MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 5, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0028

Epitope Mapping of the Anti-CD44 Monoclonal Antibody(C44Mab-46) Using Alanine-Scanning Mutagenesis
and Surface Plasmon Resonance

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Junko Takei,1,* Teizo Asano,1,* Hiroyuki Suzuki,2 Mika K. Kaneko,1 and Yukinari Kato1,2,i

CD44 is a type I transmembrane protein expressed in various kinds of normal cancer cells, including pancreatic,breast, and oral cancers. CD44 is associated with cancer progression, metastases, and treatment resistance.
CD44 consists of 20 exons, and various isoforms exist due to alternative splicing of the central 10 exons. Some
splicing variants show cancer-specific expression patterns and are related to prognosis of patients with cancer.
Therefore, CD44 targeting therapy has been attracting attention. In a previous study, we established an antiCD44 monoclonal antibody, C44Mab-46 (IgG1, kappa), useful for flow cytometry, Western blotting, and immunohistochemistry by immunizing mice with CD44v3-10 ectodomain. This study investigated the binding
epitope of C44Mab-46 using enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance(SPR) with the synthesized peptide. ELISA results using deletion mutants showed that C44Mab-46 reacted withthe amino acids (aa) of 161–180 aa of CD44. Further examination of the C44Mab-46 epitope using ELISA withpoint mutants in 161–180 aa of CD44 demonstrates that the C44Mab-46 epitope comprised Thr174, Asp177, andVal178. The SPR with point mutants in 161–180 aa of CD44 demonstrated that the C44Mab-46 epitope
comprises Thr174, Asp175, Asp176, Asp177, and Val178. Together, the C44Mab-46 epitope was determined to
be located in exon 5 of CD44.
Keywords: CD44, monoclonal antibody, epitope mapping, surface plasmon resonanceIntroductionC

D44 is a single span transmembrane glycoprotein withvarious functions, such as cell adhesion, growth, survival, differentiation, and motility.(1) The principal ligand of
CD44 is hyaluronic acid (HA), osteopontin, serglycin, collagens, fibronectin, and laminin also bind with CD44.(2) The
full-length CD44 gene consists of 20 exons. The standard
isoform (CD44s) consists of exons 1–5 and 16–20, which are
expressed in all isoforms. The other exons are alternatively
spliced to generate splicing variants (CD44v). The predicted
molecular mass of CD44s is 37–38 kDa, and posttranslational modifications, including N- and O-linked glycosylation, sulfation, and phosphorylation increase themolecular mass to 85–95 kDa. The largest possible molecularmass of CD44 is >200 kDa.(3)CD44 is expressed in various tissues, such as the central
nervous system, lung, epidermis, liver, and pancreas.(4) CD44
expression is also observed in many cancers, including pancreatic cancer,(5) breast cancer,(6) lung cancer,(7) gastriccancer,(8) colorectal cancer,(9) and head and neck squamouscell carcinoma (HNSCC).(10) Günthert et al. found CD44
variant isoforms, which contains exons v4–v7 from rat pancreatic carcinoma cell line, and its overexpression converted
the nonmetastatic cells into metastatic cells.(11) Further study
revealed that an anti-CD44v6 monoclonal antibody (mAb)
prevented the metastatic property of these cells.(12) CD44v6bearing isoforms play an essential role in metastases bypromoting c-Met activation.(13) HA binding to CD44 affects
tumor development by inhibiting apoptosis and promoting
invasion and angiogenesis.(14) CD44 also interacts with epidermal growth factor receptor (EGFR) and human epidermal
growth factor receptor 2.(15–17) The HA/CD44 interaction
leads to CD44-EGFR complex formation, and the complex
induces downstream signaling through Ras and RhoA, resulting in tumor cell growth. HA/CD44 signaling also leads
to increased cisplatin chemoresistance through EGFR signaling pathways in HNSCC.(10) In these ways, CD44 activates and modulates several cell signals that mediate tumor
progression, metastasis, and therapy resistance.

Departments of 1Antibody Drug Development, and 2Molecular Pharmacology, Tohoku University Graduate School of Medicine,
Sendai, Japan.
*Contributed equally to this study.
i
ORCID ID (https://orcid.org/0000-0001-5385-8201).

219

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220

Head and neck cancer (HNC) are the seventh most common cancer worldwide in 2020. About 930,000 people are
newly diagnosed with HNC, and 470,000 people have died ofit.(18) More than 90% of cases are pathologically diagnosed
with squamous cell carcinoma.(19) Some antibody drugs for
HNSCC have been approved: cetuximab, an anti-EGFR mAb,
was first approved in 2006,(20) and later, nivolumab and pembrolizumab, which are anti-PD-1 mAbs, were approved in2016.(21) These drugs have improved the prognoses of patientswith HNSCC; however, they have some problems. Adverse
effects such as skin toxicity and interstitial lung disease areobserved in cetuximab,(22,23) and patients with PD-L1 negative
receive little benefits.(24) Acquired resistance is also found in all
drugs. Therefore, other therapeutic targets are needed.
In HNSCC, it was reported that some CD44-splicing variants such as CD44v3, v4, v6, and v10 were expressed inaddition to CD44s.(25,26) Wang et al. reported that their expression was associated with advanced T stage (v3 and v6),
metastasis (v3 and v10), and shorter disease-free survival (v6and v10).(26) Previously, bivatuzumab mertansine, an antiCD44v6 mAb linked to a cytotoxic drug, was used in phase I
trial; however, it was discontinued because of severe skintoxicity with a fatal outcome in parallel trial.(27) Therefore,
the development of other anti-CD44 targeting drugs that havefewer side effects is desired.
We previously developed an anti-CD44 mAbs, C44Mab-46(IgG1, kappa), by immunizing mice with CD44v3-10 ectodomain. This study investigated its critical epitopes using
enzyme-linked immunosorbent assay (ELISA) and surface
plasmon resonance (SPR) with synthesized peptides.
Materials and Methods
Cell lines

A glioblastoma cell line (LN229) was obtained fromthe American Type Culture Collection (Manassas, VA).
CD44v3-10 ectodomain (CD44ec)-secreting LN229 (LN229/
CD44ec) was established by transfecting pCAG/PA-CD44ecRAP-MAP into LN229 cells using the Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA). The aminoacid (aa) sequences of the tag system in this study were as
follows: PA tag,(28) 12 aa (GVAMPGAEDDVV); RAP tag,(29)12 aa (DMVNPGLEDRIE); and MAP tag,(30) 12 aa (GDGM
VPPGIEDK). LN229 and LN229/CD44ec were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) complete
medium, including DMEM (4.5 g/L glucose) with L-Gln andwithout sodium pyruvate (cat. no: 08459-64; Nacalai Tesque,Inc., Kyoto, Japan), 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL of penicillin(Nacalai Tesque, Inc.), 100 lg/mL streptomycin (Nacalai
Tesque, Inc.), and 0.25 lg/mL amphotericin B (Nacalai
Tesque, Inc.). LN229/CD44ec was established in DMEM
complete medium, including 0.5 mg/mL of G418 (Nacalai
Tesque, Inc.). Cell lines were incubated at 37C in a humid
atmosphere containing 5% CO2.
Purification of CD44ec

After LN229/CD44ec was cultured using DMEM complete medium without G418, CD44ec was purified fromthe supernatants using RAP tag system, comprised an antiRAP tag mAb (clone PMab-2) and a RAP peptide

TAKEI ET AL.

Table 1. Identification of C44Mab-46 Epitope
Using Point Mutants by Enzyme-Linked
Immunosorbent Assay
MutantpeptideSequence

C44Mab-46

Y161A
R162A
T163A
N164A
P165A
E166A
D167A
I168A
Y169A
P170A
S171A
N172A
P173A
T174A
D175A
D176A
D177A
V178A
S179A
S180A

ARTNPEDIYPSNPTDDDVSSYATNPEDIYPSNPTDDDVSSYRANPEDIYPSNPTDDDVSSYRTAPEDIYPSNPTDDDVSSYRTNAEDIYPSNPTDDDVSSYRTNPADIYPSNPTDDDVSSYRTNPEAIYPSNPTDDDVSSYRTNPEDAYPSNPTDDDVSSYRTNPEDIAPSNPTDDDVSSYRTNPEDIYASNPTDDDVSSYRTNPEDIYPANPTDDDVSSYRTNPEDIYPSAPTDDDVSSYRTNPEDIYPSNATDDDVSSYRTNPEDIYPSNPADDDVSSYRTNPEDIYPSNPTADDVSSYRTNPEDIYPSNPTDADVSSYRTNPEDIYPSNPTDDAVSSYRTNPEDIYPSNPTDDDASSYRTNPEDIYPSNPTDDDVASYRTNPEDIYPSNPTDDDVSA

+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
+
+++
+++

+++, OD655 S 0.5; ++, 0.3 & OD655 < 0.5; +, 0.1 & OD655 <
0.3; -, OD655 < 0.1.

(GDDMVNPGLEDRIE).(29) The filtered culture supernatant(5 L) was passed through PMab-2-Sepharose (2 mL bed
volume), and the same process was repeated three times. The
beads were then washed with 100 mL of phosphate-bufferedsaline (PBS; Nacalai Tesque, Inc.), and eluted with
0.1 mg/mL a RAP peptide in a stepwise manner (2 mL · 10).

Table 2. The KD Between C44Mab-46 and Alanine
Substituted Peptides by Surface
Plasmon Resonance
Mutant peptideka (/ms)kd (/s)KD (M)

p161–180
Y161A
R162A
T163A
N164A
P165A
E166A
D167A
I168A
Y169A
P170A
S171A
N172A
P173A
T174A
D175A
D176A
D177A
V178A
S179A
S180A

1.07 · 103
1.06 · 103
1.29 · 103
1.30 · 103
2.22 · 103
1.39 · 103
1.51 · 103
1.70 · 103
1.22 · 103
1.08 · 103
1.31 · 103
1.33 · 103
1.13 · 103
1.17 · 103
ND
ND
ND
ND
ND
1.30 · 103
1.21 · 103

6.32 · 10-3
6.86 · 10-3
6.07 · 10-3
6.18 · 10-3
7.03 · 10-3
6.48 · 10-3
5.23 · 10-3
6.39 · 10-3
5.97 · 10-3
6.16 · 10-3
6.82 · 10-3
5.08 · 10-3
4.72 · 10-3
1.72 · 10-2
ND
ND
ND
ND
ND
4.32 · 10-3
7.44 · 10-3

5.92 · 10-6
6.50 · 10-6
4.72 · 10-6
4.76 · 10-6
3.16 · 10-6
4.68 · 10-6
3.46 · 10-6
3.76 · 10-6
4.88 · 10-6
5.70 · 10-6
5.20 · 10-6
3.81 · 10-6
4.16 · 10-6
1.47 · 10-5
5.46 · 10-5
4.75 · 10-4
1.35 · 10-4
1.33 · 10-4
2.00 · 10-4
3.34 · 10-6
6.16 · 10-6

ND, not determined.

EPITOPE MAPPING OF AN ANTI-CD44 MAB
CD44 peptides

CD44 peptides (accession no. X66733), including 58 deletion mutants and 20 point mutants (Table 1), were synthesizedusing PEPScreen (Sigma-Aldrich Corp., St. Louis, MO).
Enzyme-linked immunosorbent assay(Thermo Fisher Scientific, Inc.) at a concentration of 1 and10 lg/mL, respectively, for 30 min at 37C. After washingwith PBS containing 0.05% Tween20 (Nacalai Tesque, Inc.;
PBST), wells were blocked with 1% bovine serum albumincontaining PBST for 30 min at 37C. The plates wereincubated with C44Mab-46 (1 lg/mL), followed by aperoxidase-conjugated anti-mouse immunoglobulins (1:2000
diluted; Agilent Technologies, Inc., Santa Clara, CA). Enzymatic reactions were conducted by the ELISA POD

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CD44ec and synthesized CD44 peptides (Table 1) were
immobilized on Nunc Maxisorp 96-well immunoplates

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FIG. 1. (A) Schematic illustration of CD44 gene. The full-length CD44 gene consists of 20 exons (upper panel). The
standard isoform (CD44s) consists of exons 1–5 and 16–20 (middle panel). The other exons are alternatively spliced to generate
splicing variants, such as CD44v3-10, which consists of exons 1–5, v3–v10, and 16–20 (lower panel). (B) Schematic illustration of synthesized peptides. Red bars indicate the peptide detected by C44Mab-46.

222

Substrate TMB Kit (Nacalai Tesque, Inc.). Optical density
was measured at 655 nm using an iMark microplate reader(Bio-Rad Laboratories, Inc., Berkeley, CA).

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Measurement of dissociation constants (KD)between C44Mab-46 and alanine-substituted
peptides using SPR

C44Mab-46 was immobilized on the sensor chip CM5 according to the protocol (https://doi.org/10.1039/C3990000
1526), described by Cytiva (Marlborough, MA). In brief,
C44Mab-46 was diluted to 10 lg/mL by the acetate buffer(pH 4.0: Cytiva) and immobilized using amine coupling reaction. The surface of the flow cell 2 of the sensor chip CM5 wastreated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
and N-hydroxysuccinimide (NHS), followed by the injection of
C44Mab-46. Unreacted NHS-ester was blocked with ethanolamine after C44Mab-46 immobilization. The KD between
C44Mab-46 and alanine-substituted peptides shown in Table 2
were measured using Biacore X100 (Cytiva) at 25C. The
buffer used was PBS containing 0.005% (v/v) of Tween 20 and
1.1% or 4.4% (v/v) of dimethyl sulfoxide (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan). A single cycle kinetics method was used to measure the binding signals. The data
were analyzed by 1:1 binding kinetics to determine the association rate constant (ka) and dissociation rate constant (kd) andKD, or equilibrium analysis to determine KD using BIAevaluation software (Cytiva).

TAKEI ET AL.
Results
Epitope mapping using ELISA

Fifty-eight peptides were synthesized (20 aa) from theextracellular domain of CD44v3-10 (Fig. 1) and performed
ELISA. C44Mab-46 reacted with 161–180 aa (161YRTNPEDIYPSNPTDDDVSS-180) and 171–190 aa (171SNPTDDDVSSGSSSERSSTS-190) (Fig. 2). Because thereaction of C44Mab-46 to 161–180 aa is higher than that of
171–190 aa, we used 161–180 aa for alanine scanning.
A series of point mutants of 161–180 aa were synthesized(Table 1). C44Mab-46 strongly reacted with Y161A, R162A,
T163A, N164A, P165A, E166A, D167A, I168A, Y169A,
P170A, S171A, N172A, P173A, T174A, D176A, S179A,
S180A, wild type 161–180 aa, and moderately reacted with
D177A and V178A, but not with D175A (Fig. 3A). The results indicate that Asp175, Asp177, and Val178 are includedin the critical epitopes of C44Mab-46 (Fig. 3B).
Epitope mapping using SPR

We measured the KD between C44Mab-46 and a series ofpoint mutants of CD44 synthesized peptides (Table 1) using
Biacore X100. The measured values are summarized in Table 2.
The association rate constant (ka) and dissociation rate constant(kd) of T174A, D175A, D176A, D177A, and V178A were notdetermined; therefore, their KDs were calculated using equilibrium analyses. The binding affinities of T174A, D175A,
D176A, D177A, and V178A to C44Mab-46 were 9.2-, 80-, 22.8-,

FIG. 2. Determination of C44Mab-46 epitope of CD44v3-10 by ELISA using deletion mutants. CD44ec (positive control),PBS (negative control), and synthesized peptides were immobilized on immunoplates. The plates were incubated withC44Mab-46 (1 mg/mL), followed by incubation with peroxidase-conjugated anti-mouse immunoglobulins. Optical density
was measured at 655 nm. ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline.

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EPITOPE MAPPING OF AN ANTI-CD44 MAB

223

FIG. 3. (A) Determination of C44Mab-46 epitope of CD44v3-10 by ELISA using point mutants. CD44ec (positive
control) and synthesized alanine-substituted peptides were immobilized on immunoplates. The plates were incubated withC44Mab-46 (1 mg/mL), followed by incubation with peroxidase-conjugated anti-mouse immunoglobulins. Optical density
was measured at 655 nm. (B) Schematic illustration of the epitope recognized by C44Mab-46 using ELISA. Red aa indicate
the critical epitope of C44Mab-46 determined using ELISA. aa, amino acids; IC, intracellular domain; SS, signal sequence;
TM, transmembrane domain.

22.5-, and 33.8-fold decreased compared with wild type 161–
180 aa, respectively, indicating that Thr174, Asp175, Asp176,
Asp177, and Val178 are the binding epitope for C44Mab-46.
We exhibited the ratio of the KD values for each peptide to theKD value for wild type p161-180 in Figure 4.
Discussion

This study examines the C44Mab-46 epitope of CD44v310 by peptide scanning using ELISA and SPR. We determined the critical epitope as Asp175, Asp177, and Val178
using ELISA, and as Thr174, Asp175, Asp176, Asp177, andVal178 using SPR, which are coded in exon 5 of CD44 gene.
The epitope mapping result using ELISA and SPR showed
the same region of CD44v3-10 as the epitope; however,Thr174 and Asp176 are shown as the epitope only by SPR
analysis. Peptides were immobilized on immunoplates in
ELISA. In contrast, C44Mab-46 was immobilized on a sensor
chip in SPR analysis. This difference may affect the reactivityof C44Mab-46 to these peptides.
Epitope mapping by ELISA is more rapid, and the cost of
ELISA is inexpensive compared with SPR. However, it isdifficult to obtain precise quantitative information of antigen–antibody interaction by ELISA, whereas SPR reveals

each interaction much more quantitatively in real time.(31)This study showed that SPR might be a more efficient methodfor determining the epitope residues because the intensity of
the reaction of T174A and D176A to C44Mab-46 displayed
almost the same value as that of wild type p161–180 to
C44Mab-46 in ELISA.
In the previous study, we developed a novel epitope
mapping system named RIEDL insertion for epitope mapping (REMAP) method.(32) This method is for determining
the conformational epitope, which could not be determinedby alanine scanning. Using the REMAP method, we determined the critical epitope of C44Mab-46 as Thr174, Asp175,
Asp176, Asp177, and Val178 of CD44s, which are of thesame residues with the results of this study. This is the firststudy indicating that the REMAP method is useful in determining the epitope compared with conventional methods
such as ELISA and SPR.
Previously, we developed another anti-CD44 mAb, C44Mab5 (IgG1, kappa),(33) whose binding epitope was determined to be
25th to 36th aa of CD44. Both anti-CD44 mAbs are used in
detecting CD44s and CD44v. The epitope of C44Mab-46 is alittle away from the HA-binding site, which contains Arg41,
Tyr42, Arg78, Tyr79, and Arg154.(34–36) Therefore, C44Mab-46
will not block HA binding directly; however, it may affect HA

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224

TAKEI ET AL.

FIG. 4. (A) Determination of C44Mab-46 epitope of CD44v3-10 by SPR using point mutants. C44Mab-46 was immobilized on the sensor chip CM5, and peptides were injected on the sensor chip. The KDs between C44Mab-46 and each
peptide were measured using Biacore X100. The vertical axis indicates the ratio of the KD values for each peptide to the KD
value for p161-180. (B) Schematic illustration of the epitope recognized by C44Mab-46 using SPR. Red aa indicate thecritical epitope of C44Mab-46 determined using SPR. SPR, surface plasmon resonance.

binding indirectly. CD44 also has many other ligands whosebinding sites have not been mapped precisely, and C44Mab-46
may affect their interaction.(15–17) Therefore, we will study theeffect of C44Mab-46 on ligand binding in the future.
The subclass of C44Mab-46 is mouse IgG1, which does
not have antibody-dependent cellular cytotoxicity andcomplement-dependent cellular cytotoxicity; therefore, we
could not reveal its antitumor activity at this stage. In theprevious study, we converted the IgG1 subclass of C44Mab5 into an IgG2a subclass and further produced a defucosylated version, 5-mG2a-f, and revealed its antitumor activity
against mouse models of oral cancer.(37) We will convert
the subclass of C44Mab-46 into IgG2a and investigate its
effectiveness against CD44-expressing tumors in the future
study.
Authors’ Contributions

J.T., T.A., and H.S. performed experiments; M.K.K. designed the experiments; J.T., T.A., and Y.K. wrote the article.

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 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.).
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TAKEI ET AL.

Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-Machi
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: May 27, 2021
Accepted: September 1, 2021