MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 6, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0042
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Epitope Mapping of an Anti-CD20 Monoclonal Antibody(C20Mab-60) Using Enzyme-Linked Immunosorbent Assay

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Junko Takei,1 Hiroyuki Suzuki,2 Teizo Asano,1 Guanjie Li,2 Masaki Saito,2Mika K. Kaneko,1 and Yukinari Kato1,2,i

CD20 is a glycosylated transmembrane protein and is expressed on normal B cells and B cell malignancies.
Therapeutic antibodies against CD20 are developed and used in clinic. The understanding of antibody–antigen
binding by revealing the epitope is essential for future application to antibody technology. Previously, we developed
an anti-human CD20 monoclonal antibody, C20Mab-60 (IgG2a, kappa), using the Cell-Based Immunization andScreening (CBIS). C20Mab-60 can be used for flow cytometry, Western blot, and immunohistochemical analyses.
In this study, we examined the critical epitope of C20Mab-60 using enzyme-linked immunosorbent assay (ELISA)
with synthesized peptides. We performed ELISA with deletion mutants, and C20Mab-60 reacted to the 160–179
amino acids sequence of CD20. Next, we analyzed the reaction to 20 point mutants, and C20Mab-60 did notrecognize the alanine-substituted peptides of N171A, P172A, S173A, and E174A. The results indicate that thebinding epitope of C20Mab-60, developed by CBIS, includes Asn171, Pro172, Ser173, and Glu174 of CD20.
Keywords: CD20, C20Mab-60, epitope mapping, monoclonal antibody, enzyme-linked immunosorbent assay

Introduction

CD20 is a glycosylated transmembrane protein andcomprises four hydrophobic membrane domains, two
extracellular loops (*7 and 44 amino acids), and three intracellular regions including N- and C-terminus.(1) The intracellular region of CD20 is phosphorylated upon antibody
binding, thereby mediating cellular signaling.(2) CD20 is expressed on normal B cells from pre-B to mature B cell development, but not detected in early pro-B cells and terminallydifferentiated plasma cells.(3) CD20 is also detected in human
B cell malignancies including non-Hodgkin lymphoma andB-lymphoblastic leukemia/lymphoma.(4) Since CD20 is not
expressed in early pro-B cells, anti-CD20 therapy is thought
to be effective to these malignancies without affecting earlylineage of normal B cells.
CD20 was first defined by the murine monoclonal antibody(mAb) tositumomab.(5) Rituximab, a chimeric anti-CD20mAb, was developed and approved for treatment of the B cellmalignancies.(6–9) Rituximab destroys both CD20 expressed
normal B cell and malignancies through complement-dependent cytotoxicity (CDC) and antibody-dependent cellularcytotoxicity (ADCC).(10) Rituximab is also approved for autoimmune diseases including rheumatoid arthritis.(11) Although
rituximab has dramatically improved outcomes for patientswith CD20-positive malignancies and autoimmune diseases fortwo decades, the responses are not universal and resistance candevelop.(10)Through recombinant DNA technology, second and third
generation anti-CD20 mAbs were developed.(12) Amongthese, ofatumumab and obinutuzumab have been approvedfor clinical treatment of B cell malignancies, such as chronic
lymphoid leukemia and rituximab refractory follicular lymphoma.(13,14) Obinutuzumab was developed to potentiate theCDC activity and overcome resistance to rituximab.(15)
Therefore, the development of anti-CD20 antibody and theunderstanding of its molecular actions are thought to beimportant.
In our previous study, we established an anti-human
CD20 mAb (C20Mab-60) by using a Cell-Based Immunization and Screening method. C20Mab-60 is useful not
only for flow cytometry, but also for Western blot andDepartments of 1Antibody Drug Development and 2Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai,
Japan.
i
ORCID ID (https://orcid.org/0000-0001-5385-8201).

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EPITOPE MAPPING OF ANTI-CD20 MAB

251

Table 1. Identification of C20Mab-60 Epitope
Using Synthesized Peptides (Deletion Mutant)by Enzyme-Linked Immunosorbent Assay
PeptidesSequence

C20Mab-60

71_90
140_159
150_169
160_179
170_189180_190

MIPAGIYAPISVTVWYPLWGNIKISHFLKMESLNFIRAHT
ESLNFIRAHTPYINIYNSEPPYINIYNSEPANPSEKNSPSANPSEKNSPSTQYSYSIQSLTQYSYSIQSL+++-+++, OD655 S 0.5; ++, 0.3 & OD655 < 0.5; +, 0.1 & OD655< 0.3; -, OD655 < 0.1.

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immunohistochemical analyses.(16) To clarify further characteristics of C20Mab-60, we performed epitope mapping
using enzyme-linked immunosorbent assay (ELISA).
Materials and Methods
CD20 peptides

CD20 peptides (accession No.: NM_152866), including 6
deletion mutants (Table 1) and 20 point mutants (Table 2),
were synthesized by utilizing PEPScreen (Sigma-AldrichCorp., St. Louis, MO). Three cysteine residues were replacedby serine: C81S, C167S, and C183S.

blocked with 1% bovine serum albumin-containing PBSTfor 30 minutes at 37C. The plates were incubated with
C20Mab-60 (1 lg/mL), followed by a peroxidase-conjugatedanti-mouse immunoglobulins (1:2000 diluted; Agilent Technologies, Inc., Santa Clara, CA). Enzymatic reactions wereperformed using the ELISA POD Substrate TMB Kit (NacalaiTesque, Inc.). Optical density was measured at 655 nm using
an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA).
Results
Epitope mapping of C20Mab-60 using deletion mutants

To determine the binding epitope of C20Mab-60, we synthesized 6 peptides comprising 71–90 amino acids (aa), 140–
159 aa, 150–169 aa, 160–179 aa, 170–189 aa, and 180–189 aaof the CD20 protein sequence (Table 1). The results demonstrated that C20Mab-60 reacted with the 160–179 aa
sequence (160-PYINIYNSEPANPSEKNSPS-179) of CD20(Fig. 1A). We summarized the results in Figure 1B.
Epitope mapping of C20Mab-60 using point mutants

Then, we synthesized 20 alanine-substituted CD20 peptides of 160–179 aa (Table 2). C20Mab-60 showed reaction

Enzyme-linked immunosorbent assay

Synthesized CD20 peptides (Tables 1 and 2) were immobilized on Nunc Maxisorp 96-well immunoplates (ThermoFisher Scientific, Inc., Waltham, MA) at a concentrationof 10 lg/mL for 30 minutes at 37C. After washing withphosphate-buffered saline containing 0.05% Tween20(PBST; Nacalai Tesque, Inc., Kyoto, Japan), wells wereTable 2. Identification of C20Mab-60 Epitope
Using Synthesized Peptides by Enzyme-Linked
Immunosorbent AssayPeptides

Sequence

C20Mab-60

P160AY161A
I162A
N163A
I164A
Y165A
N166A
S167A
E168A
P169A
A170G
N171A
P172AS173A
E174A
K175A
N176A
S177A
P178A
S179A

AYINIYNSEPANPSEKNSPSPAINIYNSEPANPSEKNSPSPYANIYNSEPANPSEKNSPSPYIAIYNSEPANPSEKNSPSPYINAYNSEPANPSEKNSPSPYINIANSEPANPSEKNSPSPYINIYASEPANPSEKNSPSPYINIYNAEPANPSEKNSPSPYINIYNSAPANPSEKNSPSPYINIYNSEAANPSEKNSPSPYINIYNSEPGNPSEKNSPSPYINIYNSEPAAPSEKNSPSPYINIYNSEPANASEKNSPSPYINIYNSEPANPAEKNSPSPYINIYNSEPANPSAKNSPSPYINIYNSEPANPSEANSPSPYINIYNSEPANPSEKASPSPYINIYNSEPANPSEKNAPSPYINIYNSEPANPSEKNSASPYINIYNSEPANPSEKNSPA++++++++++++++++++++++++++++++++++++++++++++++++++, OD655 S 0.5; ++, 0.3 & OD655 < 0.5; +, 0.1 & OD655< 0.3; -, OD655 < 0.1.

FIG. 1. Determination of the C20Mab-60 epitope for CD20
by ELISA using deletion mutants. (A) Synthesized peptides ofCD20 were immobilized on immunoplates. The plates wereincubated with C20Mab-60 (1 mg/mL), followed by incubation
with peroxidase-conjugated anti-mouse immunoglobulins.
(B) Schematic illustration of CD20 and the C20Mab-60 epitope. Black bar indicates the peptide recognized by C20Mab60. ELISA, enzyme-linked immunosorbent assay.

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with P160A, Y161A, I162A, N163A, I164A, Y165A, N166A,S167A, E168A, P169A, A170G, K175A, N176A, S177A,P178A, S179A, and wild-type 160–179 aa (Fig. 2A). In
contrast, C20Mab-60 exhibited no reaction with N171A,P172A, S173A, and E174A (Fig. 2A), indicating that Asn171,
Pro172, Ser173, and Glu174 are included in the binding epitope of C20Mab-60. We summarized the results in Figure 2B.

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Discussion

In this study, we investigated the binding epitope ofC20Mab-60 using synthesized peptides and determined
171-NPSE-174 as an epitope (Fig. 2B). Peptide scanning is a
simple and useful technique for determining a linear epitope.
However, peptide structure is unfolded and different from
native proteins. X-ray crystallography is the most convincing
strategy to determine the epitope, however, this method requires difficult techniques for crystallization of antibody–antigen complex and costs a lot.(17)TAKEI ET AL.

In a previous study, we developed two novel epitope mapping
methods, such as REMAP method to insert RIEDL tag(18–22)
and HisMAP method to insert five His tags(23) into targeted
proteins. These methods are useful for determining both linear
and conformational epitopes. We have investigated the epitope of C20Mab-60 using HisMAP method, and the epitope
is identical to this study.(23) Therefore, this strategy is useful todetermine the critical epitope. Further studies are needed toconfirm whether the strategy is applicable to other antibodies.
Rituximab recognizes a separated epitope of 170-ANPS-173
and 182-YCYSI-186 within the extracellular domain ofCD20.(24) Especially, 170-ANPSEKN-176 is recognized byseveral anti-CD20 antibodies including ocrelizumab, obinutuzumab, tositumomab, and C20Mab-60. In contrast, ofatumumab recognizes a discontinuous epitope of 72-IPAGIYAPI-80
and 148-KMESLNFIRAHT-159. Furthermore, based on theirmechanisms, anti-CD20 mAbs are classified into Type I(CDC and ADCC) and Type II (programmed cell death[PCD] and ADCC) mAbs.(12)FIG. 2. Determination of the C20Mab-60 epitope for CD20 by ELISA using point mutants. (A) Synthesized peptides ofCD20 were immobilized on immunoplates. The plates were incubated with C20Mab-60 (1 mg/mL), followed by peroxidaseconjugated anti-mouse immunoglobulins. (B) Schematic illustration of CD20 and the C20Mab-60 epitope. The C20Mab-60epitope of CD20 involves Asn171, Pro172, Ser173, and Glu174.

EPITOPE MAPPING OF ANTI-CD20 MAB

Type I mAbs, including rituximab and ofatumumab, relocate CD20 into lipid rafts and efficiently activate thecomplement system.(25) Both Types I and II mAbs possess
ADCC. Type II mAbs, including obinutuzumab and tositumomab, induce PCD through a caspase-independent pathway.(26) Therefore, elucidation of the C20Mab-60 action
to target is also thought to be important. Furthermore, identification of the complementarity-determining regions ofC20Mab-60 is essential to understand the recognition toCD20 and future applications including chimeric antigen
receptor T cells technology.
Author Disclosure Statement

No competing financial interests exist.

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Funding Information

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED) under Grant
Nos. JP21am0401013 and JP21am0101078 (to Y.K.) and by
the Japan Society for the Promotion of Science ( JSPS)
Grants-in-Aid for Scientific Research (KAKENHI) Grant
Nos. 21K15523 (to T.A.), 21K07168 (to M.K.K.), and19K07705 (to Y.K.).
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Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: September 13, 2021
Accepted: November 17, 2021