MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 4, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0023

Development of a Novel Epitope Mapping System:RIEDL Insertion for Epitope Mapping Method

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Teizo Asano,1 Mika K. Kaneko,1 and Yukinari Kato1,2,i

To clarify the binding region of monoclonal antibodies (mAbs) to target molecules, it is very essential tounderstand the pharmacological function of each mAb. Although deletion mutants and point mutants areusefully utilized for epitope mapping, we often experience the difficulty of determining the mAb epitope against
membrane proteins. We aimed to develop a novel method to determine the binding region of mAbs using
epitope tag system. We first checked the reactivity of an anti-CD44 mAb (C44Mab-5) to several deletionmutants of CD44. We then employed the RIEDL tag system (‘‘RIEDL’’ peptide and LpMab-7 mAb). We
inserted the ‘‘RIEDL’’ peptide into the CD44 protein from the 21st to 41st amino acid (AA). The transfectants
produced were stained by LpMab-7 and C44Mab-5 in flow cytometry. C44Mab-5 did not react with 30th–361st
AA of the deletion mutant of CD44. Furthermore, the reaction of C44Mab-5 to RIEDL tag-inserted CD44 from25th to 36th AA was lost, although LpMab-7 detected most of the RIEDL tag-inserted CD44 from 21st to 41st
AA. The epitope of C44Mab-5 for CD44 was determined to be the peptide from 25th to 36th AA of CD44 using
RIEDL insertion for epitope mapping (REMAP) method. The REMAP method might be useful for determining
the critical epitope of functional mAbs against many target molecules.
Keywords: CD44, C44Mab-5, epitope mapping, monoclonal antibody, RIEDL tag

IntroductionAn epitope is a part of an antigen, which is recognized by antibodies. In general, the epitope for monoclonal antibodies (mAbs) consists of several amino acids(AAs); therefore, mAbs often might cross-react with unexpected proteins. Identification of the mAbs epitope is important to avoid unexpected cross-reactivity and is helpful forthe development of antibody drug.
Several epitope-determining methods have been reported,(1) including X-ray cocrystallography,(2) array-basedoligopeptide scanning,(3) and site-directed mutagenesis
mapping.(4,5) Although X-ray cocrystallography can clearly
determine the epitope because this method allows direct visualization of the interaction between the antigen and antibody, the crystallization of the antigen–antibody complex
usually takes time and costs a lot. In contrast, the array-basedoligopeptide scanning and site-directed mutagenesis mapping can easily determine linear epitopes, but is not appropriate for determining conformational epitopes. Therefore,
development of simple and efficient methods for determining
conformational epitopes is desired.

We developed a mAb against human CD44 (clone
C44Mab-5) by immunizing mice with CD44-overexpressed
cells. However, the epitope for C44Mab-5 has not been
determined using array-based oligopeptide scanning or sitedirected mutagenesis mapping. CD44 is related to development, metastasis, and invasion of tumor(6–9); therefore,
CD44 has been studied as a therapeutic target and biomarker
in several cancers.(10) C44Mab-5 is applicable for flow cytometry and immunohistochemistry.(11) C44Mab-5 can recognize not only CD44s, but also CD44 variants.
Recently, we converted the mouse IgG1 subclass antibody
C44Mab-5 into an IgG2a subclass antibody and further produced a defucosylated version (5-mG2a-f). The 5-mG2a-f
showed antibody-dependent cellular cytotoxicity (ADCC)
and complement-dependent cytotoxicity (CDC) against
oral squamous cell carcinoma.(12) Therefore, 5-mG2a-f may
be a useful therapy for patients with CD44-expressing oral
cancer, and determination of the epitope for C44Mab-5 isdesired.
In this study, we aimed to develop a novel epitope mapping method that is a simple and an efficient method for determining the conformational epitopes. Then, we tried to1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
ORCID ID (https://orcid.org/0000-0001-5385-8201).

2
i

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REMAP METHOD

determine the epitope region for C44Mab-5 on CD44 using thenovel epitope mapping system: RIEDL insertion for epitope
mapping (REMAP) method using RIEDL tag system.(13)Materials and Methods

163

phosphate-buffered saline, cells were treated with primarymAbs (1 lg/mL) for 30 minutes at 4C and subsequently
with Alexa Fluor 488-conjugated antimouse IgG (1:1000;
Cell Signaling Technology, Inc., Danvers, MA). Fluorescence data were collected using EC800 Cell Analyzer (SonyCorp.).

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Plasmid preparation

CD44s open reading frame (ORF) was amplified from
LN229 cDNA using HotStar HiFidelity Polymerase Kit(Qiagen, Inc., Hilden, Germany). CD44v3-10 ORF wasprovided by RIKEN BRC through the National Bio-Resource
Project of MEXT, Japan. CD44s ORF and CD44v3-10 ORF
without original signal sequence (1st–20th AAs) were subcloned into pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and a IL-2 signal sequence(MYRMQLLSCIALSLALVTNS) and PA16 tag (GLEGGVAMPGAEDDVV),(14,15) which is recognized by an antiPA16 tag mAb (NZ-1),(16) were added at the N-terminal. The
deletion mutants of CD44s or CD44v3-10 were performed
using HotStar HiFidelity Polymerase Kit with oligonucleotides containing the desired mutations. Substitutions of an
AA to alanine in CD44s sequence were conducted by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent
Technologies, Inc., Santa Clara, CA). Insertions of RIEDL
tag in CD44s sequence were performed using HotStar HiFidelity Polymerase Kit with oligonucleotides containing
RIEDL tag insertions at the desired position. For example, we
produced Gln21_RIEDL_Ile22 (Q21_R*_I22) by inserting
the RIEDL sequence between Gln21 and Ile22 of CD44s.
Polymerase chain reaction fragments bearing the desired
mutations were inserted into pCAG-Ble vector using InFusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan). The
RIEDL tag insertion mutants produced are Q21_R*_I22,
I22_R*_D23, D23_R*_L24, L24_R*_N25, N25_R*_I26,
I26_R*_T27, T27_R*_C28, C28_R*_R29, R29_R*_F30,
F30_R*_A31, A31_R*_G32, G32_R*_V33, V33_R*_F34,
F34_R*_H35, H35_R*_V36, V36_R*_E37, E37_R*_K38,
K38_R*_N39, N39_R*_G40, and G40_R*_R41.

ResultDetermination of C44Mab-5 epitope using deletionmutants of CD44s

The epitope of C44Mab-5 might exist at the extracellularregion of CD44s because C44Mab-5 was developed using
Cell-Based Immunization and Screening method.(17) First,
we produced N-terminal deletion mutants (dN30, dN40,
dN50, dN60, dN70, and dN79) with PA16 tag at theirN-terminal and investigated the reactivity between C44Mab-5
and each deletion mutant by flow cytometry analysis. The
results showed that C44Mab-5 recognized only CD44s andCD44v3-10, but did not recognize all the deletion mutants(dN30, dN40, dN50, dN60, dN70, and dN79) (Fig. 1A). In
contrast, all deletion mutants were detected by an anti-PA16
tag mAb, NZ-1 (Fig. 1B). These results show that the Nterminus of the C44Mab-5 epitope exists between 21st and29th AAs of CD44s (Fig. 1C).
Determination of C44Mab-5 epitope using point
mutants of CD44s

Next, we produced constructs of 20 point mutants within
CD44s (Q21A, I22A, D23A, L24A, N25A, I26A, T27A,
C28A, R29A, F30A, A31G, G32A, V33A, F34A, H35A,
V36A, E37A, K38A, N39A, and G40A) and performed flow
cytometry analysis using transient transfectants. All CD44s
point mutants were recognized not only by NZ-1, but also by
C44Mab-5 (Supplementary Fig. S1). These results indicatethat the epitope of C44Mab-5 will be a conformational epitope, since we could not determine the crucial epitope byalanine scanning.

Cell lines

Chinese hamster ovary (CHO)-K1 was obtained from theAmerica Type Culture Collection (ATCC, Manassas, VA).
CD44 mutation plasmids were transfected into CHO-K1 cells
using Neon Transfection System (Thermo Fisher Scientific,
Inc., Waltham, MA) and stable transfectants were sorted by
PA16 tag using a cell sorter (SH800; Sony Corp., Tokyo,
Japan). The CHO-K1 cells and transfectants were cultured in
RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan)
supplemented with 10% heat-inactivated fetal bovine serum(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 stable transfectants were cultivatedin a medium containing 0.5 mg/mL Zeocin (InvivoGen, San
Diego, CA) for selection.
Flow cytometry

Cells were harvested by brief exposure to 0.25% trypsin/
1 mM ethylenediaminetetraacetic acid (Nacalai Tesque,Inc.). After washing with 0.1% bovine serum albumin in

Determination of C44Mab-5 epitope
using REMAP method

To further investigate the critical AAs of conformational
epitope of C44Mab-5, we conducted a tag insertion scanning,which is named as REMAP method. In this method, we used
a RIEDL tag system.(13) RIEDL tag system consists of fiveAAs peptide (RIEDL tag) and an anti-RIEDL tag mAb (clone
LpMab-7). C44Mab-5 can bind to CD44s when RIEDL tag isinserted into any region, which is independent of the
C44Mab-5 epitope (Fig. 2A, upper panel). In contrast, CD44s
will not be detected by C44Mab-5 when the conformation of
the C44Mab-5 epitope is disrupted by RIEDL insertion(Fig. 2A, lower panel). We constructed 20 mutants of CD44s,
in which RIEDL tag was inserted into the expected epitope
region within CD44s (Fig. 2B). Results of the flow cytometry
analysis showed that C44Mab-5 did not detect 11 mutants(N25_R*_I26, I26_R*_T27, T27_R*_C28, C28_R*_R29,
R29_R*_F30, F30_R*_A31, A31_R*_G32, G32_R*_V33,
V33_R*_F34, F34_R*_H35, H35_R*_V36), but weakly
detected L24_R*_N25 (Fig. 3A), indicating that C44Mab-5
binds to CD44s through 12 AAs (from Asn25 to Val36). As a

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164

FIG. 1. Epitope mapping of C44Mab-5 using deletionmutants of CD44s. (A, B) Deletion mutants of CD44s wereanalyzed using flow cytometry. Deletion mutants wereexpressed on CHO-K1 cells and then incubated withC44Mab-5 (A), or anti-PA16 tag antibody (NZ-1) (B) for
30 minutes at 4C, followed by treatment with corresponding secondary antibodies. (C) Schematic illustrationof epitope mapping of CD44s, CD44v3-10, and six deletion mutants of CD44s with PA16 tag at N-termini. Deletion mutants of CD44s: dN30, dN40, dN50, dN60, dN70,
and dN79. Black bar: the positive reaction of C44Mab-5.
White bar: the negative reaction of C44Mab-5. PA16: PA16
tag. CHO, Chinese hamster ovary.

positive control, NZ-1 against N-terminal PA16 tag detected
20 mutants (Fig. 3B). LpMab-7 detected most of the mutants,
but did not detect E37_R*_K38, K38_R*_N39 (Fig. 3C),
thereby suggesting that these AAs might be hidden inside theprotein. Since RIEDL tag was not inserted in wild typeCD44s, LpMab-7 did not react with CHO/CD44s (Fig. 3C).
These results are summarized in Figure 4.

ASANO ET AL.

FIG. 2. Schematic illustration of REMAP method. RIEDL
tag is inserted into CD44s. (A) C44Mab-5 can bind to CD44s
when RIEDL tag is inserted into any region, which is independent of the C44Mab-5 epitope (upper panel). In contrast, CD44s will not be detected by C44Mab-5 when theconformation of the C44Mab-5 epitope is disrupted by
RIEDL insertion (lower panel). (B) RIEDL tag was inserted
into the expected epitope region within CD44s. REMAP,RIEDL insertion for epitope mapping.

Discussion

To investigate the epitope of mAbs, alanine-scanning mutagenesis and peptide screening are often performed.(1,11,18–30)
These methods are very useful for determining the linear
epitope; however, we could not determine the conformational epitope by these methods. For investigating the epitopeof C44Mab-5 in this study, N-terminal deletion mutant analyses showed that the N-terminus of the epitope of C44Mab-5
is located between Gln21 and Arg29 (Fig. 1). Next, weconstructed the point mutants of CD44s that were substituted
for alanine around the predicted epitope region. However, theresult of flow cytometry analysis showed that all point mutants were detected by C44Mab-5 (Supplementary Fig. S1);
therefore, we could not determine the epitope by alaninescanning mutagenesis. This is because the epitope of
C44Mab-5 will be a conformational epitope and substitutionof one AA may not disrupt the conformation of the epitope
region enough to inhibit the binding of C44Mab-5. To determine the critical AAs of the epitope of C44Mab-5, weDownloaded by Yukinari Kato from www.liebertpub.com at 08/23/21. For personal use only.

REMAP METHOD

165

FIG. 4. Schematic illustration of epitope mapping of
C44Mab-5. (A) Schematic illustration of C44Mab-5 and NZ1. Asn25, Ile26, Thr27, Cys28, Arg29, Phe30, Ala31, Gly32,
Val33, Phe34, His35, and Val36 are important for thebinding of C44Mab-5 to CD44s. (B) Schematic illustrationof CD44s and epitope of C44Mab-5. CD44s possesses hyaluronic acid-binding sites in the extracellular region and
ERM binding site and ankyrin binding site in the intracellular region. C44Mab-5 epitope is located near the
N-terminus of CD44s. SS, signal sequence; TM, transmembrane domain.

FIG. 3. Epitope mapping of C44Mab-5 using RIEDL tag
insertion mutants of CD44s. RIEDL tag was inserted into
CD44s and RIEDL tag insertion mutants were analyzed
using flow cytometry. RIEDL tag insertion mutants wereexpressed on CHO-K1 cells and incubated with(A) C44Mab-5, (B) anti-PA16 tag antibody (NZ-1), or(C) anti-RIEDL tag antibody (LpMab-7) for 30 minutes at
4C, followed by treatment with corresponding secondary
antibodies.

beta sheet(31) and this conformation will be important forthe binding of C44Mab-5 to CD44s. The crystal structuresof the extracellular hyaluronic acid (HA)-binding domainof human CD44 show that the binding epitope of C44Mab-5
is located near the HA-binding site(31,32); therefore, thebinding of C44Mab-5 to CD44s may affect the binding of
HA to CD44s.
In this study, we showed that REMAP method is useful forthe conformational epitope mapping of CD44s. RIEDL tag
consists of only five AAs and is detected by anti-RIEDL tag
mAb, LpMab-7. As shown in Figure 3C, most of the RIEDL
tag insertion mutants were detected by LpMab-7; however,E37_R*_K38 and K38_R*_N39 were not detected. The
peptide bonds between Glu37 and Lys38 and between Lys38
and Asn39 are not exposed on the surface of CD44s.(31) Since
the inserted RIEDL tags might be buried in protein structure,
RIEDL tags were not detected by LpMab-7 in those mutants.
CD44 has been studied as a therapeutic target in several
cancers and we recently developed 5-mG2a-f, which showed
ADCC and CDC activities against oral squamous cell carcinoma.(12) The epitope identification of C44Mab-5 will be
helpful for the development of antibody drug that targets
CD44s in a future study.
Author Disclosure Statement

No competing financial interests exist.
employed REMAP method (Fig. 2A). Results of the flow
cytometry analysis showed that some RIEDL tag insertion
mutants lost their reactivity to C44Mab-5 (Fig. 3A). This result suggests that insertion of RIEDL tag caused partial disruption of CD44s conformation and inhibited the bindingof C44Mab-5 to CD44s. Using REMAP method, we successfully determined that the critical epitope of C44Mab-5 islocated from Asn25 to Val36 (Fig. 4). These AAs are continuous, but may not produce a linear structure. They form aFunding Information

This research was supported, in part, by Japan Agency for
Medical Research and Development under Grant Nos.
JP21am0401013 (to Y.K.) and JP21am0101078 (to 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.), and19K07705 (to Y.K.).

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Supplementary Material

Supplementary Figure S1

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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, Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: May 14, 2021
Accepted: June 14, 2021