MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 5, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0018
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Epitope Mapping of the Anti-Human CC Chemokine
Receptor Type-2 Monoclonal Antibody (K036C2)

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

CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, and is localized
on cell surface of tumor cells and some immune cells, including monocytes and macrophages. CCR2 is a
receptor for monocyte chemoattractant protein-1/C-C motif chemokine 2, and is involved in the progressionof various diseases such as cancers. Therefore, the development of CCR2-targeted monoclonal antibody (mAb)
is desired. Its characterization, including epitope of mAb, is very important for antibody applications. In thisstudy, we investigated the critical epitope of K036C2, which is a commercially available anti-human CCR2(hCCR2) mAb. We conducted enzyme-linked immunosorbent assay (ELISA) using three N-terminal peptidesof hCCR2 and demonstrated that K036C2 recognizes 11–29 and 21–39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. K036C2 lost thereaction to the alanine-substituted peptides of D25A, Y26A, D27A, G29A, and A30G. These results indicatethat the critical binding epitope of K036C2 includes Asp25, Tyr26, Asp27, Gly29, and Ala30 of hCCR2.
Keywords: human CCR2, K036C2, epitope, monoclonal antibody, enzyme-linked immunosorbent assay

Introduction

C

hemokine receptors belong to the G protein-coupled
receptor (GPCR) with seven transmembrane regions.
Chemokines are a family of cytokines, and divided into four
different subfamilies of XC, CC, CXC, and CX3C, depending on the number and position of N-terminus cysteine residues.1,2 Chemokines orchestrate many cellular functions
including immune responses.3–5
CC chemokine receptor type-2 (CCR2) is expressed invarious cell types, including epithelial cells, macrophages,
dendritic cells, and monocytes. CCR2 is involved in theregulation of migration and positioning of immune-related
cells.6–8 CCR2 is the primary receptor of C-C motif chemokine 2 (CCL2)/monocyte chemoattractant protein-1(MCP-1). CCL2 and CCR2 play an important role in regulating the key immune regulators, including T lymphocytes,
natural killer cells, stromal cells, and monocytes.9,10 CCL2CCR2 axis is correlated with many diseases such as immune
disorders and cancer.8,11,12

CCR2-expressing cells are often involved in tissue damage at the site of inflammation.13 Interferon-g-regulated
CCR2+ monocytes become a driver of lung damage during influenza A virus infection.14 During viral and bacterial
infection in respiratory organs, the recruitment of macrophages and neutrophils mediated by CCL2-CCR2 axis contribute to the innate immune responses.15 High CCR2 levelsin blood samples have been detected in patients with severe
COVID-19.16
Furthermore, CCL2 expression has been reported to be
upregulated in several tumors, such as inflammatory breast
cancers, bladder cancers, and bone tumors.9,17,18 High CCR2
expression has been confirmed in the invasive lesion ofbreast cancers and melanoma.19 In sarcoma, CCR2 correlateswith poor prognosis by regulating the infiltration of multiple
immune and stromal cells in tumor microenvironment.20,21 In
cancer treatment, the blockade of CCR2 functions has beenreported to enhance the effectiveness of immune checkpoint
inhibitors, such as an anti-programmed-cell death-1 monoclonal antibodies (mAbs) in mouse models.18

Departments of 1Antibody Drug Development, 2Molecular Pharmacology, and 3Pharmacology, Tohoku University Graduate Schoolof Medicine, Sendai, Japan.

285

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TANAKA ET AL.

We have produced numerous mAbs against GPCRs,
including mouse CCR2,22 mouse CCR3,23–25 mouse CCR4,26
mouse CCR8,27 and human CCR9,28 and also determined
the binding epitope.29 In addition, we further determined
the binding epitope of mAbs against numerous membraneproteins, including CD20,30 CD44,31,32 CD133,33 andpodoplanin.34–36 In this study, we performed the epitope identification of anti-human CCR2 mAb (K036C2) byusing enzyme-linked immunosorbent assay (ELISA).
Materials and Methods

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Enzyme-linked immunosorbent assay

The human CCR2 peptides (Accession No. NM_
001123041), including three N-terminal peptides (Table 1)
and 20 alanine-substituted mutants (Table 2), were synthesized by utilizing PEPscreen (Sigma-Aldrich Corp., St.
Louis, MO). Each peptide was immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.,
Waltham, MA) at a concentration of 10 lg/mL for 30 minutes
at 37C. After washing with phosphate-buffered saline containing 0.05% Tween20 (PBST), wells were blocked with 1%
bovine serum albumin-containing PBST for 30 minutes at
37C. The plates were then incubated with K036C2 (BioLegend, San Diego, CA) (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 ELISA POD Substrate TMB
Kit (Nacalai Tesque, Inc., Kyoto, Japan). Optical density wasmeasured at 655 nm using an iMark microplate reader (BioRad Laboratories, Inc., Berkeley, CA).

Table 2. Identification of the K036C2 Epitope
Using Alanine-Substituted Human CC Chemokine
Receptor Type-2 Peptides
PeptidesSequences

K036C2

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

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

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

Results
Epitope determination of K036C2 using N-terminal
hCCR2 peptides

To characterize the binding epitope of anti-hCCR2 mAb,
K036C2, 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 K036C2
reacted with 11–29 aa and 21–39 aa of hCCR2 (Fig. 1A).
These results are summarized in Figure 1B.
Epitope determination of K036C2 using
alanine-substituted hCCR2 peptides

We further synthesized 20 different alanine-substituted
hCCR2 peptides (Table 2). The results of ELISA demonstrated that K036C2 reacted with point mutants, such as
T21A, T22A, F23A, F24A, Y28A, P31A, S32A, H33A,

Table 1. Identification of the K036C2 Epitope
Using N-Terminal Human CC Chemokine Receptor
Type-2 Peptides
PeptidesSequences

K036C2

1–19
11–29
21–39

MLSTSRSRFIRNTNESGEE
RNTNESGEEVTTFFDYDYG
TTFFDYDYGAPSHKFDVKQ

–
+++
+++

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

FIG. 1. Determination of the K036C2 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 K036C2 (1 mg/mL)
followed by incubation with peroxidase-conjugated antimouse immunoglobulins. (B) Schematic illustration ofhCCR2 and the K036C2 epitope. ELISA, enzyme-linked
immunosorbent assay; hCCR2, human CC chemokine
receptor type-2.

EPITOPE OF ANTI-CCR2 MAB

K34A, F35A, D36A, V37A, K38A, Q39A, and I40A as wellas the 21–40 aa wild-type sequence (positive control)(Fig. 2A). In contrast, K036C2 did not bind to alaninesubstituted hCCR2 peptides, such as D25A, Y26A, D27A,
G29A, and A30G (Fig. 2A), indicating that Asp25, Tyr26,
Asp27, Gly29, and Ala30 were determined to be the criticalaa, which are included in the K036C2 epitope. The results are
summarized in Figure 2B.

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Discussion

CCR2 is a seven-transmembrane receptor with four extracellular regions (Fig. 2B). N-terminal domains of some
GPCRs, such as CCR2, CCR3, CCR5, and CXCR1, havebeen determined as their ligand-binding sites.2 We previously developed an anti-hCCR2 mAb (clone C2Mab-9) byusing N-terminal peptide immunization method and clarified the epitope of C2Mab-9 as Phe23, Phe24, Asp25, andTyr26.37 We identified the critical epitope of K036C2 as

287

Asp25, Tyr26, Asp27, Gly29, and Ala30 using ELISA in thisstudy; therefore, the C2Mab-9 epitope was shown to be different from that of K036C2 (Fig. 2).
We have previously developed various novel epitope
mapping system, named RIEDL insertion for epitope mapping (REMAP)38,39 and histidine-tag insertion for
epitope mapping (HisMAP) method.30 These methods are
effective in determining linear and structural epitopes.
Therefore, we will try to determine the binding epitopeof K036C2 using REMAP and HisMAP methods in the
future study.
The several amino acids in the N-terminal region of
CCR2B (predominant isoform of CCR2) were reported toshow pivotal roles for CCL2-triggered cell migration andlamellipodium formation,40 indicating that anti-hCCR2
mAbs targeting N-terminal region might be advantageous
for the functional study about the CCL2-CCR2 axis. It is
expected that information on epitopes will be helpful for antiGPCR mAb drugs that have high development hurdles.41

FIG. 2. Determination of the K036C2 epitope for hCCR2 by ELISA using alanine-substituted peptides of hCCR2.
(A) Synthesized peptides of hCCR2 were immobilized on immunoplates. The plates were incubated with K036C2(1 mg/mL), followed by peroxidase-conjugated anti-mouse immunoglobulins. (B) Schematic illustration of hCCR2 and the
K036C2 epitope. The K036C2 epitope of hCCR2 involves Asp25, Tyr26, Asp27, Gly29, and Ala30.

288
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
numbers JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.),
JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), andJP21am0101078 (to Y.K.).

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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: April 28, 2022
Accepted: September 26, 2022