MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 6, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0020
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Epitope Mapping of an Anti-Mouse CCR2 Monoclonal
Antibody (C2Mab-6) Using Enzyme-Linked
Immunosorbent Assay

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Tomohiro Tanaka,1 Hiroyuki Suzuki,2 Teizo Asano,1 Guanjie Li,2 Ren Nanamiya,1 Nami Tateyama,1 Yu Isoda,1Yuki Okada,2 Hiyori Kobayashi,2 Takeo Yoshikawa,3 Mika K. Kaneko,1 and Yukinari Kato1–3

CC chemokine receptor type-2 (CCR2) is a member of the G protein-coupled receptors, and is mainly
expressed on cell surface of immune cells. CCR2 binds to its ligand, C-C motif chemokine 2 (also named as
monocyte chemoattractant protein-1), which involves in the tumor progression by modulating the tumor
microenvironment. Therefore, the monoclonal antibody (mAb) targeting CCR2 could be one of the strategies
for cancer treatment. In this study, we investigated the critical epitope of C2Mab-6, an anti-mouse CCR2(mCCR2) mAb developed by N-terminal peptides immunization. We first performed enzyme-linked immunosorbent assay (ELISA) using N-terminal peptides of mCCR2 and demonstrated that C2Mab-6 recognizes 1–19 amino acids of mCCR2. We further performed ELISA using 20 alanine-substituted peptides ofmCCR2. C2Mab-6 lost the reaction to the alanine-substituted peptides of D3A, N4A, M6A, P8A, Q9A, andF10A. These results indicate that the binding epitope of C2Mab-6 includes Asp3, Asn4, Met6, Pro8, Gln9,and Phe10 of mCCR2.
Keywords: mouse CCR2, C2Mab-6, epitope, monoclonal antibody, enzyme-linked immunosorbent assay

Introduction

C

hemokine receptors are G protein-coupled receptor(GPCR) with seven transmembrane regions, which arelocalized on the plasma membrane. Chemokines are divided into four different subfamilies of XC, CC, CXC, andCX3C, according to the number and position of conserved
N-terminus cysteine residues.1 Chemokines orchestrate thecellular function of immune cells through their interaction
with their receptors, as represented by cell migration.2
CC chemokine receptor type-2 (CCR2) is expressed invarious immune cells including T lymphocytes, natural killercells, macrophages, dendritic cells, and monocytes. CCR2 isprimarily involved in the regulation of migration and positioning of immune cells.3,4 CCR2 is the major receptor ofC-C motif chemokine 2 (CCL2)/monocyte chemoattractant
protein-1. CCL2–CCR2 axis plays pivotal roles in the regulation of immune system.5At the inflammation site, CCR2-expressing cells are associated with tissue damage. During influenza A virus infection, interferon-g-stimulated CCR2+ monocytes infiltration
has been reported as a driver of lung damage.6 High levelsof CCR2 in peripheral blood have been detected in severe
COVID-19 patients.7 Genome-wide studies suggest that
3p21.31 is involved in the risk of severe COVID-19 aggravation and is involved in upregulation of chemokine receptors, including CCR2 in monocytes and macrophages.8CCL2 expression has been reported to be upregulatedin several tumors, including breast, bladder, and bone cancers.9–11 High CCR2 expression in tumor-infiltrating immune
and stromal cells has been confirmed,12–14 and associatedwith poor prognosis.15,16 Therefore, the CCL2–CCR2 axis isthought to be an important target for cancer therapy.
We have developed monoclonal antibodies (mAbs) against
chemokine receptors, including mouse CCR2 (mCCR2),17mouse CCR3,18 mouse CCR4,19 mouse CCR8,20 human

Departments of 1Antibody Drug Development, 2Molecular Pharmacology, and 3Pharmacology, Tohoku University Graduate School ofMedicine, Aoba-ku, Sendai, Japan.

339

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TANAKA ET AL.

CCR9,21 and mouse CXCR622 by the Cell-Based Immunization and Screening method or the N-terminal peptide
immunization method. In this study, we investigated theepitope of an anti-mCCR2 mAb, C2Mab-6,17 by using
enzyme-linked immunosorbent assay (ELISA).
Materials and Methods

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Enzyme-linked immunosorbent assay

The mCCR2 peptides (accession no.: NM_009915),
including three N-terminal peptides (Table 1) and 20 point
mutants (Table 2), were synthesized by utilizing PEPscreen(Sigma-Aldrich Corp., St. Louis, MO). Each peptide wasimmobilized on Nunc Maxisorp 96-well immunoplates(Thermo Fisher Scientific, Inc., Waltham) at a concentration of 10 lg/mL for 30 minutes at 37C. After washingwith phosphate-buffered saline containing 0.05% Tween20(PBST), wells were blocked with 1% bovine serum albumincontaining PBST for 30 minutes at 37C.
The plates were then incubated with C2Mab-6 (1 lg/mL),followed by a 1:2000 (1:20000) dilution of peroxidaseconjugated anti-rat immunoglobulins (Sigma-Aldrich Corp.).
Enzymatic reactions were performed using the ELISA POD
Substrate TMB Kit (Nacalai Tesque, Inc., Japan). Opticaldensity was detected at 655 nm using an iMark microplate
reader (Bio-Rad Laboratories, Inc., Berkeley, CA).

Table 2. Identification of the C2Mab-6 Epitope
Using Alanine-Substituted Mouse CCR2 Peptides
PeptidesSequences

C2Mab-6

WT
M1A
E2A
D3A
N4A
N5A
M6A
L7A
P8A
Q9A
F10A
I11A
H12A
G13A
I14A
L15A
S16A
T17AS18A
H19A
S20A

MEDNNMLPQFIHGILSTSHS
AEDNNMLPQFIHGILSTSHSMADNNMLPQFIHGILSTSHSMEANNMLPQFIHGILSTSHS
MEDANMLPQFIHGILSTSHS
MEDNAMLPQFIHGILSTSHSMEDNNALPQFIHGILSTSHSMEDNNMAPQFIHGILSTSHSMEDNNMLAQFIHGILSTSHSMEDNNMLPAFIHGILSTSHSMEDNNMLPQAIHGILSTSHSMEDNNMLPQFAHGILSTSHSMEDNNMLPQFIAGILSTSHSMEDNNMLPQFIHAILSTSHSMEDNNMLPQFIHGALSTSHSMEDNNMLPQFIHGIASTSHSMEDNNMLPQFIHGILATSHSMEDNNMLPQFIHGILSASHSMEDNNMLPQFIHGILSTAHSMEDNNMLPQFIHGILSTSASMEDNNMLPQFIHGILSTSHA+++++++––+++–+++–––++
+++
+++
+++
+++
+++
+++
+++
+++
+++

+++, OD655 S 0.3; ++, 0.2 & OD655 < 0.3; +, 0.1 & OD655 <0.2; -, OD655 < 0.1.

Results
Epitope identification using N-terminal
mCCR2 peptides

An anti-mCCR2 mAb, C2Mab-6 (rat IgG1, kappa), wasestablished by immunization with the mixture of threekeyhole limpet hemocyanin-conjugated mCCR2 N-terminal
peptides.17 The sequences are listed in Table 1. To characterize the binding epitope of C2Mab-6, we first investigated
the reactivity of C2Mab-6 to the N-terminal synthetic peptides: 1–19 amino acids (aa), 11–29 aa, and 21–39 aa ofmCCR2 (Table 1). The results of ELISA demonstrated thatC2Mab-6 recognized 1–19 aa of mCCR2 (Fig. 1A). Theseresults are summarized in Figure 1B.
Epitope identification using alanine-substituted
mCCR2 peptides

We further synthesized 20 different alanine-substituted
mCCR2 peptides (Table 2). The results of ELISA demonstrated that C2Mab-6 bound to point mutants, such as M1A,
E2A, N5A, L7A, I11A, H12A, G13A, I14A, L15A, S16A,T17A, S18A, H19A, and S20A as well as the 1–20 aa wild
type (WT) sequence (positive control) (Fig. 2A). In contrast,
C2Mab-6 did not bind to point mutants, such as the D3A,

Table 1. Identification of the C2Mab-6 Epitope
Using N-Terminal Mouse CCR2 Peptides
PeptidesSequences

C2Mab-6

1–19
11–29
21–39

MEDNNMLPQFIHGILSTSH
IHGILSTSHSLFTRSIQELLFTRSIQELDEGATTPYDY+++––+++, OD655 S 0.3; -, OD655 < 0.1.

FIG. 1. Identification of the C2Mab-6 epitope for mCCR2
by ELISA using N-terminal peptides. (A) N-terminal synthesized peptides of mCCR2 were immobilized on immunoplates. The plates were incubated with C2Mab-6(1 mg/mL) followed by incubation with peroxidaseconjugated anti-rat immunoglobulins. (B) Schematic illustration of mCCR2 and the C2Mab-6 epitope. ELISA,
enzyme-linked immunosorbent assay; mCCR2, mouse
CCR2.

EPITOPE MAPPING OF C2MAB-6341

ity that C2Mab-9 affects the CCL2–CCR2 interaction. The
29-GAPCH-33 sequence in human CCR2 is conserved as
42-GEPCH-46 in mCCR2. Since the C2Mab-6 epitope is apartfrom the CCL2-binding sequence in mouse, further studies
are essential to evaluate the biological activity of C2Mab-6.
The inhibition of CCR2 functions has been reported toenhance the antitumor immunity of immune checkpoint
blockades including anti-programmed cell death-1 mAb inmouse model.11 C2Mab-6 could be useful for the investigation of mCCR2 expression in immune cells, and the combination therapy with immune checkpoint blockade in micetumor models.
Author Disclosure Statement

No competing financial interests exist.

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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.).
References

FIG. 2. Identification of the C2Mab-6 epitope for mCCR2by ELISA using alanine-substituted peptides. (A) Synthesized peptides of mCCR2 were immobilized on immunoplates. The plates were incubated with C2Mab-6(1 mg/mL), followed by peroxidase-conjugated anti-rat
immunoglobulins. (B) Schematic illustration of mCCR2
and the C2Mab-6 epitope. The C2Mab-6 epitope of mCCR2involves Asp3, Asn4, Met6, Pro8, Gln9, and Phe10.
N4A, M6A, P8A, Q9A, and F10A (Fig. 2A), indicating thatAsp3, Asn4, Met6, Pro8, Gln9, and Phe10 were determined
to be the critical aa, included in the C2Mab-6 epitope. The
results are summarized in Figure 2B.
Discussion

We previously developed an anti-human CCR2 mAb(clone C2Mab-9) by using N-terminal peptide immunization
method and determined the epitope of C2Mab-9 as Phe23,Phe24, Asp25, and Tyr26.23 In this study, we successfully
determined that the epitope of an anti-mCCR2 mAb (cloneC2Mab-6)17 as Asp3, Asn4, Met6, Pro8, Gln9, and Phe10(Fig. 2). The N-terminal regions of some GPCRs, including
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Furthermore, N-terminal Gly29, Ala30, Cys32, and His33 ofCCR2, closed to the C2Mab-9 epitope, are also involved inthe interaction with CCL2.24 Therefore, there is a possibil-

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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: yukinari.kato.e6@tohoku.ac.jp
Received: May 21, 2022
Accepted: September 30, 2022