MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 3, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0022

Original Article
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Epitope Mapping of an Anti-CD44v4 Monoclonal Antibody(C44Mab-108) Using Enzyme-Linked Immunosorbent Assay

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Hiroyuki Suzuki, Mayuki Tawara, Aoi Hirayama, Nohara Goto, Tomohiro Tanaka,
Mika K. Kaneko, and Yukinari Kato

CD44 is a type I transmembrane glycoprotein and possesses various isoforms which are largely classified into
CD44 standard (CD44s) and CD44 variant (CD44v) isoforms. Some variant-encoded regions play critical roles intumor progression. However, the function of CD44 variant 4 (CD44v4)-encoded region has not been fully
understood. Using peptide immunization, we developed an anti-CD44v4 monoclonal antibody, C44Mab-108,
which is useful for flow cytometry, western blotting, and immunohistochemistry. In this study, we determined thecritical epitope of C44Mab-108 by enzyme-linked immunosorbent assay (ELISA). We used the alanine(or glycine)-substituted peptides of the CD44v4-encoded region (amino acids 271–290 of human CD44v3-10) andfound that C44Mab-108 did not recognize the alanine-substituted peptides of D280A and W281A. Furthermore,these peptides could not inhibit the recognition of C44Mab-108 in flow cytometry and immunohistochemistry. The
results indicate that the critical binding epitope of C44Mab-108 includes Asp280 and Trp281 of CD44v3-10.
Keywords: CD44, CD44 variant 4, monoclonal antibody, epitope, enzyme-linked immunosorbent assay

region have not been investigated. Therefore, specific antibodies against CD44v4 are indispensable for basic research,tumor diagnosis, and therapy.
We have established anti-CD44 monoclonal antibodies(mAbs), which recognize the standard11,12—or each variant13–20—encoded region. All mAbs can be used for flow
cytometry, western blotting, and immunohistochemistry andare expected to contribute not only to basic research but also
to phathological diagnosis. We also determined the critical
epitopes of C44Mab-521 and C44Mab-46.22,23
Clinical trials of anti-CD44 mAbs have been conducted.24 A humanized anti-CD44v6 mAb BIWA4 (bivatuzumab)-mertansine drug conjugate was evaluated, but
discontinued due to the severe skin toxicities.25,26 An antipan CD44 mAb, RG7356, exhibited an acceptable safety
profile in patients with advanced CD44-expressing solid
tumors. However, the study was terminated due to no evidence of a clinical and pharmacodynamic dose–response
relationship with RG7356.27 Therefore, the developmentof anti-CD44 mAbs with more potent and fewer side effects
is required.

IntroductionC

D44 has various isoforms, which are generated by thealternative splicing of CD44 pre-mRNA.1 The mRNAof CD44 standard (CD44s) isoform is produced by constantregion exons, including the first five (1–5) and the last five
(16–20).2 The mRNAs of CD44 variant (CD44v) isoform areproduced by the assembling of variant exons (v1–v10) withthe constant region exons of CD44s.3 CD44s and CD44v
receive the posttranslational modifications, including N- or
O-glycosylation.4 Both CD44s and CD44v can attach tohyaluronic acid, which is important for cellular adhesion,
homing, and motility.5
CD44v plays important roles in the tumor progression by
specific functions of variant exon-encoded regions.6 The
heparin-binding growth factors are recruited to heparan sulphate modified in the v3-encoded region.7,8 MET, a receptor
tyrosine kinase for hepatocyte growth factor, associates withthe v6-encoded region.9,10 These functions are essential for theactivation of growth factor signaling and tumor proliferation.
However, the roles of CD44 variant 4 (CD44v4)-encoded

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
This paper was previously published in preprint.org (doi: 10.20944/preprints202311.0216.v1).

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SUZUKI ET AL.

We previously established an anti-CD44v4 mAb, C44Mab108 (IgG1, kappa) using the peptide immunization.19 To
clarify further characteristics of C44Mab-108, we performed
epitope mapping using enzyme-linked immunosorbent assay(ELISA).
Materials and Methods
PeptidesThe CD44v4 peptide (271-AFDHTKQNQDWTQWNPS
HSN-290) and 20 alanine (or glycine)-substituted peptides(Table 1) were synthesized by utilizing PEPscreen (SigmaAldrich Corp., St. Louis, MO, USA). The number of aminoacids (aa) is derived from human CD44v3-10 (Accession
No.: X66733).

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ELISA

The CD44v4 peptides were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.,
Waltham, MA, USA) at a concentration of 10 lg/mL for 30
minutes at 37C. After washing with phosphate-bufferedsaline (PBS) containing 0.05% Tween20 (PBST; Nacalai
Tesque, Inc., Kyoto, Japan), wells were blocked with 1%
bovine serum albumin (BSA)-containing PBST for 30 minutes at 37C. The plates were incubated with 10 lg/mL of
C44Mab-108, followed by a peroxidase-conjugated antimouse immunoglobulins (1:2000 diluted; Agilent Technologies, Inc., Santa Clara, CA, USA). Enzymatic reactions
were performed using the ELISA POD Substrate TMB Kit(Nacalai Tesque, Inc.). Optical density was measured at
655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA).

Table 1. Identification of the C44Mab-108 Epitope
Using Alanine (or Glycine)-Substituted CD44
Variant 4 Peptides
PeptidesWT(271–290)
A271G
F272A
D273A
H274A
T275A
K276A
Q277A
N278A
Q279A
D280A
W281A
T282A
Q283A
W284A
N285A
P286A
S287A
H288A
S289A
N290A

Sequences

C44Mab-108

AFDHTKQNQDWTQWNPSHSN+++GFDHTKQNQDWTQWNPSHSNAADHTKQNQDWTQWNPSHSNAFAHTKQNQDWTQWNPSHSNAFDATKQNQDWTQWNPSHSNAFDHAKQNQDWTQWNPSHSNAFDHTAQNQDWTQWNPSHSNAFDHTKANQDWTQWNPSHSNAFDHTKQAQDWTQWNPSHSNAFDHTKQNADWTQWNPSHSNAFDHTKQNQAWTQWNPSHSNAFDHTKQNQDATQWNPSHSNAFDHTKQNQDWAQWNPSHSNAFDHTKQNQDWTAWNPSHSNAFDHTKQNQDWTQANPSHSNAFDHTKQNQDWTQWAPSHSNAFDHTKQNQDWTQWNASHSNAFDHTKQNQDWTQWNPAHSN
AFDHTKQNQDWTQWNPSASNAFDHTKQNQDWTQWNPSHANAFDHTKQNQDWTQWNPSHSA

+++
+++
+++
+++
+++
+++
+++
+++
+++
–
–
+
+++
+
+++
+++
+++
+++
+++
+++

+++, OD655 ‡ 0.6; ++, 0.3 £ OD655 < 0.6; +, 0.1 £ OD655 < 0.3;-, OD655 < 0.1.
WT, wild type.

Flow cytometry

The N-terminal PA16-tagged CD44v3-10-overexpressed
Chinese hamster ovary-K1 (CHO/CD44v3-10)19 cells were
harvested after a brief exposure to 0.25% trypsin in 1 mM
ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque,Inc.) and washed with 0.1% BSA in PBS. C44Mab-108(10 lg/mL) was incubated with the CD44v4 peptides(10 lg/mL) for 30 minutes at 4C. The cells were further
treated with Alexa Fluor 488-conjugated anti-mouse IgG(1:2000). Fluorescence data were collected using the SA3800
Cell Analyzer (Sony Corp., Tokyo, Japan).
Immunohistochemical analysis

One formalin-fixed paraffin-embedded (FFPE) tissue oforal squamous cell carcinoma (OSCC) for peptide blocking
assay was obtained from Tokyo Medical and Dental University.28 The tissue slides were autoclaved in citrate buffer(pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20
minutes for antigen retrieval. After blocking with Super
Block T20 (Thermo Fisher Scientific, Inc.), the sections wereincubated with C44Mab-108 (10 lg/mL) in the presence orabsence of the CD44v4 peptides (10 lg/mL) and then treatedwith the EnVision+ Kit for mouse (Agilent Technologies,Inc.) for 30 minutes. The color was developed using
3,3¢-diaminobenzidine tetrahydrochloride (DAB; Agilent
Technologies, Inc.). Counterstaining was performed withhematoxylin (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan). Leica DMD108 (Leica Microsystems GmbH,
Wetzlar, Germany) was used to examine the sections andobtain images.
Results
Epitope mapping of C44Mab-108 with alanine(or glycine)-substituted CD44v4 peptides

We previously established an anti-CD44v4 mAb (C44Mab108) by peptide immunization of CD44v4 region(273-DHTKQNQDWTQWNPSHSNP-291).19 We confirmedthat C44Mab-108 recognizes only the variant 4-encoded region peptide (aa 271–290), but not other regions of CD44v310 extracellular domain.19 To identify the binding epitopeof C44Mab-108, we synthesized 20 alanine (or glycine)substituted peptides of the CD44v4 (Table 1). C44Mab-108
exhibited reaction with A271G, F272A, D273A, H274A,
T275A, K276A, Q277A, N278A, Q279A, T282A, Q283A,
W284A, N285A, P286A, S287A, H288A, S289A, N290A, andwild type (WT) (Fig. 1A). In contrast, C44Mab-108 did not reactwith D280A and W281A (Fig. 1A). This result indicated that
Asp280 and Trp281 are included in the critical epitope of
C44Mab-108. The results are summarized in Table 1. Figure 1B
shows the schematic illustration of CD44s, CD44v3-10, and thecritical aa (Asp280 and Trp281) recognized by C44Mab-108.
Flow cytometry using C44Mab-108
with alanine-substituted CD44v4 peptides

We next performed a peptide-blocking assay using flow
cytometry to confirm the importance of the C44Mab-108
epitope. As shown in Figure 2, C44Mab-108 reacted with the
CHO/CD44v3-10 cells. This reaction was completely neutralized by the WT peptide. In contrast, D280A and W281A

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EPITOPE MAPPING OF ANTI-CD44V4 MAB

FIG. 1. Determination of the C44Mab-108 epitope by
ELISA using alanine (or glycine)-substituted CD44v4 peptides.
(A) The alanine (or glycine)-substituted CD44v4 peptides in
PBS or PBS alone were immobilized on immunoplates. The
plates were incubated with C44Mab-108 (10 mg/mL), followedby peroxidase-conjugated anti-mouse immunoglobulins. Error
bars represent means – SDs. N.C. (PBS). (B) Schematic illustration of CD44s, CD44v3-10, and the C44Mab-108 epitope. The C44Mab-108 epitope includes Asp280 and Trp281
of CD44v3-10. The epitope is conserved in mouse and Chinese hamster (Cham), but not in Rat. CD44s, CD44 standard;
CD44v4, CD44 variant 4; ELISA, enzyme-linked immunosorbent assay; N.C., negative control; PBS, phosphatebuffered saline; SD, standard deviation; WT, wild type.

87

FIG. 2. Flow cytometry using C44Mab-108 and CD44v4
peptides. C44Mab-108 (10 mg/mL) plus WT, the alaninesubstituted peptides (10 mg/mL), or control (0.1% DMSO inblocking buffer, –peptide) were reacted with CHO/CD44v310 cells for 30 minutes at 4C, followed by treatment with
Alexa Fluor 488-conjugated anti-mouse IgG. The black line
represents the control. DMSO, dimethyl sulfoxide; WT,
wild type.

peptides did not block the reaction of C44Mab-108 with
CHO/CD44v3-10. The result confirmed that Asp280 and
Trp281 of CD44v3-10 are critical for detection by C44Mab108 using flow cytometry.
Immunohistochemistry using C44Mab-108
with alanine-substituted CD44v4 peptides

We also performed a peptide-blocking assay using immunohistochemical analysis. As shown in Figure 3, C44Mab-108
stained the FFPE section of OSCC, which was completely
neutralized by the WT peptide. In contrast, D280A and
W281A peptides did not neutralize the reaction. These results
were corresponding to that of Figure 2.
Discussion

In this study, the critical epitope of C44Mab-108 was determined as Asp280 and Trp281 in the CD44v4 region. Since
the reactivity of C44Mab-108 to T282A and W284A was also
reduced (Fig. 1A), Thr282 and Trp284 may contribute to the

FIG. 3. Immunohistochemistry using C44Mab-108 andCD44v4 peptides. The FFPE sections of OSCC were incubated with C44Mab-108 (10 mg/mL) plus WT, the alaninesubstituted peptides (10 mg/mL), or control (0.1% DMSO inblocking buffer, –peptide), followed by that with the Envision + Kit. Scale bar = 100 mm. FFPE, formalin-fixedparaffin-embedded; OSCC, oral squamous cell carcinoma.

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88

recognition partially. Figure 1B shows the homology of thev4 region among human, mouse, Chinese hamster, and rat
sequences. The Asp280 and Trp281 are conserved in human,
mouse, Chinese hamster, but not in rat. Furthermore, Thr282
and Trp284 are also conserved in human and mouse. Although the result suggests that C44Mab-108 might recognize
both human and mouse CD44v4, it did not react with mouse
CD44v4 using flow cytometry (data not shown).
Since C44Mab-108 was established by the peptide immunization, C44Mab-108 can recognize the definite peptide structureof the variant 4-encoded region. In contrast, CD44 is predicted
to carry 146 O-glycosylation sites in the variant region. Amongthem, 41 of these sites have already been experimentally confirmed.29 Thr282 is also a confirmed O-glycosylation site.30
Further studies are required to reveal whether the O-glycan
at Thr282 affects the recognition by C44Mab-108.
In our previous study, C44Mab-108 could detect CD44v310-overexpressed cells such as CHO/CD44v3-10, but notdetect endogenous CD44v4 in several cancer cell lines inflow cytometry.19 In contrast, C44Mab-108 could detect endogenous and membranous CD44v4 in immunohistochemistry.19 These results suggest that the variant 4-encoded
region is folded into the inside of protein in living cells, but
exposed by antigen retrieval in immunohistochemistry. Recently, we provided a potential strategy for developing
cancer-specific antibodies that target locally misfolded cellsurface receptors such as human epidermal growth factor
receptor 2.31,32 Further studies are needed to investigate
whether C44Mab-108 is involved in the recognition of specific CD44v4 type and/or specific condition of cells.
Authors’ Contributions

H.S., M.T., A.H., N.G., and T.T. performed the experiments. M.K.K. and Y.K. designed the experiments. H.S. and
M.K.K. analyzed the data. H.S. and Y.K. wrote the manuscript. All authors have read and agreed to the publishedversion of the manuscript.
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) under Grant
Nos.: JP23ama121008 (to Y.K.), JP23am0401013 (to Y.K.),
23bm1123027h0001 (to Y.K.), and JP23ck0106730(to Y.K.), and by the Japan Society for the Promotion of
Science ( JSPS) Grants-in-Aid for Scientific Research (KAKENHI) Grant Nos. 22K06995 (to H.S.), 21K07168(to M.K.K.), and 22K07224 (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
Miyagi 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: November 7, 2023
Accepted: January 17, 2024