MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 3, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0012
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Epitope Mapping of the Anti-Human CCR2
Monoclonal Antibody C2Mab-9

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

CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, localized oncell surface of some immune-related cells, including monocytes and macrophages. CCR2 and its ligand CCL2
are involved in the progression of various diseases such as cancers. Therefore, CCR2-targeted monoclonal
antibodies (mAbs) are needed for treatment and diagnosis. Previously, we successfully developed an antihuman CCR2 (hCCR2) mAb, C2Mab-9 (mouse IgG1, kappa), which is applicable for flow cytometry andimmunocytochemistry. In this study, we investigated the critical epitope of C2Mab-9. We conducted enzymelinked immunosorbent assay (ELISA) using several N-terminal peptides of hCCR2, and demonstrated that
C2Mab-9 recognizes 11–29 and 21–39 amino acids of hCCR2. We further performed ELISA using 20 peptides,
which include alanine substitution of hCCR2. C2Mab-9 lost the reaction to the alanine-substituted peptides of
F23A, F24A, D25A, Y26A, and D27A. Among them, F23A, F24A, D25A, and Y26A did not block the C2Mab9 reaction with U937 cells in flow cytometry. These results indicate that the critical binding epitope of C2Mab-9
includes Phe23, Phe24, Asp25, and Tyr26.
Keywords: human CCR2, C2Mab-9, epitope, monoclonal antibody, enzyme-linked immunosorbent assay, flow
cytometry
Introduction

C

hemokines play pivotal roles in cellular function,
including immune responses.(1,2) They are divided
into four different subfamilies of XC, CC, CXC, and CX3C,
depending on the number and position of cysteine residues
at the N-terminus.(3,4) Chemokine receptors are G proteincoupled receptor (GPCR) with seven transmembraneregions.
CC chemokine receptor type-2 (CCR2) is expressed in
multiple cells, including epithelial cells, monocytes, macrophages, and dendritic cells. CCR2 is involved in the regulation of migration and infiltration of immune-related cells.(5–8)
CCR2 is the primary receptor of C-C motif chemokine 2(CCL2)/monocyte chemoattractant protein-1. CCL2 also
plays an important role in attracting the key immune regulators, including T lymphocytes, natural killer cells, andmonocytes.(5,9) CCL2-CCR2 axis is correlated with many
diseases such as immune disorders and cancers.(10,11)

In respiratory organs, CCL2 and CCR2-expressing macrophages and neutrophils contribute to the innate immune responses during viral and bacterial infection.(12)
CCR2 induces neutrophil organ entry in sepsis.(13) HighCCR2 levels in blood samples have been detected in patientswith severe COVID-19.(14) Furthermore, CCL2 has beenreported to be increased in several tumors, such as bladder,(15) bone,(16) and inflammatory breast cancers.(17) HighCCR2 expression has been confirmed in the invasive lesionof breast cancers as well as CCL2 expression.(18) Inhibitionof CCR2 has been reported to enhance the effectiveness of
immune checkpoint inhibitors, such as an anti-programmedcell death-1 monoclonal antibody (mAb) in mouse tumor
models, and clinical trials are ongoing.(15,19)
We have produced numerous mAbs against membraneproteins, including EGFR,(20) HER2,(21) HER3,(22) CD20,(23)CD44,(24) CD133,(25) and podoplanin,(26–30) and determined
their epitopes.(31–34) Moreover, we have also developed
anti-GPCR mAbs, including anti-mouse CCR2,(35) mouse

Departments of 1Antibody Drug Development and 2Molecular Pharmacology, Tohoku University Graduate School of Medicine,
Sendai, Japan.
*These authors contributed equally.

150

DETERMINATION OF C2MAB-9 EPITOPE

151

Table 1. Identification of the C2Mab-9
Epitope Using N-Terminal Human CC
Chemokine Receptor 2 Peptides

Table 2. Identification of the C2Mab-9 Epitope
Using Alanine-Substituted Human CC
Chemokine Receptor 2 Peptides

PeptidesSequences

C2Mab-9

PeptidesSequences

C2Mab-9

1–19
11–29
21–39

MLSTSRSRFIRNTNESGEE
RNTNESGEEVTTFFDYDYG
TTFFDYDYGAPSHKFDVKQ

+++
+++

T21A
T22A
F23A
F24A
D25A
Y26A
D27A
Y28A
G29A
A30G
P31A
S32A
H33A
K34A
F35A
D36A
V37A
K38A
Q39A
I40A

ATFFDYDYGAPSHKFDVKQITAFFDYDYGAPSHKFDVKQITTAFDYDYGAPSHKFDVKQITTFADYDYGAPSHKFDVKQITTFFAYDYGAPSHKFDVKQI
TTFFDADYGAPSHKFDVKQI
TTFFDYAYGAPSHKFDVKQI
TTFFDYDAGAPSHKFDVKQI
TTFFDYDYAAPSHKFDVKQI
TTFFDYDYGGPSHKFDVKQI
TTFFDYDYGAASHKFDVKQI
TTFFDYDYGAPAHKFDVKQI
TTFFDYDYGAPSAKFDVKQI
TTFFDYDYGAPSHAFDVKQI
TTFFDYDYGAPSHKADVKQI
TTFFDYDYGAPSHKFAVKQI
TTFFDYDYGAPSHKFDAKQITTFFDYDYGAPSHKFDVAQI
TTFFDYDYGAPSHKFDVKAI
TTFFDYDYGAPSHKFDVKQA

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

+++, OD655S0.3; -, OD655 < 0.1.

CCR3,(36) mouse CCR4,(37) mouse CCR8,(38) and human
CCR9,(39) and also determined the binding epitope.(40) In
this study, we performed the epitope mapping of C2Mab-9
by using enzyme-linked immunosorbent assay (ELISA) andflow cytometric methods.

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Materials and Methods
Cell lines

The U937 (human histiocytic lymphoma) cell line wasobtained from the Japanese Collection of Research Bioresources (Osaka, Japan). U937 cells were cultured in a
Roswell Park Memorial Institute 1640 medium (Nacalai
Tesque, Inc., Kyoto, Japan) that was supplemented with
10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific, Inc., Waltham, MA), 100 units/mL penicillin,+++, OD655S0.3; -, OD655 < 0.1.

FIG. 1. Determination of the C2Mab-9 epitope for hCCR2 by ELISA using N-terminal peptides. (A) N-terminal synthesized peptides of hCCR2 were immobilized on immunoplates. The plates were incubated with C2Mab-9 (1 mg/mL)
followed by incubation with peroxidase-conjugated anti-mouse immunoglobulins. (B) Schematic illustration of hCCR2 andthe C2Mab-9 epitope. ELISA, enzyme-linked immunosorbent assay; hCCR2, human CC chemokine receptor 2.

152

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.
ELISA

were then incubated with C2Mab-9 (1 lg/mL), followed
by a 1:2000 dilution of peroxidase-conjugated anti-mouse
immunoglobulins (Agilent Technologies, Inc., Santa Clara,
CA). Enzymatic reactions were performed using the ELISAPOD Substrate TMB Kit (Nacalai Tesque, Inc.). Opticaldensity was measured at 655 nm using an iMark microplate
reader (Bio-Rad Laboratories, Inc., Berkeley, CA).

Flow cytometry

U937 cells were washed with 0.1% BSA in PBS. C2Mab-9(1 lg/mL) was incubated with each peptide (10 lg/mL) for 30
minutes at 4C. U937 cells were treated with C2Mab-9+each
peptide, and further with Alexa Fluor 488-conjugated antimouse IgG (1:1000). The fluorescence data were collectedusing a BD FACSLyric (BD Biosciences, Franklin Lakes, NJ).

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The human CCR2 (hCCR2) peptides (Accession No.
NM_001123041), including 3 N-terminal peptides (Table 1)
and 20 point mutants (Table 2), were synthesized by utilizing PEPScreen (Sigma-Aldrich Corp., St. Louis, MO). Eachpeptide was immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at a concentration of 10 lg/mL for 30 minutes at 37C. After washing withphosphate-buffered saline (PBS) containing 0.05% Tween20(PBST), wells were blocked with 1% bovine serum albumin(BSA)-containing PBST for 30 minutes at 37C. The plates

TANAKA ET AL.

FIG. 2. Determination of the C2Mab-9 epitope for hCCR2 by ELISA using point mutants. (A) Synthesized peptides ofhCCR2 were immobilized on immunoplates. The plates were incubated with C2Mab-9 (1 mg/mL), followed by peroxidaseconjugated anti-mouse immunoglobulins. (B) Schematic illustration of hCCR2 and the C2Mab-9 epitope. The C2Mab-9
epitope of hCCR2 involves Phe23, Phe24, Asp25, Tyr26, and Asp27.

DETERMINATION OF C2MAB-9 EPITOPE
Results
Epitope mapping using N-terminal hCCR2 peptides

Epitope mapping using alanine-substituted
hCCR2 peptides

We further synthesized 20 different alanine-substituted
hCCR2 peptides (Table 2). The results of ELISA demonstrated that C2Mab-9 reacted with point mutants, such as
T21A, T22A, Y28A, G29A, A30G, P31A, S32A, H33A,
K34A, F35A, D36A, V37A, K38A, Q39A, and I40A, as
well as the 21–40 aa wild-type sequence (positive control)(Fig. 2A). In contrast, C2Mab-9 did not bind to point mutants,
such as the F23A, F24A, D25A, Y26A, and D27A (Fig. 2A),
indicating that Phe23, Phe24, Asp25, Tyr26, and Asp27 were
determined to be the critical aa, which are included in the
C2Mab-9 epitope. The results are summarized in Figure 2B.

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In our previous study, we developed an anti-hCCR2
mAb (clone C2Mab-9) using synthetic peptide immunization method.(41) C2Mab-9 is suitable for in vitro applications, including flow cytometry and immunocytochemistry.
To characterize the binding epitope of C2Mab-9 for hCCR2,
we synthesized three N-terminal peptides: 1–19 amino acids(aa), 11–29 aa, and 21–39 aa (Table 1). The results of ELISA
demonstrated that C2Mab-9 reacted with 11–29 aa and 21–39
aa of hCCR2 (Fig. 1A). These results are summarized in
Figure 1B.

153

FIG. 3. Flow cytometry using C2Mab-9 and peptides of hCCR2. (A) C2Mab-9 (1 mg/mL), C2Mab-9 (1 mg/mL) plus
peptides (T22A, F23A, F24A, D25A, Y26A, and D27A; 10 mg/mL), or control (blocking buffer) were reacted with U937
cells for 30 min at 4C, followed by the addition of secondary antibodies. (B) Schematic illustration of hCCR2 and theC2Mab-9 epitope. The C2Mab-9 epitope of hCCR2 involves Phe23, Phe24, Asp25, and Tyr26.

154
Flow cytometry using C2Mab-9 and alanine-substituted
hCCR2 peptides

We then performed a blocking assay using flow cytometry. C2Mab-9 reacted with the U937 cell line (Fig. 3A).
This reaction was completely neutralized by T22A andD27A. In contrast, F23A, F24A, D25A, and Y26A did not
block the reaction of C2Mab-9 for U937 cells (Fig. 3A),
indicating that Phe23, Phe24, Asp25, and Tyr26 of hCCR2
are critical for C2Mab-9 detection. The results are summarized in Figure 3B.

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Discussion

Design of immunogen and epitope identification are
essential for the development of mAbs and vaccines.(42,43)
Furthermore, the determination of the mAb epitope can leadto the development of peptide tag systems for protein extraction, purification, or detection assays.(44–47)
We previously developed an anti-human CCR9 mAb(clone C9Mab-1) by using Cell-Based Immunization andScreening method and clarified that the epitope of C9Mab-1
is located on the N-terminus of the human CCR9.(39,40)
Then, we have developed several anti-GPCR mAbs, including anti-mouse CCR2,(35) human CCR2,(41) mouse CCR3,(48)and mouse CCR4(37) by immunizing mice or rats with the
N-terminal peptides of GPCRs.
In this study, we identified the critical epitope of C2Mab-9
as Phe23, Phe24, Asp25, Tyr26, and Asp27 using ELISA(Fig. 2). In contrast, blocking assay using flow cytometry revealed that Phe23, Phe24, Asp25, and Tyr26 of hCCR2 werecritical epitope of C2Mab-9 (Fig. 3). This discrepancy might becaused by the condition of peptides: solid phase of ELISA or
liquid phase of flow cytometry. We have previously developed
various novel epitope mapping system, named RIEDL insertion for epitope mapping (REMAP)(49–52) and histidine-tag
insertion for epitope mapping (HisMAP) method.(53) Thesemethods could be applied to determine linear and structural
epitopes. Therefore, we should determine the binding epitopeof C2Mab-9 using REMAP and HisMAP methods in the future
study.
CCR2 is a seven-transmembrane receptor with four
extracellular regions (Figs. 2B and 3B). N-terminal domains of some GPCRs, such as CCR2, CCR3, CCR5, andCXCR1, have been determined as their ligand-bindingsites.(54) The several aa in the N-terminal region of
CCR2B (predominant isoform of CCR2) were reported to
be important for CCL2-triggered cell migration and lamellipodium formation,(55) indicating that C2Mab-9
might be advantageous for the functional study about the
CCL2-CCR2 axis.
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 (to Y.K.), JP22am0401013 (to Y.K.),
JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), andJP21am0101078 (to Y.K.).

TANAKA ET AL.
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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 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: February 24, 2022
Accepted: April 19, 2022