MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 1, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0051
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Epitope Mapping of the Anti-CD20 Monoclonal Antibodies(C20Mab-11 and 2H7) Using HisMAP Method

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Hiroyuki Suzuki,1 Teizo Asano,2 Tomohiro Tanaka,2 Mika K. Kaneko,2 and Yukinari Kato1,2

CD20, which is expressed on B lymphocytes, has been studied as a therapeutic target for B cell lymphomas
and autoimmune disorders. Identifying the binding epitopes of monoclonal antibodies (mAbs) can contributeto our understanding of their functions. We have previously developed an anti-CD20 mAb (clone C20Mab11) using a Cell-Based Immunization and Screening (CBIS) method. In this study, we aimed to determine
the binding epitopes of anti-CD20 mAbs, such as C20Mab-11 and 2H7, using the His-tag insertion for
epitope mapping (HisMAP). The results showed that 171-NPSE-174 and 168-EPANPSE-174 in the second loop of
CD20 were essential for C20Mab-11 binding and 2H7 binding, respectively. Although we developed many mAbs
that recognize conformational epitopes using the CBIS method, there are many difficulties in epitope mapping
for these mAbs. HisMAP could be useful for determining the conformational epitopes of other mAbs against
membrane proteins.
Keywords: CD20, epitope mapping, monoclonal antibody, His tag

these mAbs, including their specific binding epitopes, affinities of target binding, and particular conformations, areimportant to understand their molecular activity. Epitope
identification is particularly important for avoiding unexpected cross-reactivity and understanding the overall pharmacological function of mAbs. Nevertheless, determining theconformational epitopes of mAbs is challenging.
In our previous study, we established an anti-human CD20
mAb, C20Mab-11 (mouse IgM, kappa), by using a Cell-Based
Immunization and Screening (CBIS) method. C20Mab11 is useful not only for flow cytometry but also for Western
blotting and immunohistochemical analyses.(15) In this study,
we aimed to determine the binding epitopes of C20Mab-11,
developed by the CBIS method, and comparing it with acommercially available anti-human CD20 mAb (clone 2H7),
using a His-tag insertion for epitope mapping (HisMAP).

Introduction

C

D20, a glycosylated transmembrane protein with
two small and large extracellular loops (*7 and 44
amino acids), four transmembrane domains, and intracellular N- and C-terminal regions,(1,2) is expressed on normal B
cells from pre-B to mature B cells, but not on pro-B cells
and plasma cells.(3) CD20 is also detected in human B cellmalignancies, including non-Hodgkin’s lymphoma(4) and B
lymphoblastic leukemia.(5) Since CD20 is not expressed onearly pro-B cells, anti-CD20 monoclonal antibodies (mAbs),
including rituximab, ofatumumab, and obinutuzumab, candestroy B cell malignancies through complement-dependent
cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) without affecting the early lineage of normal B
cells.(6–8)
Rituximab also affects CD20-expressing normal B cells
and is approved for autoimmune disorders, including rheumatoid arthritis,(9,10) granulomatosis with polyangiitis,(11,12)
and microscopic polyangiitis.(13,14) These mAbs have dramatically improved the prognosis of patients with CD20positive malignancies and autoimmune diseases in the past
two decades. Therefore, analyses of the specific nature ofMaterials and Methods
Cell lines and antibodies

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

Departments of 1Molecular Pharmacology and 2Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai,
Japan.

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EPITOPE MAPPING OF ANTI-CD20 MAbs

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FIG. 1. Schematic illustration of HisMAP. The 5 · H* isinserted into CD20. (A) C20Mab-11 could bind to CD20
when 5 · H* was inserted into any region, which was independent of the C20Mab-11 epitope. (B) C20Mab-11 did
not bind to CD20 when the conformation of the C20Mab-11
epitope was disrupted by the 5 · H* tag insertion. HisMAP,
His-tag insertion for epitope mapping.
study.(15,16) CD20 mutation plasmids were transfected into
CHO-K1 cells using Lipofectamine LTX with Plus Reagent(Thermo Fisher Scientific, Inc., Waltham, MA). 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).
Anti-CD20 mAb (clone 2H7) was purchased from BioLegend (San Diego, CA).
Establishment of an anti-His-tag mAb

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 sub-FIG. 2. The 5 · H* tag was inserted into the large extracellular region of CD20.
unit) (Cat. No. 230-01103; RayBiotech Life, Inc., Norcross,GA), 50 lg per 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
SARS-CoV-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 per mouse), followed
by a final booster injection of SARS-CoV-2 spike protein(50 lg per 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/mLof streptomycin, and 0.25 lg/mL of amphotericin B, 5 lg/mLof plasmocin (InvivoGen), and hypoxanthine/aminopterin/
thymidine (HAT; Thermo Fisher Scientific, Inc.). N-terminal‰
FIG. 3. Epitope mapping of C20Mab-11 using 5 · H* insertion mutants of CD20. The 5 · H* inserted into CD20 and its
mutants were analyzed using flow cytometry. Each mutant was expressed on CHO-K1 cells and incubated with HisMab-1(A) or C20Mab-11 (B) for 30 minutes at 4C, followed by treatment with a secondary antibody. Red lines: treated with
HisMab-1 or C20Mab-11, black lines: without first antibodies as negative controls. CHO, Chinese hamster ovary.

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EPITOPE MAPPING OF ANTI-CD20 MAbs

His-tag-positive wells were selected by enzyme-linked
immunosorbent assay. After limiting dilution, HisMab-1(mouse IgG2b, kappa), which can detect His-tag ofN-terminus and C-terminus, was established.
Establishment of a recombinant anti-His-tag mAb

To produce recombinant HisMab-1, we subcloned VH and
CH of cDNAs of HisMab-1 into the pCAG-Neo vector, alongwith VL and CL cDNAs of HisMab-1 into the pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan), respectively. Vectors of HisMab-1 were transfected
into ExpiCHO cells using the ExpiCHO Expression System (Thermo Fisher Scientific, Inc.). The resulting mAb(recHisMab-1) was purified using Ab-Capcher (ProteNova,
Kagawa, Japan).

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Plasmid preparation

DNA encoding the CD20 gene (IRAL012D02) was provided by RIKEN BRC through the National BioResource
Project of MEXT, Japan.(15,16) The open reading frame ofCD20 was subcloned into a pCAG-Ble vector (FUJIFILM
Wako Pure Chemical Corporation). Insertions of 5-histidinetag (5 · H*) in the second loop of CD20 were performed
using the HotStar HiFidelity Polymerase Kit with oligonucleotides containing 5 · H* insertions at the selected position.
Lys142_5 · H*_Ile143 (K142_5 · H*_I143) was produced
for instance by inserting the 5-histidine tag between Lys142
and Ile143 of CD20. Polymerase chain reaction fragments
bearing the desired mutations were inserted into the pCAGBle vector using the In-Fusion HD Cloning Kit (Takara Bio,Inc., Shiga, Japan).
The 5 · H* insertion mutants produced were the following:
K142_5 · H*_I143, I143_5 · H*_S144, S144_5 · H*_H145,
H145_5 · H*_F146, F146_5 · H*_L147, L147_5 · H*_
K148, K148_5 · H*_M149, M149_5 · H*_E150, E150_5 ·H*_S151, S151_5 · H*_L152, L152_5 · H*_N153, N153_
5 · H*_F154, F154_5 · H*_I155, I155_5 · H*_R156, R156_
5 · H*_A157, A157_5 · H*_H158, H158_5 · H*_T159,
T159_5 · H*_P160, P160_5 · H*_Y161, Y161_5 · H*_I162,
I162_5 · H*_N163,
N163_5 · H*_I164,
I164_5 · H*_
Y165, Y165_5 · H*_N166, N166_5 · H*_C167, C167_5 ·H*_E168, E168_5 · H*_P169, P169_5 · H*_A170, A170_5 ·H*_N171, N171_5 · H*_P172, P172_5 · H*_S173, S173_
5 · H*_E174, E174_5 · H*_K175, K175_5 · H*_N176,
N176_5 · H*_S177, S177_5 · H*_P178, P178_5 · H*_
S179, S179_5 · H*_T180, T180_5 · H*_Q181, Q181_5 ·H*_Y182, and Y182_5 · H*_C183.

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Results
Production of His-tag-inserted CD20 mutants

We conducted HisMAP to investigate the C20Mab-11
epitope. C20Mab-11 can bind to CD20 when 5 · H* is inserted in any region independently on the C20Mab-11 epitope(Fig. 1A). Contrastingly, C20Mab-11 was not able to detect
CD20 when 5 · H* was inserted into the C20Mab-11 epitope
region because the insertion of 5 · H* disrupted the conformation of the C20Mab-11 epitope (Fig. 1B).
After 5 · H* was inserted into the large extracellular loop
region of CD20, 41 mutant plasmids of CD20 were constructed. The mutant plasmids were transfected and transiently expressed on CHO-K1 cells (Fig. 2).
Determination of C20Mab-11 epitopes using HisMAP

Mutant protein expressing CHO-K1 cells were treated with
HisMab-1 (Fig. 3A) or C20Mab-11 (Fig. 3B) and were analyzed using flow cytometry. Flow cytometry showed that
HisMab-1, used as a positive control, was detected in all
mutants (Fig. 3A). Thus, all mutant proteins were expressedon CHO-K1 cells. HisMab-1 did not react with CHO/CD20
because 5 · H* was not inserted into the wild type CD20
(Fig. 3A). Contrarily, C20Mab-11 did not detect the threemutants (CHO/N171_5 · H*_P172, CHO/P172_5 · H*_S173,
and CHO/S173_5 · H*_E174) (Fig. 3B). Thus, four aminoacids (Asn171, Pro172, Ser173, and Glu174) of CD20 wereobserved to be important for C20Mab-11 binding (Fig. 4).
Determination of 2H7 epitope using HisMAP

Mutant protein expressed in CHO-K1 cells was treatedwith 2H7 (Fig. 5A), and analyzed using flow cytometry.
Treatment with 2H7 was not able to detect the six mutants(CHO/E168_5 · H*_P169, CHO/P169_5 · H*_A170, CHO/
A170_5 · H*_N171, CHO/N171_5 · H*_P172, CHO/P172_
5 · H*_S173, and CHO/S173_5 · H*_E174) (Fig. 5A). Thus,
seven amino acids (Glu168, Pro169, Ala170, Asn171,
Pro172, Ser173, and Glu174) of CD20 were observed to beimportant for 2H7 binding (Fig. 5B).
Discussion

In this study, we determined the binding epitope of
C20Mab-11 and 2H7 as 171-NPSE-174 and 168-EPANPSE-174
in the second loop, respectively (Figs. 3–5). The epitopes

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) or EC800 Cell
Analyzer (Sony Biotechnology Corp.) for HisMAP analyses.

FIG. 4. Schematic illustration of epitope mapping of
C20Mab-11. Schematic illustration of C20Mab-11 and its epitope. Four amino acids (Asn171, Pro172, Ser173, and Glu174)
of CD20 were important for the binding of C20Mab-11 to CD20.

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FIG. 5. (A) The 5 · H* was inserted into CD20, its mutants were analyzed using flow cytometry. Each mutant was
expressed on CHO-K1 cells and incubated with 2H7 for 30 minutes at 4C, followed by treatment with a secondary antibody. Red lines: treated with 2H7, black lines: without first antibodies as negative controls. (B) Schematic illustration of
2H7 and its epitope. Seven amino acids (Glu168, Pro169, Ala170, Asn171, Pro172, Ser173, and Glu174) of CD20 are
important for the binding of 2H7 to CD20.

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EPITOPE MAPPING OF ANTI-CD20 MAbsof several anti-CD20 mAbs have been determined.(17–21)
Rituximab recognizes a separated epitope of 170-ANPS-173
and 182-YCYSI-185 in the second loop.(17) 170-ANPSEKN-176
is recognized by ocrelizumab,(18,19) obinutuzumab,(20) andC20Mab-60.(21) Ofatumumab also recognizes a separated epitope of 72-IPAGIYAPI-80, in the first loop, and 148-KMESLNFIRAHT-159, in the second loop.(19) CD20 mAbs are classified into type I (CDC and ADCC) and type II (programmed
cell death and ADCC).(8,22)
Type I mAbs, including rituximab and ofatumumab,
stimulate the CD20 translocation into lipid rafts and potently
activate CDC.(23) Both type I and II mAbs have ADCC. Type
II mAbs, including obinutuzumab and tositumomab, can induce caspase-independent cell death.(24) Since the isotype
of C20Mab-11 is IgM, a class switch to IgG is required toelucidate the functions of C20Mab-11. Furthermore, the identification of the complementarity-determining regions of
C20Mab-11 is essential in understanding the key structuralfeatures of its recognition to CD20.
We have developed two novel epitope mapping methods,
a RIEDL insertion for epitope mapping (REMAP) and
HisMAP, to determine the epitopes of anti-EGFR mAbs
(EMab-51 and EMab-134)(25,26) and an anti-CD44 mAb(C44Mab-46)(27) using the REMAP. The targets are type I
transmembrane proteins and have conformational epitopes
that cannot be determined by conventional methods, including deletion mutant and alanine scanning analyses. On thecontrary, CD20 possesses four transmembrane domains andtwo extracellular loops (Fig. 1).
Previously, we reported that HisMab-1 can detect 4 · H*,
5 · H*, and 6 · H* inserted into a target protein, using flow
cytometry.(21) We further optimized HisMAP and determinedthat 5 · H* insertion was minimally required. 5 · H* was inserted into the large extracellular loop of CD20 (Fig. 2). Although the reactivity of HisMab-1 tended to decrease uponinsertion of 5 · H* close to the transmembrane domain(Fig. 3A), the epitopes of C20Mab-11 and 2H7 were successfully determined. The 5 · H* insertion did not affect the exposure of the extracellular loop of CD20 and, therefore,
HisMAP could be applied to determine the epitopes of proteins with several transmembrane domains. Further studies areneeded to reveal the usefulness of HisMAP for the determination of the conformational epitopes of the other mAbs.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by Japan Agency
for Medical Research and Development (AMED) underGrant Nos. JP21am0401013 (to Y.K.) and JP21am0101078(to Y.K.), and by the Japan Society for the Promotionof Science ( JSPS) Grants-in-Aid for Scientific Research(KAKENHI) Grant Nos. 21K15523 (to T.A.), 21K20789(to T.T.), 21K07168 (to M.K.K.), and 19K07705 (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
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: October 14, 2021
Accepted: November 30, 2021