MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 4, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0015
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Identification of the Binding Epitope of an Anti-mouse CCR4
Monoclonal Antibody, C4Mab-1

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

C-C chemokine receptor 4 (CCR4) is one of G protein-coupled receptors, and interacts with chemokines, CCL17
and CCL22. CCR4 is expressed on T cells such as helper T type 2 cells, regulatory T cells, and interleukin 17producing T helper cells. CCR4 is associated with T cells trafficking into the tumor microenvironment, and isassociated with tumor progression or metastasis. Therefore, CCR4 may be a potential thera-peutic option for T
cell malignancies. C4Mab-1 is a novel anti-mouse CCR4 (mCCR4) monoclonal antibody produced by
mCCR4 N-terminal peptide immunization. C4Mab-1 is useful for flow cytometric analysis. In this study, we
conducted the epitope mapping of C4Mab-1 using enzyme-linked immunosorbent assay (ELISA) and peptide
blocking assay. The result of ELISA indicated that Thr7, Asp8, and Gln11 of mCCR4 are the critical amino acids
for the C4Mab-1 binding. Furthermore, peptide blocking assay by flow cytometry showed that Thr7, Asp8, andGln11 of mCCR4 are essential for C4Mab-1 binding to mCCR4-overexpressed Chinese hamster ovary-K1(CHO/mCCR4) cells, and Val6, Thr9, and Thr10 are involved in the C4Mab-1 binding to CHO/mCCR4 cells.
These results indicate that the critical binding epitope of C4Mab-1 includes Thr7, Asp8, and Gln11 of mCCR4.
Keywords: human CCR4, C4Mab-1, epitope, monoclonal antibody, enzyme-linked immunosorbent assay, flow
cytometry

Introduction

C

hemokines are a family of small cytokines secreted
by cells, and act as a chemoattractant to guide the migration of cells that induce directional movement of leukocytes to sites of inflammation or injury.(1,2) The chemokines
also play fundamental roles in function of the immune system.(3) Chemokines interact with chemokine receptor, and thisinteraction transduces extracellular signals to intracellularsignals. Chemokine receptors belong to G protein-coupled
receptors, which is characterized by a seven-transmembrane
receptor. Because of their crucial role in cell migration, chemokine receptors are regarded as important therapeutic targets for inflammatory diseases and cancer.(4)C-C chemokine receptor 4 (CCR4) is one of a chemokine
receptor family. CCR4 is a receptor for CC chemokine ligand
17 (CCL17; thymus and activation-regulated chemokine) andCC chemokine ligand 22 (CCL22; macrophage-derived

chemokine).(5) CCR4 is expressed on helper T type 2 (Th2)cells and is upregulated by T cell receptor activation.(6,7)
CCR4 is also expressed on other T cell subsets such as regulatory T (Treg) cells and interleukin 17-producing helper T(Th17) cells.(7–10) It has been reported that CCR4 is associated with T cells trafficking into the tumor microenvironment,(11) and is overexpressed on malignant T cells.(12,13)
Moreover, high CCR4+ Treg levels are found in murine
and human solid tumors, and/or are associated with tumor
progression or metastasis.(14–22) Therefore, CCR4 could be adiagnostic and prognostic marker, and targeting CCR4 has
become a promising therapeutic option for T cell malignancies such as adult T cell leukemia/lymphoma (ATLL), andcutaneous T cell lymphomas (CTCLs).(23,24)
Mogamulizumab is a humanized anti-CCR4 monoclonal
antibody (mAb) with a defucosylated Fc region.(25,26) Mogamulizumab has an antibody-dependent cellular cytotoxicity (ADCC) activity, and is an effective antibody drug

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

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EPITOPE MAPPING OF ANTI-CCR4 MAB

215

for patients with CCR4-positive ATLL and CTCLs.(27,28)
Moreover, mogamulizumab binds to the N-terminal residues
from 12th to 29th of human CCR4.(28) Furthermore, it has
been reported that the N-terminus of the chemokine receptor,
such as CCR2, CCR3, CCR5, and CXCR1, is important for
the ligand binding.(2,29) Therefore, N-terminus of CCR4 is an
attractive target region for producing anti-CCR4 mAbs.
Previously, we produced a novel anti-mouse CCR4(mCCR4) mAb, C4Mab-1 (rat IgG1, kappa), using the mCCR4
N-terminal peptide immunization.(30) C4Mab-1 reacted with
mCCR4-overexpressed Chinese hamster ovary-K1 (CHO/
mCCR4) cells, P388 (mouse lymphoid neoplasma) cells andJ774-1 (mouse macrophage-like) cells in flow cytometry.
However, critical amino acids of mCCR4 for C4Mab-1 binding
has not been determined. Epitope identification of mAbs isimportant to elucidate the pharmacological function of mAbs
and is essential to avoid unexpected cross-reactivity. In thisstudy, we conducted the determination of the binding epitopeof C4Mab-1 on mCCR4 using enzyme-linked immunosorbent assay (ELISA) and peptide blocking assay using flow
cytometry.
Materials and Methods
Cell lines

CHO-K1 was obtained from the American Type Culture
Collection (ATCC; Manassas, VA). CHO/mCCR4 was previously established by transfecting plasmid that encodes
mCCR4 with PA tag(31–34) at N-terminus and RAP tag(35,36)
and MAP tag(37,38) at C-terminus into CHO-K1 cells using
the Neon transfection system (Thermo Fisher Scientific, Inc.,
Waltham, MA). CHO/mCCR4 cells were cultured in RPMI
1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) that was
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.). Cells were grown in a humidified
incubator, which was supplied with 5% CO2 and 95% air
at 37C.
Enzyme-linked immunosorbent assay

The mCCR4 peptides (Accession No.: NM_009916), including 20 point mutants (Table 1), were synthesizedby utilizing PEPScreen (Sigma-Aldrich Corp., St. Louis,
MO).(39–61) Each peptide was immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.)at a concentration of 10 lg/mL for 30 minutes at 37C. Afterwashing with phosphate-buffered saline (PBS) containing
0.05% Tween 20 (PBST), wells were blocked with 1% bovine serum albumin (BSA)-containing PBST for 30 minutes
at 37C.
The plates were then incubated with C4Mab-1 (1 lg/mL),followed by a 1:10,000 dilution of peroxidase-conjugatedanti-rat immunoglobulins (Sigma-Aldrich Corp.). 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).
Peptide blocking assay

CHO/mCCR4 cells were washed with 0.1% BSA in PBS.
C4Mab-1 (1 lg/mL) was incubated with each peptide(10 lg/mL) for 30 minutes at 4C. CHO/mCCR4 cells weretreated with peptide mixed C4Mab-1, and further with
Alexa Fluor 488-conjugated anti-rat IgG (1:1000). The
fluorescence data were collected using the SA3800 Cell
Analyzer (Sony Corp., Tokyo, Japan).

Table 1. Identification of the C4Mab-1 Epitope Using Alanine-Substituted Mouse
C-C Chemokine Receptor 4 Peptides
PeptidesSequence

Reactivity
with C4Mab-1

WT
M1A
N2A
A3G
T4A
E5A
V6A
T7A
D8A
T9A
T10A
Q11A
D12A
E13A
T14A
V15A
Y16A
N17A
S18A
Y19A
Y20A

MNATEVTDTTQDETVYNSYYANATEVTDTTQDETVYNSYYMAATEVTDTTQDETVYNSYYMNGTEVTDTTQDETVYNSYYMNAAEVTDTTQDETVYNSYYMNATAVTDTTQDETVYNSYYMNATEATDTTQDETVYNSYYMNATEVADTTQDETVYNSYYMNATEVTATTQDETVYNSYYMNATEVTDATQDETVYNSYYMNATEVTDTAQDETVYNSYYMNATEVTDTTADETVYNSYYMNATEVTDTTQAETVYNSYYMNATEVTDTTQDATVYNSYYMNATEVTDTTQDEAVYNSYYMNATEVTDTTQDETAYNSYYMNATEVTDTTQDETVANSYYMNATEVTDTTQDETVYASYYMNATEVTDTTQDETVYNAYYMNATEVTDTTQDETVYNSAYMNATEVTDTTQDETVYNSYA

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

+++, OD655 S 0.6; ++, 0.3 & OD655 < 0.6; -, OD655 < 0.1.

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ASANO ET AL.

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critical epitope of C4Mab-1 on mCCR4 (Table 1). Eachalanine-substituted mutant peptide was immobilized. Noneof peptide was immobilized as a negative control (NC). The
results of ELISA showed that C4Mab-1 reacted with 16
alanine-substituted mutant peptides (M1A, N2A, A3G, T4A,
E5A, T9A, T10A, D12A, E13A, T14A, V15A, Y16A, N17A,
S18A, Y19A, Y20A) as well as the 1–20 amino acids wildtype sequence (WT; positive control) (Fig. 1).
In contrast, C4Mab-1 did not react with three mutant
peptides, T7A, D8A, and Q11A, as well as the NC (Fig. 1),
These results indicated that Thr7, Asp8, and Gln11 of
mCCR4 are the critical amino acids for the C4Mab-1 binding.
The results are summarized in Table 1.

FIG. 1. Determination of the C4Mab-1 epitope for
mCCR4 by ELISA using point mutant peptides. Synthesizedpeptides of mCCR4 were immobilized on immunoplates.
The plates were incubated with C4Mab-1 (1 mg/mL), followed by incubation with peroxidase-conjugated anti-rat
immunoglobulins. ELISA, enzyme-linked immunosorbent
assay; NC, negative control; mCCR4, mouse C-C chemokine receptor 4.
Results
Epitope mapping by ELISA

C4Mab-1 was established using the mCCR4 N-terminal
peptide immunization. Therefore, we synthesized 20 alaninesubstituted mCCR4 N-terminal peptides to investigate thePeptide blocking assay by flow cytometry

We then performed a peptide blocking assay by flow
cytometry. C4Mab-1 was mixed with each peptide or
blocking buffer (–peptide). The reactivity between
CHO/mCCR4 and peptide mixed C4Mab-1 was measured
by flow cytometry. C4Mab-1 reacted with CHO/mCCR4(Fig. 2A, –peptide). This reaction was completely neutralized by mixing WT, M1A, N2A, A3G, T4A, E5A, D12A,
E13A, T14A, V15A, Y16A, N17A, S18A, Y19A, and Y20A
peptide with C4Mab-1, and almost neutralized by mixing
V6A, T9A, and T10A peptide with C4Mab-1. In contrast,
T7A, D8A, and Q11A peptides did not block the reaction of
C4Mab-1 with CHO/mCCR4 (Fig. 2A). These results

FIG. 2. Determination of the C4Mab-1 epitope for mCCR4 by peptide blocking assay. (A) C4Mab-1 (1 mg/mL) plus eachpoint mutant peptide (10 mg/mL), control (blocking buffer; –peptide) was reacted with CHO/mCCR4 cells for 30 minutes at
4C, followed by the addition of secondary antibodies. (B) Schematic illustration of mCCR4 and the C4Mab-1 epitope.
Underlines indicate the critical amino acids for C4Mab-1 binding. CHO, Chinese hamster ovary.

EPITOPE MAPPING OF ANTI-CCR4 MAB

indicated that Thr7, Asp8, and Gln11 of mCCR4 are essential for C4Mab-1 binding to CHO/mCCR4. The results
are summarized in Figure 2B.

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Discussion

Epitope identification of mAbs is important to elucidate
the pharmacological function of mAbs and is essential to
avoid unexpected cross-reactivity. We previously developed
an anti-mCCR4 mAb (clone C4Mab-1) by immunizing themCCR4 N-terminal peptide.(30) C4Mab-1 can be applicable
to flow cytometric analysis. However, its epitope has not been
determined. In this study, we identified the epitope of C4Mab1 using two methods, ELISA and peptide blocking assay. The
results of ELISA and peptide blocking assay indicated thatthree amino acids, Thr7, Asp8, and Gln11 of mCCR4, arecritical for C4Mab-1 binding (Figs. 1 and 2).
Molecular targeted therapy is one of the strategies forcancer treatment, and mAb is an important modality for it.
The main antitumor mechanisms of mAbs are neutralizationof growth factors, ADCC, and complement-dependent cytotoxicity (CDC). In addition, mogamulizumab can deplete
Treg cells, which is expected for the application to solid
tumors as an immunomodulator.(62,63) To confirm whether
C4Mab-1 can be available for cancer therapy, further investigations are required. For investigation of ADCC and CDC activities, C4Mab-1 needs to be changed into mouse IgG2a.(64–70)
A neutralizing mAb blocks interaction between a receptor
and its ligand. The ligand-binding region of CCR4 to its ligands has not been reported; in contrast, the N-terminal regions of several chemokine receptors, such as CCR2, CCR3,
CCR5, and CXCR1, have been determined to be critical forthe ligand–receptor binding.(29) Thus, the N-terminus of
CCR4 may also be important for the ligand binding. C4Mab-1
binds to N-terminus region of mCCR4; therefore, C4Mab-1 isa potential neutralizing mAb. In a future study, we will investigate neutralization activity of C4Mab-1.
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. JP22ama121008 (Y.K.), JP21am0401013 (Y.K.), andJP21am0101078 (Y.K.).
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ASANO ET AL.

Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 30, 2022
Accepted: June 28, 2022