorganized into lipid rafts. Complement lysis is either potentiated,
as in the case of anti-CD20 and some other anti–B-cell mAbs
(current work), or it is diminished, as can occur with anti-BCR and
anti-TL Abs in certain cells. Further work will be required to
understand the processes that control these molecular decisions.
Recently, Polyak and Deans26 showed that recognition of CD20
by most mAbs depends on an alanine and a proline at positions 170
and 172, respectively, in the extracellular loop of human CD20.
Simply introducing these 2 residues into the equivalent positions of
the mouse sequence reconstituted the binding of a range of mAbs,
including B1 and Ritux. However, although this work underlined
the subtleties in epitope specificity for anti-CD20 mAbs and
showed that mAbs with apparently similar specificities can have
quite distinct functions, it did not reveal which features of a mAb
controlled translocation of CD20 into rafts. CD20 is an unusual
plasma membrane molecule in terms of its ability to translocate
into lipid rafts following ligation by mAbs.40,45 Most surface
molecules either are constitutively associated with rafts, such as
GPI-linked receptors, or remain excluded from rafts even following
mAb ligation. In the current work we confirmed CD52 to be
constitutively present in rafts, while CD37, CD38, and MHCII are
excluded even after ligation by mAbs. There are only 2 published
examples, B1 and Bly1, of mAbs that do not deliver CD20 into
rafts.26 However, data presented here suggest that following
hyper–cross-linking with polyclonal F(ab⬘)2 anti-Ig, B1 was at least
partially translocated into the detergent-insoluble fraction of the
cell. Simultaneously, B1 became more active in CDC and interestingly, hyper–cross-linking other B-cell targets in this way, such as
MHCII, CD37, and CD38, also delivered them into a detergentinsoluble fraction and improved CDC activity. Thus, movement of
membrane proteins into rafts appears directly related to the degree
of cross-linking provided by the ligating agents, and we suggest
that this unusual property of CD20 in part relates to the degree of
cross-linking that most anti-CD20 mAbs are able to provide to
CD20 on the surface of B cells.
An alternative explanation for the unusual activity of CD20 may
relate to its topology within the plasma membrane. Evidence from a
number of studies has suggested that CD20 exists as a multimer in the
surface membranes of B cells, with 4 self-associated CD20 monomers
or with a mixture of CD20 monomers and other plasma membrane
molecules.26,27 Polyak et al45 identified a short stretch of membrane
proximal sequence in the cytoplasmic carboxyl tail of CD20 that
appears important for redistribution into rafts following mAb ligation.
One postulated function for such a motif is promoting oligomerization
of CD20, perhaps via protein-protein interactions. Thus, one may
envisage that CD20 resides in nonraft regions of the membrane, perhaps
as multimers, as inactive or incomplete ion channels. Ligation by most
1051
anti-CD20 mAbs causes efficient cross-linking of neighboring molecular complexes, resulting in aggregation of CD20 molecules and translocation into rafts. However, perhaps in the case of rare mAbs such as B1,
the recognized epitope favors a different mode of binding. Studies using
radiolabeled Fab⬘ (not shown) and F(ab⬘)2 fragments (Figure 3) show
that B1 binds to cells bivalently. The most logical explanation for our
findings is that B1 binds 2 CD20 molecules within a single CD20
multimer, while other anti-CD20 mAbs tend to engage neighboring
CD20 multimers. A similar situation has been described for anti–V
region mAb binding to surface IgM, with some mAbs preferring to bind
between BCR and others engaging both Fab arms on the same BCR.56
The functional consequences of such binding were seen in the readiness
with which these 2 types of reagent caused redistribution of the BCR
into patches and caps and in the propensity with which they evoked
apoptosis.57 Interestingly, in the current work, binding studies showed
that at equilibrium, approximately half as many radiolabeled B1
molecules bound as did Ritux and 1F5 (Figure 3A), supporting the
proposal that B1 binds in an unusual configuration.
It is not known at this time whether translocation of CD20 into
membrane rafts is important for the activity of anti-CD20 mAbs in
patients. Although the potency of Ritux indicates that mAbs that
move CD20 into rafts can be highly therapeutic, there is currently
little convincing evidence that complement plays a significant part
in this activity.14,15 However, what is not clear is whether a
humanized version of B1, because it does not move CD20 into
rafts, would provide a more or less potent reagent. While B1 has
been used extensively in clinical trials, its role has been as a carrier
for radioactive 131I, tositumomab, with little consideration for its
potential intrinsic therapeutic activity.58 Only an extremely limited
amount of patient data are available for unlabeled B1, acquired as
part of a study to measure the contributions of the mAbs and the
targeted radioisotope in the therapeutic activity of radioiodinated
B1.59 Interestingly, the results show that, even as a “naked” mAb,
B1 is capable of inducing objective responses in NHL patients,
including a number of complete responses. Thus, although the
study also showed that B1 generated a number of human anti–
mouse antibody responses, it indicated a surprising level of activity
for a rodent mAb. For the future, a humanized version of B1 might
be an interesting reagent for development, since it should have
many of the beneficial properties of Ritux, including the ability to
induce ADCC29,60 and apoptosis7,8,29,60 (and M.S.C. et al., unpublished results, June 2001), but might avoid some of the adverse
toxic activity that arises when Ritux activates complement.14
Acknowledgments
We wish to express our gratitude to all members of the Tenovus
Cancer Laboratory who provided expert technical support and
valuable discussion. We are also indebted to Drs Richard Miller,
Geoff Hale, and Fabio Malavasi for generously providing mAbs.
We wish to thank Mr Roger Alston (Biomedical Imaging Unit) for
assistance with confocal microscopy.
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