MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 4, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0012

Epitope Mapping of the Anti-CD44 Monoclonal Antibody(C44Mab-46) Using the REMAP Method

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Teizo Asano,1 Mika K. Kaneko,1 Junko Takei,1 Nami Tateyama,1 and Yukinari Kato1,2,i

CD44 functions as a major hyaluronan receptor on most cell types, with roles in cell adhesion, migration,
proliferation, differentiation, and survival. The CD44 gene comprises 20 exons, with alternative splicing
producing many different isoforms. CD44 variant isoforms exhibit tissue-specific expression patterns and havebeen studied as therapeutic targets for several cancers; therefore, anti-CD44 monoclonal antibodies (mAbs) areuseful for investigating CD44 expression in various cancers. Previously, we established an anti-CD44 mAb,
C44Mab-46 (IgG1, k), by immunizing mice with the CD44v3–10 ectodomain. Although C44Mab-46 recognized
all CD44 isoforms, the binding epitope of C44Mab-46 has not been determined. In this study, we first checked
the reactivity of C44Mab-46 to several CD44v3–10 deletion mutants such as dN79, dN124, dN147, and dN224.
We found the N-terminus of the C44Mab-46-binding epitope between residues 147 and 224 of CD44v3–10. We
next investigated this epitope using a novel mapping system: RIEDL insertion for epitope mapping (REMAP)
method. We constructed 31 CD44 standard (CD44s) mutants where the RIEDL tag was inserted into the
expected epitope region in CD44s. We observed that the C44Mab-46 epitope constituted five amino acids: 174TDDDV-178 of CD44s. Thus, the REMAP method could be used to determine mAb binding epitopes formembrane proteins.
Keywords: CD44, monoclonal antibody, epitope mapping, REMAP method

IntroductionC

D44 is a type I transmembrane glycoprotein widely
expressed on the surface of many cell types, including
epithelial, fibroblast, and leukocyte cells.(1) CD44 functions asa hyaluronan (HA) receptor, with CD44–HA interactions
mediating cell adhesion and migration in several physiologicaland pathological processes.(2,3) The CD44 structure comprises
extracellular, transmembrane, and intracellular domains. The
extracellular domain contains an HA-binding region(4) and analternative splicing site. The CD44 gene comprises 20 exons,
with alternative splicing producing many different isoforms,
such as CD44 standard (CD44s) that lacks variant exons, andCD44v3–10, with exons v3 to v10.(5) The intracellular domain
contains two ligand binding sites: an ezrin, radixin, and moesin(ERM) binding site and an ankyrin binding site. CD44 links
HA and the membrane cytoskeleton via these binding sites to
ERM and ankyrin.(6–9) Moreover, the intracellular domain of
CD44 is also necessary for the binding of soluble HA; therefore, this domain potentially mediates ligand binding at theextracellular domain.(10)CD44 is related to development, metastasis, and tumor
invasion(11–14); therefore, it is a biomarker and therapeutictarget in several cancers.(15) We previously developed several
anti-CD44 monoclonal antibodies (mAbs),(16) including
clone C44Mab-46, by immunizing mice with the CD44v3–10
ectodomain. C44Mab-46 recognized not only CD44s but also
CD44 variants. However, the C44Mab-46 epitope remains
undetermined.
To determine molecular epitopes, several methods may be
used.(17) X-ray cocrystallography allows the direct visualization of interactions between antigens and antibodies;
however, the approach takes time and is expensive. By
contrast, array-based oligopeptide scanning and site-directedmutagenesis mapping can easily determine linear epitopes,
but these methods are inappropriate for conformational epitopes. Recently, we developed a novel epitope mapping
system: the RIEDL insertion for epitope mapping (REMAP)
method using the RIEDL tag system.(18) This approach issimple and efficient for the determination of conformational
epitopes. In this study, we identified the epitope of C44Mab46 for CD44 using the REMAP method.

1

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

156

EPITOPE MAPPING OF AN ANTI-CD44 MAB
Materials and Methods
Plasmid construction

signal sequence (MYRMQLLSCIALSLALVTNS) andPA16 tag (GLEGGVAMPGAEDDVV),(16,19–23) which isrecognized by an anti-PA16 tag mAb (NZ-1),(24) were added
at each N-terminus. CD44v3–10 deletion mutants wereconstructed using the HotStar HiFidelity Polymerase
Kit (Qiagen Inc.), using oligonucleotides containing the desired mutations. Insertion of RIEDL tags into CD44s sequences was performed with oligonucleotides containing
RIEDL tag insertions at the desired position. For example, byinserting the RIEDL sequence (R*) between Glu160 and
Tyr161 of CD44s, we constructed Glu160_RIEDL_Tyr161

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The CD44s open reading frame (ORF) was amplified from
LN229 complementary DNA using the HotStar HiFidelity
Polymerase Kit (Qiagen Inc., Hilden, Germany).(16) The
CD44v3–10 ORF was provided by the RIKEN BioResource
Research Center via the National Bioresource Project of
MEXT, Japan.(16) Both CD44s and CD44v3–10 ORFs wereseparately subcloned into the pCAG-Ble vector (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan), and a

157

FIG. 1. Epitope mapping of C44Mab-46 using CD44v3–10 deletion mutants. (A) The CD44 gene and its 20 exons. The
smallest isoform is the standard CD44 form (CD44s), and other isoforms include CD44 variants, such as CD44v3–10. (B,C) CD44v3–10 deletion mutants were analyzed using flow cytometry. They were expressed in CHO-K1 cells and incubatedwith C44Mab-46 (B) or anti-PA16 tag antibody (NZ-1) (C) for 30 minutes at 4C, followed by a corresponding secondary
antibody treatment. Black lines: without 1st antibody in blocking buffer (negative control). (D) Schematic illustration;
epitope mapping of CD44v3–10, with four CD44v3–10 deletion mutants with a PA16 tag at N-termini. CD44v3–10 deletionmutants; dN79, dN124, dN147, and dN224. Black bar: the positive reaction of C44Mab-46. White bar: the negative reactionof C44Mab-46. PA16: PA16 tag.

158

(E160_R*_Y161). PCR fragments bearing the desired mutations were inserted into pCAG-Ble using an In-Fusion HD
Cloning Kit (Takara Bio, Inc., Shiga, Japan). The RIEDL tag
insertion mutants were as follows: E160_R*_Y161,
Y161_R*_R162, R162_R*_T163, T163_R*_N164, N164_
R*_P165, P165_R*_E166, E166_R*_D167, D167_R*_I168, I168_R*_Y169, Y169_R*_P170, P170_R*_S171,
S171_R*_N172, N172_R*_P173, P173_R*_T174, T174_
R*_D175, D175_R*_D176, D176_R*_D177, D177_R*_
V178, V178_R*_S179, S179_R*_S180, S180_R*_G181,
G181_R*_S182, S182_R*_S183, S183_R*_S184, S184_
R*_E185, E185_R*_R186, R186_R*_S187, S187_R*_
S188, S188_R*_T189, T189_R*_S190, and S190_R*_G191.

ASANO ET AL.

CHO-K1 parental cells (Figs. 1B, C). These results indicatedthat the N-terminus of the C44Mab-46 epitope existed between
the 147th and 224th amino acids of CD44v3–10 (Fig. 1D).
Determination of the C44Mab-46 epitope using
REMAP method

To further investigate the C44Mab-46 epitope, we performed the REMAP method. For this, we used the RIEDL tag

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Cell lines

Chinese hamster ovary (CHO)-K1 cells were purchasedfrom the America Type Culture Collection (ATCC, Manassas, VA). CD44 mutant plasmids were transfected into cells
using the Neon Transfection System (Thermo Fisher Scientific, Inc., Waltham, MA), and stable transfectants were
sorted using NZ-1 with a cell sorter (SH800; Sony Corp.,
Tokyo, Japan). Cells were cultured in the RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented
with 10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific Inc.), 100 U/mL penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.)at 37C in a humidified atmosphere containing 5% CO2.
Transfectants were cultivated in a medium containing
0.5 mg/mL Zeocin (InvivoGen, San Diego, CA).
Flow cytometry

Cells were harvested by brief exposure to 0.25%
trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai
Tesque, Inc.). After washing in 0.1% bovine serum albuminin phosphate-buffered saline (blocking buffer), cells weretreated with primary mAbs, such as C44Mab-46 (mouse IgG1,
kappa), NZ-1 (rat IgG2a, lambda), or LpMab-7 (mouse IgG1,
kappa) at a concentration of 1 lg/ml for 30 minutes at 4C;
subsequently, with Alexa Fluor 488-conjugated anti-mouse
IgG or Alexa Fluor 488-conjugated anti-rat IgG (1:1000; Cell
Signaling Technology, Inc., Danvers, MA). Fluorescencedata were collected using a BD FACSLyric (Becton, Dickinson and Company, Franklin Lakes, NJ) instrument for deletion mutant analysis, or the EC800 Cell Analyzer (SonyCorp.) for REMAP analyses.
Results
Determination of the C44Mab-46 epitope using
CD44v3–10 deletion mutants

The CD44 gene comprises 20 exons. The smallest isoform isthe standard form, CD44s, and other variant isoforms include
CD44v3–10 (Fig. 1A). The CD44 extracellular domain is encoded by exons 1–17. First, we constructed CD44v3–10 Nterminal deletion mutants (dN79, dN124, dN147, and dN224),
with the PA16 tag at their N-terminus. Reactions between
C44Mab-46 and deletion mutants were characterized by flow
cytometry and showed that C44Mab-46 recognized CD44v3–
10, dN79, dN124, and dN147, but not dN224 (Fig. 1B). By
contrast, all deletion mutants were detected by the anti-PA16 tag
mAb, NZ-1 (Fig. 1C). C44Mab-46 and NZ-1 did not recognize

FIG. 2. RIEDL insertion for the REMAP method. RIEDL
tags were inserted into CD44s. (A) C44Mab-46 bound toCD44s when RIEDL tags were inserted into any region,
which was independent of the C44Mab-46 epitope (upper
panel). By contrast, C44Mab-46 lost reactivity to CD44s
when the C44Mab-46 epitope structure was disrupted by
RIEDL insertion (lower panel). (B) RIEDL tags were inserted into expected epitope regions in CD44s to generate 31
mutants. REMAP, RIEDL insertion for epitope mapping.

EPITOPE MAPPING OF AN ANTI-CD44 MAB

where the RIEDL tag was inserted into the expected epitope
region in CD44s (Fig. 2B). Our flow cytometry results
showed that C44Mab-46 did not detect four mutants(T174_R*_D175, D175_R*_D176, D176_R*_D177, and
D177_R*_V178) and weakly detected P173_R*_T174(Fig. 3A), suggesting C44Mab-46 bound to CD44s at fiveamino acids (from Thr174 to Val178). As positive controls,
NZ-1 against the N-terminal PA16 tag and LpMab-7 against

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system,(18) which comprised a five-amino acid peptide(RIEDL tag) and an anti-RIEDL tag mAb (clone LpMab-7).
C44Mab-46 bound to CD44s when the RIEDL tag was inserted into any region, which was independent of the
C44Mab-46 epitope (Fig. 2A, upper panel). By contrast,
CD44s was not detected by C44Mab-46 when C44Mab-46
epitope conformation was disrupted by RIEDL insertion(Fig. 2A, lower panel). We constructed 31 CD44s mutants

159

FIG. 3. Epitope mapping of C44Mab-46 using RIEDL-tagged CD44s insertion mutants. RIEDL tags were inserted into
CD44s, with mutants analyzed by flow cytometry. RIEDL tag insertion mutants were expressed in CHO-K1 cells andincubated with (A) C44Mab-46, (B) anti-PA16 tag antibody (NZ-1), or (C) anti-RIEDL tag antibody (LpMab-7) for 30
minutes at 4C, followed by a corresponding secondary antibody treatment. Black lines: without 1st antibody (negative
control).

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ASANO ET AL.
Funding Information

This research was supported, in part, by the Japan Agency
for Medical Research and Development (AMED) under grantnos: JP21am0401013 (Y.K.) and JP21am0101078 (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.), and 19K07705(to Y.K.).

FIG. 4. Summary of epitope mapping of C44Mab-46.
Thr174, Asp175, Asp176, Asp177, and Val178 are criticalamino acids for C44Mab-46 binding to CD44s.
the RIEDL tag detected all mutants (Fig. 3B, C). Since the
RIEDL tag was not inserted into wild-type CD44s, LpMab-7
did not react with CHO/CD44s (Fig. 3C). These results are
summarized (Fig. 4).

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Discussion

To investigate mAb binding epitopes, alanine-scanningmutagenesis and peptide screening methodologies are generally used.(17) These methods are simple and effective for
identifying linear epitopes; however, they are unsuited toconformational epitopes. To determine these epitopes, wedeveloped the REMAP method.(18)
We previously developed several anti-CD44 mAbs, including C44Mab-46 and C44Mab-5 by immunizing mice with
CD44v3–10 ectodomain and CD44v3-10-overexpressed
CHO-K1 cells, respectively.(16) To investigate the C44Mab46 epitope, we conducted N-terminal deletion mutant analyses. Our data showed that the N-terminus of the C44Mab-46
epitope was located between amino acids 147 and 224 of
CD44v3–10 (Fig. 1). This epitope was also located in CD44s.
Using additional deletion mutants, we further found that theC44Mab-46 epitope was located between amino acids 161
and 190 of CD44s (data not shown). To identify criticalamino acids, we further used the REMAP method (Fig. 2).
Flow cytometry analyses showed that NZ-1 and LpMab-7
recognized all RIEDL tag insertion mutants. By contrast,
C44Mab-46 lost its reactivity to four mutants of CD44s (Fig. 3).
RIEDL tag insertion may disrupt the structure of the epitope
region and inhibit the binding of C44Mab-46 to CD44s. We
successfully determined the C44Mab-46-binding epitope forCD44s as 174-TDDDV-178 (Fig. 4). These amino acids wereencoded in exon 5 (Fig. 1); therefore, C44Mab-46 could detect
not only CD44s but also all CD44 variant isoforms. Identifying
the C44Mab-46 epitope will help in the development of aCD44-targeting therapeutic antibody in the future.
LpMab-7 can detect the RIEDL tag, which is inserted into
the loop structure of protein.(18) Therefore, the REMAP
method could be advantageous for determination of theconformational epitope of various membrane proteins, even
if the epitope cannot not be determined by the alanine scanning method in the future study.
Authors’ Contributions

T.A., J.T., and N.T. performed experiments; M.K.K. designed the experiments; and T.A. and Y.K. wrote the article.
Author Disclosure Statement

The authors have no conflict of interest.

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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, Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 21, 2021
Accepted: May 17, 2021