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IMMUNOBIOLOGY
Alanine-170 and proline-172 are critical determinants for extracellular CD20
epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies
is defined by additional requirements imposed by both amino acid
sequence and quaternary structure
Maria J. Polyak and Julie P. Deans
In vivo ablation of malignant B cells can
be achieved using antibodies directed
against the CD20 antigen. Fine specificity
differences among CD20 monoclonal antibodies (mAbs) are assumed not to be a
factor in determining their efficacy because evidence from antibody-blocking
studies indicates limited epitope diversity with only 2 overlapping extracellular
CD20 epitopes. However, in this report a
high degree of heterogeneity among antihuman CD20 mAbs is demonstrated. Mutation of alanine and proline at positions
170 and 172 (AxP) (single-letter amino acid
codes; x indicates the identical amino acid
at the same position in the murine and
human CD20 sequences) in human CD20
abrogated the binding of all CD20 mAbs
tested. Introduction of AxP into the equivalent positions in the murine sequence, which
is not otherwise recognized by antihuman
CD20 mAbs, fully reconstituted the epitope
recognized by B1, the prototypic anti-CD20
mAb. 2H7, a mAb previously thought to
recognize the same epitope as B1, did not
recognize the murine AxP mutant. Reconstitution of the 2H7 epitope was achieved with
additional mutations replacing VDxxD in the
murine sequence for INxxN (positions 162166 in the human sequence). The integrity of
the 2H7 epitope, unlike that of B1, further
depends on the maintenance of CD20 in an
oligomeric complex. The majority of 16 antihuman CD20 mAbs tested, including rituximab, bound to murine CD20 containing the
AxP mutations. Heterogeneity in the fine
specificity of these antibodies was indicated by marked differences in their ability
to induce homotypic cellular aggregation
and translocation of CD20 to a detergentinsoluble membrane compartment previously identified as lipid rafts. (Blood. 2002;
99:3256-3262)
© 2002 by The American Society of Hematology
Introduction
CD20, a nonglycosylated 33- to 35-kd integral membrane protein
expressed at high density only on B lymphocytes, has proven to be
an effective target for immunotherapeutic removal of malignant B
cells. Rituximab, a chimeric monoclonal antibody (mAb) directed
against CD20, is an approved treatment for low-grade nonHodgkin lymphoma (for a review, see Gopal and Press1). Mechanisms involved in anti–CD20-mediated B-cell depletion include
complement-mediated lysis and antibody-dependent cellular cytotoxicity, but apoptotic cell death occurring as a result of signaling
events induced by CD20 cross-linking is also believed to play a
role.2-4 It is generally assumed, based on antibody blocking
studies,5 that CD20 bears only 2 extracellular epitopes, one that is
recognized by the vast majority of CD20 mAbs, and a second that
is recognized by a single antibody (1F5) with unusual activating
properties. Consequently, little attention has been given to the
possibility that heterogeneity in the fine specificity of anti-CD20
mAbs could result in differences in signal initiation, potentially
modulating clinical outcome.
The complementary DNA (cDNA) sequence of CD20 predicts a
tetraspan protein with intracellular termini and a single extracellular loop between the third and fourth transmembrane domains.6-8 The membrane orientation and topology of CD20 was
confirmed using antisera generated against peptides near the amino
and carboxyl termini and by proteolytic digestion of extracellular regions.9 CD20 forms oligomers on the cell surface, as
indicated by chemical cross-linking studies,10 and appears to
function as a component or regulator of a voltage-independent
calcium channel.10,11
CD20-directed mAbs exert a variety of biochemical and
biologic effects on B cells, including activation of tyrosine kinases
leading to c-myc induction and homotypic aggregation,12,13 translocation of CD20 to lipid rafts,9,14 down-regulation of antigen and
CD23 receptors,15,16 inhibition of antibody production by mitogenactivated B cells,17,18 and induction of apoptosis.2-4,19,20 With the
exception of the 1F5 mAb, which appears to be unique in its ability
to activate resting B cells,21-25 there have been few indications that
CD20 mAbs differ in their effects on B cells. However, we
observed differences between 2 mAbs, B1 and 2H7, in their ability
to induce intracellular calcium release,13 raft association of CD2014
homotypic aggregation of B cells (unpublished data, December
1997), and coprecipitate src family kinases,13,26 suggesting that
these antibodies recognize distinct epitopes. We therefore sought to
characterize CD20 epitopes in greater detail as a means of
increasing our understanding of CD20 structure and function.
Because antibodies directed against extracellular epitopes on human
CD20 do not bind to murine B cells, we took advantage of differences in
From the Department of Biochemistry and Molecular Biology, Immunology
Research Group, University of Calgary, Calgary, Alberta, Canada.
Reprints: Julie Deans, Department of Biochemistry and Molecular Biology,
University of Calgary, Health Sciences Center, 3330 Hospital Dr NW, Calgary,
Alberta, T2N 4N1, Canada; e-mail: [email protected].
Submitted July 27, 2001; accepted December 4, 2001.
Supported by the Canadian Institutes of Health Research, the Natural Sciences
and Engineering Research Council of Canada, and the Weiss Memorial
Endowment Fund. J.P.D. is a senior scholar of the Alberta Heritage Foundation
for Medical Research.
3256
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.
© 2002 by The American Society of Hematology
BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
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BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
the amino acid sequences of the extracellular domains of human and
mouse CD206-8,27 in a homolog scanning mutagenesis strategy to
characterize the fine specificity of CD20 mAbs.
EPITOPE MAPPING OF CD20
3257
extracellular domain (residues 142-151; ESLNFIRAHT; single-letter amino
acid codes), for 30 minutes. Immunofluorescence was measured using a
FACScan cytometer (Becton Dickinson).
Biochemical analyses
Materials and methods
Cells and antibodies
Raji and Ramos lymphoblastoid B cells were grown in RPMI/5% fetal
bovine serum (FBS) and HEK293 cells in Dulbecco modified Eagle
medium (DMEM)/10% FBS. 1F5 and 2H7 mAbs were provided by Dr J.
Ledbetter, and NK1 by Dr A. Hekman.28 B1 mAb was purchased from
Coulter (Miami, FL), L27 from Becton Dickinson (San Jose, CA) and
rituximab from IDEC Pharmaceuticals (San Francisco, CA). Other CD20
mAbs were obtained from the human leukocyte differentiation workshops
IV, VI, and VII. Affinity-purified rabbit antiserum directed against a peptide
near the amino terminus of CD20 (anti-CD20N) was previously described.9
Human IgG and murine isotype control antibodies IgG1, IgG2a, and IgG2b
were purchased from Caltag Laboratories (Burlingame, CA), Sigma (St
Louis, MO), and Southern Biotechnology Association (Birmingham, AL).
Fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG and goat
antihuman IgG were from Southern Biotechnology Association and Caltag
Laboratories, respectively.
Mutagenesis
Mutations were made using overlap extension polymerase chain reaction
(PCR) using human CD20 cDNA template,7 internal primer pairs encoding
the desired mutations, and outside primers 5⬘-ATAATGAATTCATTGAGCCTCTTT-3⬘ and 5⬘-AATCACTTAAGGAGAGCT-3⬘ encoding unique
EcoRI and AflII restriction sites at positions 451 and 983, respectively, of
the CD20 cDNA. PCR fragments were digested with EcoRI and AflII and
cloned into pBluescript containing human CD20 cDNA from which the
EcoRI/AflII fragment had been excised. To generate a chimera containing
the extracellular sequence of murine CD20 with the transmembrane and
intracellular regions of human CD20 (h/m CD20), the extracellular region
of human CD20 cDNA was converted to murine using overlap extension
PCR. The h/m chimera cDNA was then used as a template to introduce
mutations into the murine sequence, generating constructs A through D
described in “Results.” Construct C was subsequently used as a template to
generate construct E, and construct E was the template for constructs F and
G. The sequences of all constructs were confirmed prior to subcloning into
the pCDM8 mammalian expression vector.
Transfections
HEK293 cells grown to approximately 50% confluence were transiently
transfected with CD20 wild-type and mutant cDNA constructs using the
calcium phosphate method. DNA in a 250-mM solution of CaCl2 was
slowly bubbled into an equal volume of 2 times Hepes buffer (280 mM
NaCl, 50 mM Hepes, 1.5 mM Na2HPO4) and left for no more than 5
minutes to precipitate before transferring to a HEK293 culture plate.
Transfection conditions were optimized by monitoring the expression of
titrated amounts of CD20 wild-type cDNA construct at 1, 2, 3, and 4 days
after transfection. Maximal expression was observed after 2 to 3 days using
10 ␮g DNA. No difference in expression was observed using 10, 20, or 30
␮g cDNA, but to minimize expression variability between samples, we
routinely used 20 ␮g cDNA per sample. Two to 3 days after transfection,
cells were washed twice with phosphate-buffered saline (PBS; 140 mM
NaCl, 2.5 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4), and lifted off the
culture plates for analysis of CD20 expression and antibody binding.
Cells were washed, pelleted, and lysed on ice for 15 minutes in lysis buffer
containing protease inhibitors (1 ␮g/mL aprotinin, 1 ␮g/mL leupeptin, 1
mM NaMoO4, 1 mM NaVO4, 1 mM phenylmethylsulfonyl fluoride
[PMSF], 1 mM EDTA) and either 1% Triton X-100 or 1% digitonin as
indicated. Lysates were cleared of nuclei and other insoluble material by
centrifugation at 13 000g for 15 minutes at 4°C. Where indicated, residual
detergent-insoluble material was cleared using a Beckman TL-100 ultracentrifuge at 100 000g for 1 hour at 4°C. For immunoprecipitations, lysates
were incubated with appropriate antibodies and then with protein A
Sepharose (Repligen, Cambridge, MA) for 1 to 2 hours at 4°C. The beads
were washed with lysis buffer and precipitated protein was eluted in sodium
dodecyl sulfate (SDS) sample buffer.
CD20 translocation experiments were performed as described.14 Briefly,
cells were treated before lysis with anti-CD20 mAbs for 15 minutes at 37°C
and both the cleared lysates and the insoluble pellets were collected for
analysis by CD20 immunoblotting. Equal cell equivalents from the lysate
and pellet samples were loaded onto gels and the relative amount of
insoluble CD20 in each sample was estimated and scored as follows: 75%
to 100%, ⫹⫹⫹⫹; 50% to 75%, ⫹⫹⫹; 25% to 50%, ⫹⫹; 5% to 25%, ⫹;
and less than 5%, ⫺.
For density gradient centrifugation analysis, 100 mM iodoacetamide
(Sigma) was included in the lysed samples to prevent postlysis disulfide
bond formation. Cleared lysates were layered onto a 5% to 40% sucrose
gradient containing either 1% Triton X-100 or 1% digitonin, as appropriate,
and centrifuged at 170 000g for 17 hours at 4°C using a SW41 rotor
(Beckman). Fractions (0.5 mL) were collected from the top of the gradients.
Protein molecular weight standards (Sigma), run simultaneously on an
adjacent gradient, were treated similarly. Equal aliquots of each fraction
were mixed with SDS sample buffer. Western blot analysis and Coomassie
staining were used to identify the fractions containing CD20 and the
molecular weight standards, respectively.
To confirm cell surface expression of constructs transiently expressed in
HEK293 cells, proteinase K digests were performed as described.9 Briefly,
cells were incubated alone or with 12.5 ␮M proteinase K for 15 minutes on
ice. Protease inhibitors (4 mM Pefabloc [Boehringer Mannheim, Laval,
Quebec, Canada], 1 ␮g/␮L aprotinin, 1 ␮g/␮L leupeptin, and 1 mM PMSF)
were added to halt digestion. Samples were rapidly centrifuged, supernatants aspirated, and the cell pellets lysed directly in SDS sample buffer.
Samples in SDS sample buffer were heated at 100°C for 5 minutes.
Proteins were separated by SDS–polyacrylamide gel electrophoresis (SDSPAGE) and transferred to Immobilon P membranes (Millipore, Bedford,
MA). Membranes were blocked, incubated with anti-CD20N, washed, and
bound antibodies detected with horseradish peroxidase conjugated to
protein A (Bio-Rad, Richmond, CA). Proteins were visualized using
enhanced chemiluminescence (Pierce, Rockford, IL) recorded on Kodak
X-OMAT film (Eastman Kodak, Rochester, NY) or Fluor-S Max MultiImager (Bio-Rad).
Homotypic aggregation
Ramos B cells, 1 ⫻ 106 in 1 mL tissue culture medium, were placed into
wells of 24-well flat-bottomed plates with 1 ␮g anti-CD20 or control
antibody, and incubated at 37°C for 4 hours. All assays were carried out in
duplicate. Extent of cellular aggregation was scored as follows: ⫹/⫺, less
than 10% of cells were clustered; ⫹, 10% to 25% of cells were in small
clusters; ⫹⫹, less than 50% of cells were clustered into medium-sized
aggregates; ⫹⫹⫹, 50% to 75% of cells were in medium to large
aggregates; and ⫹⫹⫹⫹, 75% to 100% of cells were in large aggregates.
Immunofluorescence
Cells were incubated with anti-CD20 or control mAbs in PBS/2% FBS.
After washing, bound antibodies were detected using appropriate FITCconjugated secondary antibodies. As a component of the epitope mapping
studies, 1F5, 2H7, and B1 mAbs (1 ␮g) were preincubated with 10 ␮g
peptide containing a stretch of the least conserved amino acids in the
Results
For the initial epitope mapping studies, mAbs 1F5, 2H7, and B1
were selected for analysis; B1 is the prototypic CD20 mAb, 2H7 is
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3258
POLYAK and DEANS
BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
representative of mAbs previously thought to recognize the same
epitope as B1, and 1F5 has distinct specificity. None of these mAbs
was capable of detecting CD20 by immunoblot (data not shown),
indicating that their epitopes are conformational.
Mutagenesis of the human CD20 extracellular region
The human CD20 cDNA sequence predicts a protein that is 73%
identical to murine CD20 with regions of greatest similarity in the
transmembrane domains.6-8,27 The extracellular region is less
conserved, differing at 16 of the approximate 43 amino acid
residues (Figure 1A). Among these 16 residues are 8 nonconservative differences, most of which are located within a 10-amino acid
stretch (ESLNFIRAHT). A synthetic peptide with this sequence
failed to block binding of mAbs 1F5, 2H7, and B1 to Raji B cells
(data not shown), suggesting that either these residues are not
represented within the corresponding epitopes or that the secondary
structure assumed by the soluble peptide was insufficient for
antibody binding.
A mutation strategy was then designed in which extracellular
residues in the human CD20 sequence that differ from those in the
mouse were replaced with those from the equivalent positions in
the murine sequence. As indicated in Figure 1A, 7 constructs were
generated. In construct 1, for example, KI in the human sequence
was replaced with TL, but all other residues remained unchanged.
The constructs were expressed in HEK293 cells, and the cells
collected for analysis 2 to 3 days after transfection. All constructs
were expressed, as shown by immunoblotting using a polyclonal
antibody generated against a cytoplasmic peptide (Figure 1B),
although there was some variability in the degree of expression.
Recognition of the constructs by mAbs directed against extracellular epitopes was assessed by flow cytometry (Figure 1C). The only
construct not recognized by mAbs 1F5, 2H7, or B1, was construct
6. Extracellular protease digestion of cells transfected with construct 6 led to loss of the full-length 33- to 35-kd protein,
confirming its expression at the cell surface (Figure 1B, far right,
lanes 6 and 6*).
Mutagenesis of the murine CD20 extracellular region
The murine sequence was then mutated in a complementary
strategy, replacing residues that differ from human CD20 with
those from the equivalent positions in the human sequence (Figure
2A). First, a chimera was constructed in which the entire extracellular region in the human sequence was replaced with that from the
mouse (h/m CD20). By constructing a chimera it was possible to
use existing antisera against cytoplasmic peptides in human CD20
to confirm expression of the chimeric proteins. As expected, mAbs
directed against extracellular epitopes of human CD20 did not
recognize the h/m chimera (Figure 2C). Initially, 4 constructs were
generated (Figure 2A); one in which SNS in the murine sequence
was replaced with ANP (construct C); a second in which only the
proline residue was introduced (construct D); a third in which
VDxxD in the mouse was replaced with INxxN from the human
(construct B); fourth, a construct that accommodated all of the
remaining differences in the amino-terminal half of the sequence
(construct A). These constructs were transfected into HEK293 cells
and expression was confirmed in all cases by immunoblot (Figure
2B). There was no binding of mAbs 1F5, B1, or 2H7 to cells
expressing either construct A or construct B, as assessed by flow
cytometry (Figure 2C). Cell surface expression of these constructs,
as well as the h/m chimera, was confirmed by protease digestion as
described for construct 6 in Figure 1 (data not shown). Remarkably,
the epitope recognized by the B1 mAb was completely recovered
Figure 1. Binding of CD20 mAbs is abolished by mutation of alanine-170 and
proline-172. (A) The human CD20 extracellular sequence was mutated toward the
murine sequence to produce constructs 1 through 7. Mutated residues in constructs 1
through 7 are highlighted in bold. (B) Wild-type (WT) human CD20 and constructs 1
through 7 were expressed in HEK293 cells and equimolar amounts of cell lysates
were tested for CD20 expression by immunoblot analysis using a polyclonal antibody
generated against a cytoplasmic peptide (anti-CD20N). To confirm cell surface
expression of construct 6, transfected cells were either untreated or treated with
proteinase K before lysis (lanes 6 and 6*, far right). (C) Binding of anti-CD20 mAbs
1F5, 2H7, and B1 to transfected cells was monitored by flow cytometry (top); mean
fluorescence values, with the isotype control values subtracted, are shown in the bar
graph below. Results are representative of 6 independent experiments.
by construct C, and partially by construct D. There was partial
recovery of 1F5 binding by construct C. Binding of 2H7 was not
rescued to a significant extent by any of the mutations in constructs
A through D, although there was detectable, albeit extremely low,
binding of 2H7 to construct C.
Figure 2 shows that replacement of residues SxS in the mouse
sequence with AxP was sufficient to completely reconstitute the
epitope of the B1 mAb but not that of 2H7 or 1F5. Additional
mutant constructs were then generated to investigate the requirements for 2H7 and 1F5 binding. Because AxP was a minimum
requirement for 1F5 and 2H7 binding (Figure 1), SxS was replaced
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BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
EPITOPE MAPPING OF CD20
3259
construct C (AxP) or E (AxP/Y), but was fully recovered by
construct F (INxxN/AxP/Y).
To assess whether any of the remaining amino acid differences
contributed to epitopes recognized by those mAbs that did not bind
well to constructs C or E, mutations were made to introduce the
relevant human residues into the equivalent positions in the murine
chimeric construct E (AxP/Y). Mutation of QTSK to RAHT
(construct G) or RR to ES (construct I) increased the binding of
mAbs 2H7 and CAT 13.6E12 compared to construct E. Mutation of
EL to NF (construct H) increased the binding of 2H7 but not CAT
13.6E12. None of these mutations increased binding of 1F5, which
was the only epitope not fully reconstituted by any of the constructs
generated in this study.
Additional fine specificity differences among CD20 mAbs
suggested by their biologic activities
Figure 2. Conversion of SNS in the murine CD20 sequence to ANP recovers
binding of B1. (A) The extracellular region of human CD20 was replaced with that
from the murine sequence to generate the h/m chimera. Residues in the murine
sequence were then mutated to produce constructs A through D. Mutated residues in
constructs A through D are highlighted in bold. (B) Wild-type human CD20 (WT), h/m
CD20, and constructs A through D were expressed in HEK293 cells and CD20
expression assessed by immunoblot analysis as in Figure 1. (C) Binding of anti-CD20
mAbs 1F5, 2H7, and B1 to transfected cells was monitored by flow cytometry (top);
mean fluorescence values, with the isotype control values subtracted, are shown in
the bar graph below. Results are representative of 4 independent experiments.
with AxP in all of the mouse/human CD20 chimeric constructs
subsequently produced (Table 1). In construct E, Y284, located at
the carboxyl-terminal end of the extracellular human sequence,
replaced N at the equivalent position in the mouse sequence. This
mutation had no significant effect on antibody binding compared to
construct C and was included in all subsequent constructs. Next,
VDxxD in the murine sequence was replaced with INxxN (construct F). The protein product from this construct completely
recovered binding of the 2H7 mAb and enhanced the binding of
1F5 (Table 1). Also shown in Table 1 is the analysis of 4 additional
mAbs: CAT 13.6E12, LT20, L27, and rituximab. Epitopes recognized by LT20, L27, and rituximab, like that of B1, were fully
reconstituted in construct C (AxP). The epitope recognized by CAT
13.6E12 was similar to that of 2H7 in that it was not recovered by
A panel of 16 CD20-reactive mAbs was screened and divided into
4 groups according to their ability to bind to construct C (AxP;
Table 2). Only one antibody, CAT 13.6E12, mentioned earlier, was
consistently and completely unable to recognize the protein
product of this construct (group I in Table 2). This mAb, like most
CD20 mAbs, failed to induce Ramos cell aggregation but strongly
induced translocation of CD20 to the detergent-insoluble fraction.
The majority of CD20 mAbs was similar to B1 insofar as the AxP
mutation in the murine sequence largely or completely reconstituted the epitope (group IV in Table 2). However, fine specificity
differences among these mAbs can be inferred from marked
variance in their ability to induce homotypic aggregation of Ramos
B cells and to induce detergent insolubility of CD20. Only B1 and
Bly1 induced strong aggregation of Ramos cells. Interestingly, B1
and Bly1 were the only antibodies that were ineffective at inducing
CD20 translocation (Table 2). Thus, Bly1 and B1 probably
recognize an identical epitope. Rituximab fell into group IV but
was unusual in that it induced a low level of aggregation and also
strongly induced CD20 translocation. The remaining mAbs in
group IV did not induce aggregation, strongly induced CD20
translocation, and could not be differentiated from one another
using these parameters.
Two mAbs were identified, which, like 2H7, bound very weakly
to construct C (AxP) (group II, Table 2). One of these, AT80, was
the only mAb in the entire panel that strongly induced both cellular
aggregation and CD20 translocation, and therefore likely has a
unique fine specificity. Finally, 1F5 and 2 other mAbs showed an
intermediate degree of reactivity with construct C (AxP; group III,
Table 2). Further differentiation of fine specificity among these
mAbs could not be determined, because all 3 mAbs strongly
induced CD20 translocation and did not induce cellular aggregation. Antibodies in the panel were of various IgG isotypes but this
did not correlate with or account for any of the differences in
reactivity observed.
The CD20 epitope recognized by mAb 2H7 is presented only on
oligomeric complexes
Altogether, 7 different patterns of reactivity were distinguished
when 16 mAbs were compared for their ability to bind to the AxP
construct C and to induce homotypic aggregation or CD20
translocation: 2 in group II, 3 in group IV, and those in groups I and
III. It seemed unlikely that the small extracellular loop of CD20
could present the variety of epitopes necessary to account for this
array of fine specificity differences. We hypothesized that some
epitopes are comprised of residues contributed by different CD20
molecules adjacent to one another in a homo-oligomeric complex.
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3260
BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
POLYAK and DEANS
Table 1. Antibody reactivity with CD20 extracellular loop mutants
Anti-CD20 mAbs
CD20 construct
Extracellular loop mutations*
B1
2H7
1F5
CAT 13.6E12
Rituxan
L27
LT20
Hu
KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCY
⫹⫹⫹⫹†
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
NT
#6
----------------------------S-S------------
⫺
⫺
⫺
NT
NT
NT
Mu
TL------RR-EL-QTSK--VD--D---S-S-----------N
⫺
⫺
⫺
⫺
⫺
⫺
⫺
D
TL------RR-EL-QTSK--VD--D---S-P-----------N
⫹⫹
⫺
⫹/⫺
NT
NT
NT
NT
C
TL------RR-EL-QTSK--VD--D---A-P-----------N
⫹⫹⫹⫹
⫹/⫺
⫹⫹
⫺
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
E
TL------RR-EL-QTSK--VD--D---A-P-----------Y
⫹⫹⫹⫹
⫹/⫺
⫹⫹
⫺
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
F
TL------RR-EL-QTSK--IN--N---A-P-----------Y
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
G
TL------RR-EL-RAHT--VD--D---A-P-----------Y
⫹⫹
⫹⫹
⫹⫹
⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹⫹⫹
H
TL------RR-NF-QTSK--VD--D---A-P-----------Y
⫹⫹
⫹⫹
⫹
⫺
⫹⫹⫹⫹
⫹⫹
⫹⫹⫹⫹
I
TL------ES-EL-QTSK--VD--D---A-P-----------Y
⫹⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹
J
KI------RR-EL-QTSK--VD--D---A-P-----------Y
⫹⫹
⫹
⫹
⫺
⫹⫹
⫹⫹
⫹⫹
NT indicates not tested. All antibodies were tested at least 3 times against each construct.
*Black letters are human sequence; gray letters are murine sequence; black dashes are identical to human.
†Reactivity is shown on a scale of ⫹ (weak positive) to ⫹⫹⫹⫹ (maximum positive). ⫹/⫺ indicates borderline positive.
Others have reported the detection of CD20 dimers and tetramers
after chemical cross-linking of cell surface proteins10; therefore, we
sought detergent lysis conditions that would retain the integrity of
such complexes. Raji B cells were lysed either in Triton X-100 or in
digitonin and the postnuclear cleared lysates were layered onto 5%
to 40% linear sucrose density gradients and centrifuged to equilibrium; fractions were collected and probed for the presence of CD20
by immunoblot. CD20 migrated on the gradient as a monomer
when the cells were lysed in Triton X-100 (Figure 3A). In contrast,
when the cells were lysed in digitonin, CD20 migrated to a position
corresponding to about 200 kd (Figure 3A). CD20 protein formed
the bulk of the mass of the approximate 200-kd complex, as
demonstrated by its immunoprecipitation from digitonin lysates
followed by silver staining (not shown). These data are consistent
with chemical cross-linking experiments,10 and indicate that the
CD20 complex includes multiple (probably 4) CD20 molecules
and additional minor component(s).
The 2H7 mAb precipitates only a small subset of CD20 protein
from Triton X-100 lysates compared to B1 and 1F5 (Figure 3B),
even though analysis by flow cytometry indicates that it binds at
least as well as B1 and 1F5 to CD20 at the cell surface14 (Figure 1).
We predicted that this CD20 subset was associated with membrane
rafts, because 2H7 coprecipitates a 75- to 80-kd tyrosine phosphorylated protein that we subsequently identified as PAG/Cbp, an
adaptor protein only found in lipid rafts29,30 (unpublished data, June
2000). We reasoned that the 2H7 mAb can only recognize CD20 in
its native oligomeric form, which would be preserved in Tritoninsoluble membrane rafts, whereas it cannot recognize the monomeric form of CD20 in the soluble fraction of Triton X-100 lysates.
To test this hypothesis, CD20 immunoprecipitations were perTable 2. Reactivity of expanded panel of CD20 mAbs
Group
AxP
reactivity
Antibody designation
Homotypic
aggregation
CD20
translocation
I
None
CAT 13.6E126
⫺
⫹⫹⫹⫹
II
Weak
2H76, 1C0-1652
⫺
⫹⫹⫹⫹, ⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
III
Intermediate
1F56, B-H202, MEM-972
⫺
⫹⫹⫹
IV
High
B16, Bly12
AT802
Rituxan3
NK12, PDR782, F4B13661,
LT205, L275, CAT 13.7H82
⫹⫹⫹⫹
⫺
⫹⫹
⫹⫹⫹⫹
⫺
⫹⫹⫹ to
⫹⫹⫹⫹
Superscripts indicate the number of times each antibody was tested for reactivity
against the m/h AxP chimera (construct C). At least 2 homotypic aggregation and
translocation experiments were performed for each antibody, and were scored as
described in “Materials and methods.”
Figure 3. Differential sensitivity of CD20 epitopes to detergent lysis. (A) Raji B
cells were lysed in either 1% Triton X-100 or 1% digitonin and cleared lysates were
layered on top of 5% to 40% linear sucrose density gradients and centrifuged to
equilibrium. Fractions were collected from the top of the gradient and probed by
immunoblot using a polyclonal antibody raised against a cytoplasmic CD20 peptide
(anti-CD20N). Densitometry analysis was performed and the results plotted to
generate the graphs shown. Molecular weight standards were run in parallel samples
(arrows). (B) Immunoprecipitation of CD20 by mAbs 1F5, 2H7, and B1 from Raji B
cells lysed in 1% Triton X-100. The mAbs were added to cleared (13 000g) lysates as
indicated to immunoprecipitate CD20. Samples were analyzed by anti-CD20N
immunoblot. (C) Immunoprecipitation of CD20 by mAbs 2H7 and B1 from Raji B cells
lysed in either 1% Triton X-100 or in 1% digitonin. The mAbs were added to cleared
(13 000g) lysates as indicated. Anti-CD20N immunoblots were analyzed by densitometry and the relative density of the CD20 bands was expressed as the percent of the
amount precipitated by B1. (D) As in panel C, except that the lysates were cleared at
100 000g for 1 hour. Note the loss of 2H7-precipitable CD20 from Triton lysates.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
formed from cell lysates prepared with either digitonin or Triton
X-100. Immunoprecipitation of CD20 by 2H7 was indeed as
effective as B1 when cells were lysed in digitonin (Figure 3C).
Ultracentrifugation of Triton X-100 lysates depleted 2H7precipitable CD20 (Figure 3D), confirming that this subset of
CD20 protein was associated with detergent-insoluble membranes.
Discussion
The B1 epitope and AxP
Previously, the B1 and 2H7 mAbs were thought to recognize the
same epitope. Indeed, the existing evidence suggested that all
antihuman CD20 mAbs, with the exception of 1F5, bound to a
single epitope.5 This report shows that 2H7 and B1 unequivocally
recognize distinct epitopes and demonstrates a high degree of
heterogeneity among 16 mAbs tested. Rituximab was most like the
B1 mAb with respect to its reactivity with the AxP h/m chimera
(construct C), but was distinct from B1 with respect to its ability to
induce CD20 translocation (Table 2).
It is clear from these studies that the sequence AxP at positions
170-172 in human CD20 is a critical determinant in the secondary
structure of the extracellular loop because its mutation destroyed
the epitopes of all 3 CD20 mAbs tested. Replacement of the second
serine in the equivalent mouse sequence SxS with a proline residue
was sufficient to allow binding of B1, although replacement of both
serines with alanine and proline was necessary to fully reconstitute
the B1 epitope. Site-directed mutagenesis, although a powerful tool
in epitope mapping studies, cannot define contact sites, and it is not
possible to conclude that AxP are contact residues in the B1
epitope. Crystallographic determinations have shown that there are
at least 15 contact residues in the binding regions of antigenantibody complexes.31 It can be inferred from creation of the B1
binding site in the murine CD20 sequence by replacement of only 2
residues, that most, if not all, contact residues for the B1 mAb are
shared between human and mouse CD20. Proline residues introduce kinks into polypeptide backbones, often interrupting regions
of defined secondary structure. Whether or not AxP is included in
the contact site, it is likely that this sequence defines the conformation of contact residues at neighboring or distant sites.
The 2H7 epitope and INxxN
The 2H7 epitope was recreated in the AxP murine chimera by the
replacement of VDxxD with INxxN. This was a surprising finding
because these are conservative mutations. Again, it cannot be
determined whether INxxN are contact residues in the 2H7 binding
site or residues that are essential for defining the conformational
epitope at a distant site. The INxxN sequence is not critical for
maintenance of the 2H7 epitope in human CD20, because its
mutation did not abrogate antibody binding (Figure 1). Further,
INxxN is insufficient to recreate the 2H7 epitope in the background
of the murine sequence (Figure 2). However, INxxN was an
essential determinant in the creation of the 2H7 epitope when AxP
was included in the murine sequence (Table 1).
B1 versus 2H7 epitopes
Others have reported,5 and we have confirmed (unpublished data,
March 2000), that 2H7 blocks the binding of B1 to its epitope. It is
possible that the paratopes of the 2 mAbs have overlapping
footprints or that there is steric hindrance from the Fc regions of the
antibodies. In support of the latter we have found that Fab
fragments of 2H7 do not effectively block the binding of B1,
EPITOPE MAPPING OF CD20
3261
suggesting that the epitopes are spatially distinct (unpublished data,
March 2000). Although neither B1 nor 2H7 is capable of recognizing denatured epitopes, there is a marked difference in the
sensitivity of the epitopes to detergent lysis. B1 efficiently precipitates CD20 from Triton X-100 lysates, whereas 2H7 does not.
Density gradient centrifugation analyses indicated that Tritonsoluble CD20 exists in monomeric form (Figure 3A). Therefore,
the ability of B1 to precipitate CD20 from Triton lysates indicates
that the B1 epitope is retained on CD20 monomers. A small
proportion of CD20 molecules are constitutively associated with
Triton-insoluble lipid rafts in unstimulated cells, and our data are
consistent with the interpretation that it is this population that is
recognized by 2H7 in Triton X-100 lysates. The 2H7 epitope is
retained in digitonin lysates in which CD20 is solubilized as an
approximate 200-kd complex. Together these data provide strong
evidence that retention of the 2H7 epitope requires the integrity of
the multimeric complex.
The 1F5 epitope
The molecular basis for the activating properties of the 1F5 mAb
was not revealed by this analysis, but this mAb was the only one
among 7 tested against the full panel of h/m mutants, for which full
binding was not recovered by any of the mutations made in the
murine sequence (Table 1). The epitope that it recognizes is
therefore unique. 1F5 falls between B1 and 2H7 with respect to its
ability to precipitate CD20 from Triton lysates (Figure 3B),
indicating that its epitope is likely to be partially dependent on the
integrity of the CD20 oligomeric complex. Recently, analyses of
the genomic databases have revealed a large family of CD20
related genes.32,33 Several members of this MS4A gene family are
expressed in hematopoietic cells and at least 3 of them, in addition
to CD20, are expressed in B cells.33 Whether or not heterologous
associations between members of this family can form oligomeric
complexes remains to be determined, but would be expected to
further increase the array of possible epitopes.
Nonconservative amino acid differences between human
and mouse CD20
The creation of B1 and 2H7 human CD20 epitopes in the mouse
sequence by replacement of residues that differ only conservatively
underscores the fact that antibody binding sites cannot be predicted
based on sequence differences between homologs. Most residues in
the human CD20 sequence that are radically different at the
equivalent positions in the murine sequence were not essential for
mAb recognition (Figure 1). However, it is likely that some of these
residues are included in the epitopes of some CD20 mAbs, because
their introduction into the AxP murine chimera enhanced the
binding of some antibodies (Table 1).
Most mAbs generated against native proteins are directed
against nonlinear determinants, and antihuman CD20 mAbs are
clearly no exception. Nevertheless, considering the small size of the
extracellular region of CD20, it is remarkable that we could discriminate
at least 7 different patterns of reactivity among 16 CD20 mAbs. This
analysis revealed that the induction of homotypic aggregation is an
unusual characteristic among CD20 mAbs, in contrast to the relatively
common ability to induce CD20 insolubility and translocation into lipid
rafts. We, and others, have shown that the B-cell antigen receptor also
translocates into lipid rafts after ligation.34-36 Colocalization of the BCR
and CD20 in rafts may serve to localize calcium influx (through the
putative CD20 calcium channel) to regions proximal to antigen engagement. No ligand for CD20 has yet been identified, but it is possible that
ligand binding mediates CD20 translocation to rafts in vivo. Results
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
3262
BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
POLYAK and DEANS
from this study predict that binding sites for such a ligand would be
created by neighboring CD20 molecules in an oligomeric complex.
It has been suggested that CD20 delivers an apoptotic signal
responsible for some of the cell death underlying therapeutic
efficacy of rituximab. Because lipid rafts concentrate src-family
kinases and other signaling effectors, anti–CD20-induced signals
may be secondary to raft aggregation occurring as a consequence of
CD20 translocation. Interestingly, in support of this idea, aggregation of rafts by cross-linking either glycosphingolipid GM1 or
glycosylphosphatidylinositol-linked proteins, which are constitutively localized to rafts, was shown to lead to apoptosis.37 Rafts
exclude most integral membrane proteins and antibody-mediated
translocation of CD20 into rafts is unusual; ligation of several other
surface proteins expressed in B cells did not result in their
redistribution into Triton-insoluble rafts.36 Raft association of
antibody-bound CD20 may partly explain the particular efficacy of
CD20 as an immunotherapeutic target, and those antibodies such as
rituximab, which most efficiently induce CD20 translocation, may
confer some added benefit.
Acknowledgments
We thank L. Robertson for assistance with flow cytometry; Dr L. Ayer
for assistance with cellular aggregation and CD20 translocation experiments; T. Kryzanowski for secretarial assistance; C. Mutch, R. Petrie,
and Dr C. Brown for comments on the manuscript; and Dr J. Ledbetter
and Dr A. Hekman for providing antibodies.
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2002 99: 3256-3262
doi:10.1182/blood.V99.9.3256
Alanine-170 and proline-172 are critical determinants for extracellular CD20
epitopes; heterogeneity in the fine specificity of CD20 monoclonal
antibodies is defined by additional requirements imposed by both amino
acid sequence and quaternary structure
Maria J. Polyak and Julie P. Deans
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