MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 6, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0035
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Epitope Mapping of an Anti-CD20 Monoclonal Antibody(C20Mab-60) Using the HisMAP Method

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

CD20 is one of the B-lymphocyte antigens and an effective target for the detection and treatment of B cell lymphomas; specific and sensitive monoclonal antibodies (mAbs) are required thus for their diagnosis. Recently, wedeveloped a novel anti-CD20 mAb (clone C20Mab-60), which is not only useful for flow cytometry but also forWestern blot and immunohistochemical analyses. However, the epitope of C20Mab-60 has not been determined. To
clarify the binding region of mAbs against their target molecules, it is essential to understand the pharmacological
function of each mAb. In this study, we aimed to identify the epitope of C20Mab-60 for CD20 using the novel
histidine tag (His-tag) insertion for epitope mapping (HisMAP) method. We first established an anti-His-tag mAb,
HisMab-1 (mouse IgG2b, kappa), by immunizing mice with recombinant proteins containing an N-terminal His-tag.
Although HisMab-1 detected the 4x, 5x, and 6xHis tag-inserted CD20 proteins using flow cytometry, 5xHis tag wasselected. While HisMab-1 recognized all the 5xHis tag-inserted CD20 from the 142nd to the 183rd amino acid (aa),C20Mab-60 did not react with the 5xHis tag-inserted CD20 from the 171st to the 174th aa. These results indicatethat the main epitope of C20Mab-60 for CD20 is a peptide from 171st to 174th aa of CD20. HisMAP method could
be advantageous in the determination of the critical epitope of functional mAbs against many target molecules.
Keywords: CD20, C20Mab-60, epitope mapping, monoclonal antibody, His tag
Introduction

C

D20 is a transmembrane protein containing four transmembrane domains, and consists of 297 amino acids(aa).(1) CD20 is expressed on B cells from pre-B to mature B
cell development, and is also detected in many types of nonHodgkin lymphoma,(2–4) implicated in B cell activation,differentiation, and regulation of calcium influx.(5,6) CD20 isexpressed in 50% of B lymphoblastic leukemia/lymphoma
originating from pre-B cells, but it is not detected in terminally differentiated plasma cell malignancies.(7,8) CD20 has
been studied as a therapeutic target of B cell lymphomas andautoimmune disorders.(9–11)
Specific and sensitive monoclonal antibodies (mAbs) are
critical for the diagnosis of many types of cancer. Using the
Cell-Based Immunization and Screening method, we havesuccessfully produced a specific and sensitive anti-CD20
mAb (clone C20Mab-60) that can be used not only for flow

cytometry but also for Western blot and immunohistochemical analyses.(12) The epitope of C20Mab-60 has not been,
however, elucidated.
In general, the epitope consists of several amino acids;
mAbs often might therefore cross-react with other proteinsbesides their epitope. Determination of the epitope is important to avoid unexpected cross-reactivity and is crucial in thedevelopment of antibody drugs. Several methods have beenpreviously reported for the characterization of epitopes, including X-ray cocrystallography, array-based oligo-peptide
scanning, and site-directed mutagenesis mapping.(13)The array-based oligopeptide scanning and site-directedmutagenesis mapping are useful for determining linear epitopes, but are not suitable for determining conformational
epitopes. In contrast, X-ray cocrystallography can accurately
determine epitopes as direct visualization of the interaction
between the antigen and antibody can be achieved; however,the crystallization of the antigen–antibody complex usuallyDepartments 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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takes a lot of time and is expensive. The development ofsimple and efficient methods for determining conformational
epitopes would thus be crucial.
We previously developed a novel epitope mapping method
named as the pentapeptide Arg79, Ile80, Asp81, Glu82, andLeu83 (RIEDL) insertion for epitope mapping (REMAP)(14,15)
using a RIEDL tag system.(16) REMAP method is a simple
and an efficient method for linear and conformational epitopes. In this study, we developed a histidine tag (His-tag)
insertion for epitope mapping (HisMAP) method, aiming todetermine the epitope region of C20Mab-60 for CD20.

Corp., Tokyo, Japan). CHO-K1 cells and transfectants were
cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto,
Japan) supplemented with 10% heat-inactivated fetal bovine
serum (FBS) (Thermo Fisher Scientific, Inc.), 100 U/mL of
penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified
atmosphere containing 5% CO2. The transfectants were cultivated in a medium containing 0.5 mg/mL Zeocin (InvivoGen, San Diego, CA).

Materials and Methods

Female BALB/c mice (6 weeks old) were purchased from
CLEA Japan (Tokyo, Japan). The animals were housed underpathogen-free conditions. All animal experimentation procedures were approved by the Animal Care and Use Committeeof Tohoku University. Two mice were immunized with
N-terminal His-tagged SARS-CoV-2 spike protein (S2 subunit) (cat. no. 230-01103; RayBiotech Life, Inc., Norcross,GA) and 50 lg/mouse of using Imject Alum (Thermo Fisher
Scientific, Inc.) to develop an anti-His-tag mAb, although our
initial goal was to develop the anti-S2 spike protein of SARSCoV-2 (www.med-tohoku-antibody.com/topics/001_paper_
antibody_PDIS.htm#SARS-CoV-2). The procedure included three additional immunizations with SARS-CoV-2
spike protein (50 lg/mouse), followed by a final booster injection of SARS-CoV-2 spike protein (50 lg/mouse) 2 days
before harvesting splenic cells. Splenocytes were subsequently fused with P3U1 cells using polyethylene glycol
1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas
were then grown in RPMI-1640 supplemented with 10%
FBS, 100 U/mL of penicillin, 100 lg/mL streptomycin, and0.25 lg/mL amphotericin B, 5 lg/mL plasmocin (InvivoGen), and hypoxanthine/aminopterin/thymidine (Thermo
Fisher Scientific, Inc.). N-terminal His-tag-positive wells wereselected by Enzyme-Linked ImmunoSorbent Assay. After
limiting dilution, HisMab-1 (IgG2b, kappa), which can detect
His-tag of N-terminus and C-terminus, was established.

Plasmid preparation

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ASANO ET AL.

DNA encoding the CD20 gene (IRAL012D02) was provided by RIKEN BRC through the National BioResource
Project of MEXT, Japan. The open reading frame of CD20
was subcloned into a pCAG-Ble vector (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan). Insertions of
4xHis-tag, 5xHis-tag, and 6xHis-tag (4xH*, 5xH*, and 6xH*)
between Pro169 and Ala170 of CD20 sequence were performed using the HotStar HiFidelity Polymerase Kit (Qiagen,Inc., Hilden, Germany) with oligonucleotides containing
His-tag insertions, resulting in the production of CD20–
P169_4xH*_A170, CD20–P169_5xH*_A170, and CD20–
P169_6xH*_A170 plasmids.
Insertions of 5xH* in the extracellular region of CD20
were performed using the HotStar HiFidelity Polymerase Kit
with oligonucleotides containing 5xH* insertions at the selected position. Lys142_5xH*_Ile143 (K142_5xH*_I143)
was produced for instance by inserting the 5-histidine sequence between Lys142 and Ile143 of CD20. Polymerase
chain reaction fragments bearing the desired mutations wereinserted into the pCAG-Ble vector using the In-Fusion HD
Cloning Kit (Takara Bio, Inc., Shiga, Japan). The 5xH*
insertion mutants produced were the following: K142_
5xH*_I143, I143_5xH*_S144, S144_5xH*_H145, H145_
5xH*_F146,
F146_5xH*_L147,
L147_5xH*_K148,
K148_5xH*_M149, M149_5xH*_E150, E150_5xH*_S151,
S151_5xH*_L152, L152_5xH*_N153, N153_5xH*_F154,
F154_5xH*_I155, I155_5xH*_R156, R156_5xH*_A157,
A157_5xH*_H158, H158_5xH*_T159, T159_5xH*_P160,
P160_5xH*_Y161, Y161_5xH*_I162, I162_5xH*_N163,
N163_5xH*_I164, I164_5xH*_Y165, Y165_5xH*_N166,
N166_5xH*_C167, C167_5xH*_E168, E168_5xH*_P169,
P169_5xH*_A170, A170_5xH*_N171, N171_5xH*_P172,
P172_5xH*_S173, S173_5xH*_E174, E174_5xH*_K175,
K175_5xH*_N176, N176_5xH*_S177, S177_5xH*_P178,
P178_5xH*_S179, S179_5xH*_T180, T180_5xH*_Q181,
Q181_5xH*_Y182, and Y182_5xH*_C183.
Cell lines

Chinese hamster ovary (CHO)-K1 and P3U1 cells wereobtained from the America Type Culture Collection (ATCC,
Manassas, VA). CHO/CD20 was produced in our previous
study.(17) CD20 mutation plasmids were transfected into
CHO-K1 cells using Lipofectamine LTX with Plus Reagent(Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants of CHO/CD20–P169_4xH*_A170, CHO/CD20–
P169_5xH*_A170, and CHO/CD20–P169_6xH*_A170 wereselected using a cell sorter (SH800; Sony Biotechnology

Establishment of an anti-His-tag mAb

Flow cytometry

Cells were harvested after brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque,Inc.). After washing with 0.1% bovine serum albumin in
phosphate-buffered saline, cells were treated with primarymAbs (1 lg/mL) for 30 minutes at 4C and subsequently with
Alexa Fluor 488-conjugated anti-mouse IgG (1:1000; Cell
Signaling Technology, Inc., Danvers, MA). Fluorescencedata were collected using a BD FACSLyric (Becton, Dickinson and Company, Franklin Lakes, NJ) for characterizationof HisMab-1, or the EC800 Cell Analyzer (Sony Biotechnology Corp.) for HisMAP analyses.
Results
Characterization of HisMab-1 using flow
cytometry analysis

Flow cytometry was performed using HisMab-1 against
CHO/CD20–P169_4xH*_A170, CHO/CD20–P169_5xH*_
A170, and CHO/CD20–P169_6xH*_A170. HisMab-1
recognized CHO/CD20–P169_4xH*_A170, CHO/CD20–
P169_5xH*_A170, and CHO/CD20–P169_6xH*_A170
cells, but not parental CHO-K1 cells and CHO/CD20 cells

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FIG. 1. Characterization of HisMab-1 using poly-histidine-inserted CD20. 4x, 5x, and 6xHis-inserted CD20 were expressedon CHO-K1 cells and then incubated with HisMab-1 (A) or anti-CD20 mAb (C20Mab-11) (B) for 30 minutes at 4C, followed
by treatment with a secondary antibody. (C) C20Mab-60 can bind to CD20 when 5xHis is inserted into any region, which is
independent of the C20Mab-60 epitope (upper panel). In contrast, C20Mab-60 will not bind to CD20 when the conformation of
the C20Mab-60 epitope is disrupted by 5xHis insertion (lower panel). mAb, monoclonal antibody. H*, histidine.

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Determination of C20Mab-60 epitope using
HisMAP method

To investigate the C20Mab-60 epitope, we conducted a
His-tag insertion scanning, known as the HisMAP method. In

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(Fig. 1A). In contrast, an anti-CD20 mAb (clone C20Mab11(17)) that was used as a positive control reacted with allcells, except for parental CHO-K1 cells (Fig. 1B). These
results indicate that HisMab-1 can detect 4xHis-tag, 5xHistag, and 6xHis-tag in flow cytometry analysis.

ASANO ET AL.

FIG. 2. Schematic illustration of HisMAP method. 5xHis was inserted into the expected epitope region within CD20.
HisMAP, histidine tag insertion for epitope mapping.

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FIG. 3. Epitope mapping of C20Mab-60 using His-tag insertion mutants of CD20. 5xHis-inserted CD20 mutants wereanalyzed using flow cytometry. Each mutant was expressed in CHO-K1 cells and incubated with HisMab-1 (A) or C20Mab60 (B) for 30 minutes at 4C, followed by treatment with a secondary antibody. Red lines: treated with HisMab-1 or
C20Mab-60; black lines: no primary antibodies, used as negative controls. CHO-K1, Chinese hamster ovary-K1.

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this method, we utilized a 5xHis-tag (5xH*) and an anti-Histag mAb (clone HisMab-1). C20Mab-60 can bind to CD20
when 5xH* is inserted in any region, independent of the
C20Mab-60 epitope (Fig. 1C, upper). In contrast, C20Mab-60
did not detect CD20 when 5xH* is inserted into the C20Mab60 epitope region, while the conformation of the C20Mab-60
epitope is disrupted by 5xH* insertion (Fig. 1C, lower).
We then constructed 41 mutants of CD20, in which 5xH*
was inserted into the second extracellular loop region of
CD20 (Fig. 2). Mutant proteins were transiently expressed on
CHO-K1 cells, and analyzed using flow cytometry analysis.
Results of the flow cytometry analysis showed that HisMab-1
used as a positive control detected all mutants (Fig. 3A). This
result shows that all mutant proteins were expressed on CHOK1 cells. Since 5xH* was not inserted in wild-type CD20,
HisMab-1 did not react with CHO/CD20 (Fig. 3A). In contrast, C20Mab-60 did not detect the three mutants (CHO/
N171_5xH*_P172, CHO/P172_5xH*_S173, and CHO/S173_
5xH*_E174) (Fig. 3B), indicating that the main epitope of
C20Mab-60 contains four amino acids (Asn171, Pro172,
Ser173, and Glu174). These results are summarized in Figure 4.

ASANO ET AL.

to C20Mab-60 (Fig. 3B). This result suggests that insertionof 5xH* causes partial disruption of the CD20 conformationand inhibits binding of C20Mab-60 to CD20.
CD20 has two extracellular regions. The first loop between
helix one and helix two is relatively small, and it is unlikely
to protrude extensively, while the second loop between helix
three and helix four consists of more than 40 amino acids.(34,35) We hypothesized thus that the epitope of C20Mab60 might be located at the second extracellular loop regionof CD20. Using HisMAP method, we successfully determined that the critical epitope of C20Mab-60 is located in
Asn171, Pro172, Ser173, and Glu174 in the second extracellular loop region of CD20 (Fig. 4).
This work demonstrated that HisMAP method is useful aswell as straightforward for epitope mapping of CD20. The
epitope identification of C20Mab-60 will be critical for thedevelopment of an antibody drug that targets CD20 in futurestudies. Interestingly, there are a number of mAbs for which
the epitope has not been identified and could be characterizedusing the HisMAP method.
Author Disclosure Statement

Discussion

To identify the epitope of mAbs, it is important to avoid
unexpected cross-reactivity, while it is essential to understand the pharmacological function of mAbs. Alaninescanning mutagenesis and peptide screening are commonlyused in the process of investigating new epitopes.(13,18–31)
Although these methods are very useful in determining a linear
epitope, a conformational epitope could not be determined bythese methods. X-ray cocrystallography can accurately determine the epitope. Crystallization of the antigen–antibody
complex, however, is costly and takes a lot of time. Development of a simple and efficient method for the determination of conformational epitopes would be thus essential.
Recently, we developed a novel epitope mapping method
named REMAP method(14,15) using RIEDL tag system.(16)
REMAP method is useful in determining conformational aswell as linear epitopes.(14,15) In this study, we developed anovel epitope mapping system, named as HisMAP method.
In the HisMAP method, we employed a His-tag, which iscommonly used for protein purification and detection as
an alternative of RIEDL tag.(32,33) Flow cytometry analysis
showed that some 5xH* insertion mutants lose their reactivity

FIG. 4. Schematic illustration of epitope mapping of
C20Mab-60. Four amino acids (Asn171, Pro172, Ser173, andGlu174) are important for binding of C20Mab-60 to CD20.

No competing financial interests exist.
Funding Information

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED) under grant nos.
JP21am0401013 (Y.K.) and JP21am0101078 (Y.K.), and by
the Japan Society for the Promotion of Science Grants-in-Aid
for Scientific Research (KAKENHI) grant nos. 21K15523(to T.A.), 21K07168 (to M.K.K.), and 19K07705 (to Y.K.).
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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: August 5, 2021
Accepted: September 24, 2021